Microbiology

The textbook includes theoretical and practical sections. The theoretical sections

provide information about the development of microbiology, the properties of
microorganisms, the infectious process, immunity and allergies. In practical sections,
according to a strictly defined scheme, the main methods of microbiological research
in individual infections are described. The textbook complies with the program
approved by the Ministry of Health of the USSR and is intended for students of
paramedical laboratory and sanitary paramedical departments of medical schools.

 About the book

 Foreword
 Introduction - L. B. Bogoyavlenskaya
 Brief essay on the history of microbiology - L. B. Bogoyavlenskaya
 Part I. General microbiology
o Chapter 1. Microbiological laboratory - L. B. Bogoyavlenskaya
o Chapter 2. Microscope and microscopic methods of research -
M. Ya. Korn
o Chapter 3. Fundamentals of classification and morphology of
microorganisms - O. B. Orleanskaya
o Chapter 4
o Chapter 5. The influence of environmental factors on
microorganisms - N. A. Belskaya
o Chapter 6. The spread of microorganisms in nature - L. B.
Bogoyavlenskaya
o Chapter 7. Nutrient media and microbiological research - G. I.
Katz
o Chapter 8. Phages - G. I. Katz
o Chapter 9 Chemoprophylaxis and chemotherapy
o Chapter 10. Genetics of microorganisms - F. K. Cherkes
o Chapter 11. The doctrine of infection - F. K. Cherkes
o Chapter 12 immune reactions. Immunoprophylaxis and
immunotherapy of infectious diseases - L. B. Bogoyavlenskaya,
G. I. Katz
o Chapter 13. Allergy - L. B. Bogoyavlenskaya
 Part II. Private microbiology
o Pathogenic cocci - F.K. Cherkes
o Chapter 14
o Chapter 15
o Chapter 16
o Chapter 17
o family of intestinal bacteria
o Chapter 18. Escherichia - L. B. Bogoyavlenskaya, F. K. Cherkes
o Chapter 19. Salmonella - L. B. Bogoyavlenskaya, F. K. Cherkes
o Chapter 20. Shigella - F.K. Cherkes
o Conditionally pathogenic bacteria - L. B. Bogoyavlenskaya
o Chapter 21
o Chapter 22
o Chapter 23
o Chapter 24
o The causative agents of especially dangerous infections - F. K.
Cherkes
o Chapter 25
o Chapter 26
o Chapter 27
o Chapter 28
o Chapter 29
o Chapter 30
o Bordetella - F.K. Cherkes
o Chapter 31
o Pathogenic corynebacteria - F. K. Cherkes
o Chapter 32
o Pathogenic mycobacteria - F. K. Cherkes
o Chapter 33
o Pathogenic anaerobes - F. K. Cherkes
o Chapter 34
o Chapter 35
o Chapter 36
o Pathogenic spirochetes - F. K. Cherkes
o Chapter 37
o Chapter 38
o Chapter 39
o Chapter 40
o Rickettsia - F.K. Cherkes
o Chapter 41
o Chapter 42
o Viruses
 o RNA viruses
o Chapter 43
o Chapter 44
o Chapter 45
o DNA containing viruses
o Chapter 46
o Unclassifiable viruses
o Chapter 47
o Oncogenic viruses
 Part III. Sanitary microbiology
o Sanitary-bacteriological study of water - F.K. Cherkes
o Sanitary and bacteriological study of the soil - N. A. Belskaya
o Sanitary and bacteriological examination of air - F. K. Cherkes
o Sanitary and bacteriological examination of milk and dairy
products F. K. Cherkes, N. A. Belskaya
o Sanitary and bacteriological examination of cream and cream
products - F. K. Cherkes
o Sanitary and bacteriological study of sausage and meat
products of industrial production - F. K. Cherkes
o Sanitary and bacteriological examination of canned food - F. K.
Cherkes
o Sanitary and bacteriological examination of swabs - F. K.
Cherkes
o Sanitary and bacteriological examination of dressing and
surgical material for sterility - F.K. Cherkes
o Sanitary and bacteriological study of soft drinks - F. K. Cherkes
 Brief Dictionary of Microbiological Terms - G. I. Katz

Source:
Cherkes F.K., Bogoyavlenskaya L.B., Belskaya N.A. 'Microbiology' - Moscow:
Medicine, 1986 - p.512
 About the book

Frida Karlovna Cherkes, Lina Borisovna Bogoyavlenskaya, Natalya Alexandrovna

Belskaya - Microbiology

The textbook includes theoretical and practical sections. The theoretical sections

provide information about the development of microbiology, the properties of
microorganisms, the infectious process, immunity and allergies. In practical sections,
according to a strictly defined scheme, the main methods of microbiological research
in individual infections are described.
The textbook complies with the program approved by the Ministry of Health of the
USSR and is intended for students of paramedical laboratory and sanitary
paramedical departments of medical schools.
Educational literature
For medical students
F. K. Cherkes, L. B. Bogoyavlenskaya, N. A. Belskaya
Microbiology
Edited by F.K. Cherkes
Approved by the Main Directorate of Educational Institutions of the Ministry of
Health of the USSR as a textbook for students of paramedical laboratory and sanitary
paramedical departments of medical schools
Moscow. "Medicine". 1986
LBC 52.6
Ch48
UDC 579(075.8)
Reviewers: A. M. Smirnova, Professor-Consultant of the Department of Microbiology,
II MMI. N. I. Pirogov; I. L. Zaborskaya, Chairman of the Subject Commission on
Microbiology and Epidemiology of the Leningrad Medical School No. 1.
Cherkes F. K., Bogoyavlenskaya L. B., Belskaya N. A.
Ch48 Microbiology / Ed. F. K. Cherkes. - M.: Medicine, 1986. - 512 p., ill.
In lane: 1 p. 40 k. 150,000 copies.
4107000000-241

H 94-86

039(01)-87

© Publishing house "Medicine", Moscow, 1986.

Frida Karlovna Cherkes,
Lina Borisovna Bogoyavlenskaya,
Natalia Aleksandrovna Belskaya
Microbiology
Editor A. M. Smirnova. Editor of the publishing house I. V. Voitekhova. Hood. editor
T. K. Vinokurova. Hardcover by the artist V. S. Sergeeva. Technical editor S. P.
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 Foreword
The decisions of the XXVII Congress of the CPSU and the resolutions of the Central
Committee of the CPSU and the Council of Ministers of the USSR "On measures to
further improve public health" (1977) and "On additional measures to improve public
health" (1982) provide for a set of measures aimed at combating infectious
diseases. The success of these activities largely depends on the training and
qualifications of the middle medical level, who owns modern methods of diagnosing
infectious diseases.
This textbook is the first to combine a theoretical course with detailed methods of
microbiological and virological research.
Knowledge of the theory is necessary to understand the choice of the application of
certain methods of laboratory diagnostics and the correct development of practical
skills, a high level of performance of diagnostic studies can be achieved if workers
know the methods of taking material and the course of the study, therefore, the
authors tried to cover in detail and fully the main methods of microbiological and
virological studies . Their description is based on unified methods that have received
universal recognition and are available for work in laboratories of a wide network.
The textbook contains three parts: general, private and sanitary microbiology. Each
section ends with control questions that help the assimilation and consolidation of
the material covered. A number of chapters have tasks for independent work of
students, which contributes to the development of thinking and skills in
microbiological technology. Identification of the selected pathogen is accompanied
by color schemes that clearly illustrate the methods described. The textbook is
provided with tables and figures. At the end of the textbook there is a glossary of the
main microbiological terms mentioned in the text, which will facilitate the work of
students.
The authors hope that this textbook will help the middle medical level to participate
more qualified in the fight against infectious diseases in our country.
The authors express their deep gratitude to the specialists of the Research Institute
of Epidemiology and Microbiology named after N.N. N. F. Gamalei Academy of
Medical Sciences of the USSR, TsKVI im. V. G. Korolenko Ministry of Health of the
USSR, Institute of Molecular Biology of the Academy of Sciences of the USSR, GISK
im. L. A. Tarasevich of the Ministry of Health of the USSR and Medical School No. 14,
who provided great assistance in working on the textbook.
All comments made on the content and design of the textbook will be accepted by
the authors with gratitude.
 Introduction - L. B. Bogoyavlenskaya
From the first days of the creation of the Soviet state, the protection of the health of
the population has become one of the main tasks facing the party and the
government. Measures aimed at reducing morbidity, including infectious diseases,
are constantly decreed by laws and resolutions of the Central Committee of the
CPSU, the Supreme Soviet and the Council of Ministers of the USSR.
The resolution of the Central Committee of the CPSU and the Council of Ministers of
the USSR "On measures to further improve public health" (1968) sets out the
principles in accordance with which medical institutions are fighting for human
health. In the Fundamentals of the Law of the Legislation of the USSR and the Union
Republics on Public Health (1969), this resolution is expanded and developed. Laws
protecting water sources and natural wealth of our country are specially provided
for: "Fundamentals of Water Legislation of the USSR and the Union Republics"
(1970), "On Strengthening Nature Protection and Improving the Use of Natural
Resources" (1972).
Anti-epidemic and preventive measures, advances in medical science and
technology, and an increase in the standard of living and culture of the population
led to the elimination of a number of serious infectious diseases in the USSR. In our
country, not a single case of smallpox has been registered for a long time; no plague
and no epidemic relapsing fever. The incidence of diphtheria, whooping cough,
measles and other infections has been reduced.
However, influenza epidemics, outbreaks of intestinal diseases, nosocomial
infections still cause damage to the national economy and public health, so the fight
against infectious diseases is one of the priorities of public health.
Ways to solve this problem were outlined by the 26th Congress of the CPSU, at which
it was noted that "the knowledge of the mechanism ... of the immunological
processes of human vital activity, the improvement of methods for the prevention,
diagnosis and treatment of the most common diseases"* is one of the most
important tasks of medical science .
*
 ( Materials of the XXVI Congress of the CPSU. - M .: Politizdat, 1981, p. 146.  )
To achieve success in reducing and eliminating infectious diseases, it is necessary to
know well the ecology and biology of their pathogens, the features of the interaction
of microorganisms with the host organism (human, animal) and the patterns of
infection spread. Such knowledge makes it possible to create a system of measures
aimed at preventing the emergence and spread of infectious diseases.
The success of these events is largely ensured by the activities of paramedical
personnel, and the quality of the work of paramedics, laboratory assistants, and
nurses depends on their knowledge and training in the field of medical microbiology.
Subject and tasks of microbiology . Microbiology is a branch of biology that studies
the patterns of life and development of microorganisms in their unity with the
environment. This science studies the properties of microorganisms, as well as the
processes that they cause in a macroorganism (in particular, in the human body) and
various environmental objects. The properties of microorganisms useful for humans
are used in various sectors of the national economy.
Microorganisms are all around us. They live in soil, water, humans and animals. With
the help of some microorganisms, the circulation of substances in nature takes place
- the environment is cleansed (decaying organic waste under the influence of
microorganisms turns into inorganic substances that are absorbed by plants); others
cause disease in humans and animals.
Microbiology deals with a wide range of topics and is subdivided into a number of
disciplines.
General microbiology studies the structure and vital activity of microorganisms, their
distribution in nature, heredity and variability.
Medical microbiology studies microorganisms that cause human diseases and the
processes that occur in the body when pathogens invade. The task of medical
microbiology is the development of methods for the laboratory diagnosis of
infectious diseases, the creation of immunobiological medicines for their prevention
and treatment.
The course of medical microbiology includes general and private parts.
General medical microbiology considers the properties of microorganisms and their
interaction with the host organism, private - characterizes the causative agents of
individual diseases and methods for their laboratory diagnosis.
Currently, the following are distinguished from medical microbiology: virology (the
science of viruses); protozoology (studies protozoa); mycology (studies
fungi); immunology (considers the protective processes occurring in the
body); sanitary microbiology (engaged in microorganisms living in the external
environment); space microbiology (studies the influence of space conditions on
microorganisms and changes in human microbial flora in space).
Veterinary microbiology is the study of microorganisms that cause disease in
animals. This branch of science is closely related to medical microbiology, since many
microorganisms are pathogens of both humans and animals.
Industrial microbiology studies microorganisms that are used in the production of
food, antibiotics and other medicinal substances, creates ways to protect food and
various materials from the harmful effects of microorganisms.
Agromicrobiology studies microorganisms that play a role in the formation of soil
structures, increasing soil fertility, and creating bacterial fertilizers.

Brief essay on the history of microbiology - L. B.

Bogoyavlenskaya
Long before the discovery of microorganisms, a person was faced with the processes
of their vital activity. Since time immemorial, people have used fermentation of
dough, fermentation of milk, fermentation of grape juice. It has long been familiar to
mankind and epidemic diseases that carried away the population of entire villages
and cities. Already in the writings of the physician of ancient Greece, Hippocrates,
there are assumptions about the connection between infectious diseases and special
pathogenic fumes, which he called "miasms". A similar hypothesis was put forward
by the Italian physician Girolamo Fracastoro (XVI century). He created the doctrine of
the living "contagion" - "the smallest and inaccessible to our senses particles", which,
penetrating into the human body, cause illness. The opinion that living beings are the
cause of contagious diseases was expressed at the beginning of the 17th century by
Athanasius Kircher.
The final confirmation of these assumptions was received by the Dutch naturalist
Anthony van Leeuwenhoek (1632-1723). Grinding glass, he was able to make
biconvex lenses that gave an increase of 160 times or more. Considering infusions,
rainwater, plaque and other objects in the optical system he created, he saw,
sketched and described "living animals", which today can be considered as the main
forms of microorganisms: spherical, convoluted, rod-shaped, mold, yeast.
 Anthony van Leeuwenhoek (1632-1723)

Assumptions about the causes of infectious diseases have not been proven, but the
practice of doctors since antiquity confirmed them.
The laws of the Hindus (1800-800 BC) provided for the first "anti-epidemic"
measures - a patient with consumption was equated in terms of the degree of
danger to those around him to a leper, and a Brahmin was forbidden to marry a girl
who had consumptives among her ancestors. The prescriptions for the isolation of
lepers, the disinfection of their belongings and dwellings (1700-500 BC) are widely
known.
In 1771-1772. During the plague epidemic in Moscow, military doctor Danila
Samoylovich, believing that "the plague is caused by a special and completely
excellent creature", for the first time disinfects the things of plague patients,
inoculates "a contagious weakened onset of the plague" to healthy people who came
into contact with the sick.
One of the most interesting chapters in the history of medicine is the development
of the method of inoculation, which has long been practiced by the Turks, Persians
and Chinese. The use of vaccinations was based on the belief that with artificial
infection, the disease proceeds more easily than with natural infection. Smallpox
produced monstrous devastation. Only in 1723 in Paris 20 thousand people died of
smallpox, in Naples in 1768 - 16 thousand people in a few weeks.
The English physician Edward Jenner noticed that many people who did not have
smallpox did not become infected by contact with the sick. Especially often this
phenomenon was observed in milkmaids who became infected while milking animals
with cowpox. This was the starting point of his research, which lasted more than 10
years. In 1796, Jenner inoculated a healthy boy with the contents of a purulent
vesicle from a cow with smallpox. A month and a half later, he inoculated the same
boy with material from a person with smallpox. The boy didn't get sick. Since then,
vaccinations against smallpox have brought humanity relief from this terrible disease
that claimed millions of human lives.
Many years later. The microscopic creatures discovered by Leeuwenhoek were not
associated with the occurrence of infectious diseases. And only in the XIX century,
the brilliant work of the French scientist Louis Pasteur (1822-1895) provided
scientific evidence of the importance of microorganisms in the occurrence of
infectious diseases and the rationale for the development of methods to combat
them.
 Louis Pasteur (1822-1895)

A chemist by education, Pasteur showed that microorganisms can change their

environment and cause its chemical transformations. But he did not dwell on
chemical phenomena, but described the processes of fermentation and putrefaction
and showed that they are caused by microorganisms.
He was the first to identify microorganisms that can exist without access to oxygen
(anaerobes).
One of the most important merits of Pasteur is Proof of the impossibility of
spontaneous generation of living beings. This question has troubled the minds of
scientists for a long time.
Although back in 1775 the Russian researcher M. M. Terekhovsky expressed the
opinion that microscopic creatures ("ciliates") do not arise by spontaneous
generation, the question of the emergence of living things from non-living things
remained controversial.
Pasteur's experiments showed that microorganisms enter the nutrient medium only
from the air. Having boiled the broth in a flask with a neck elongated in the shape of
the Latin letter S, he kept the broth transparent (sterile), since the microorganisms,
penetrating into the tube along with air, settled under the action of gravity in its
curved knee and did not reach the broth at the bottom of the flask.
The scope of Pasteur's research is very wide. Having proved the role of
microorganisms in the development of fermentation and decay processes, he began
to study diseases of silkworms, which at that time caused great damage to the
French economy. And in this work, he was able to find the cause of the defeat of
worms - their diseases were also caused by microorganisms, and Pasteur found a
means of combating them.
For the first time in the history of science, Pasteur developed methods for the
destruction of microorganisms when exposed to high temperatures. This method of
decontaminating the environment is called sterilization.
For food products that change their properties when boiled, he proposed a more
"soft" sterilization called pasteurization. Products are processed several times at a
relatively low temperature.
Pasteur did not stop at studying the processes in the world around us. He showed
the role of microorganisms in human life, proving for the first time that contagious
human diseases are also the result of the vital activity of microorganisms. This
discovery played a huge role in the development of biology and gave rise to a new
science - medical microbiology.
The next step in Pasteur's work was the creation of vaccines (from Latin vacca -
cow). By this term, Pasteur called weakened cultures of microorganisms, the
introduction of which does not cause disease, but makes people and animals
immune to it.
He prepared a vaccine from a culture of chicken cholera pathogens weakened by
heating, inoculated it into chickens, causing a non-fatal disease in them, and after
they recovered, re-infected them with a very "strong" culture of the pathogen. The
chickens are alive and well. According to the same principle, an anthrax vaccine was
created in animals - preliminary vaccinations made cows and sheep insensitive to this
disease.
In 1885, Pasteur proposed vaccination against rabies; Since the causative agent of
rabies could not be detected either under a microscope or on an artificial medium,
Pasteur used the brain of a rabid dog to make a vaccine, which he successively
transplanted from rabbit to rabbit many times, reducing the period from infection to
disease and weakening the strength of the grafting material. The use of such
vaccines on dogs gave good results, and Pasteur decided to vaccinate a child bitten
by a rabid dog and saved him from certain death. The next step is the vaccination of
Russian peasants who arrived in Paris on foot after being bitten by a rabid wolf.
The success of vaccination gave rise to the confidence of doctors and the public in a
new means of combating the most terrible infections of the past.
Based on the discoveries of Pasteur, the English surgeon D. Lister (1867) created an
antiseptic method - washing wounds and dressings with a solution of carbolic acid to
prevent the development of microorganisms in wounds.
The discoveries made by Louis Pasteur are summarized on a plaque on the building
of his first laboratory:
"Pasteur's laboratory was here:
1857 - fermentation
1860 - spontaneous generation
1865 - diseases of wine and beer
1868 - diseases of silkworms
1881 - contagion and vaccines
1885 - protection against rabies".
Pasteur's scientific research showed the role of microorganisms in causing
infection. He learned to grow them in artificial nutrient media, but did not know how
to detect the pathogen in each individual case of infection.
The German scientist Robert Koch (1843-1910) was the first to introduce dense
nutrient media into practice, which made it possible to obtain individual colonies and
a pure culture of microorganisms. Koch was the first to propose aniline dyes for
staining microorganisms, introduced illumination during microscopy (Abbe
condenser), applied an immersion system and microphotography.
 Robert Koch (1843-1910)

He discovered the causative agent of tuberculosis (1882), named after the scientist
"Koch's wand" and the causative agent of cholera (1883).
Koch's methods for growing and isolating microorganisms led to a number of
discoveries. At the end of the 19th century, the causative agents of diphtheria (E.
Klebs and F. Leffler), typhoid fever (K. Ebert and G. Gaffki), tetanus (A. Nikolaier and
S. Kitazato), dysentery (A. V. Grigoriev, K. . Shiga) and many others.
The discovery of the causative agents of some infections is associated with dramatic
pages in the history of medicine. So in 1874, a professor at Kazan University, G. N.
Minkh, infected himself with the blood of a patient with relapsing fever. He fell ill,
proving by this that the causative agents of relapsing fever are in the blood. Minh
suggested that pathogens are transmitted through blood-sucking insects. Two years
later, the doctor O. O. Mochutkovsky, repeating Minkh's experiment, infected
himself with the blood of patients with typhus and relapsing fever and confirmed
Minkh's assumption that the causative agent of this disease is in the blood.
D. I. Ivanovsky (1864-1920) in the same years studied the mosaic disease of tobacco
leaves and came to the conclusion that it was caused by the smallest agent. It does
not grow on nutrient media and passes through filters. Ivanovsky came to this
conclusion by causing a disease of healthy plants with the juice from the affected
leaves of tobacco, after filtering it through the smallest pores (which do not allow
other microorganisms to pass through). This was the first work to prove the viral
nature of infectious diseases.

D. I. Ivanovsky (1864-1920)

In the history of microbiology, several periods can be conditionally distinguished:

morphological, which is characterized by a description of microorganisms (A.
Levenguk); physiological, the development of which is associated with the works of L.
Pasteur and R. Koch, who revealed the essence of the vital activity of microorganisms
and the laws of their development; immunological. During this period, I. I.
Mechnikov lived and worked, and his research laid the foundation for the doctrine of
immunity.
Essay on the history of Russian microbiology . Domestic scientists have made a great
contribution to the development of microbiology.
Back in 1698, while in Holland, Peter I met A. Leeuwenhoek, who showed him a
microscope and showed him a number of microscopic objects.
During the formation of microbiology, L. S. Tsenkovsky (1822-1887) published the
work "On lower algae and ciliates", in which he first attributed bacteria to plant
organisms. He, in accordance with the principles of Pasteur, received a weakened
version of anthrax bacilli and used it to vaccinate animals.
Along with Pasteur and Koch, one of the founders of microbiology is the great
Russian scientist I. I. Mechnikov (1845-1916). His classical, still comprehensive theory
of immunity is based on the recognition of the role of cellular defense in the
development of resistance to infectious diseases. He showed that many cells of the
body (leukocytes, cells of the spleen, bone marrow, etc.) are able to capture and
digest various foreign elements, including bacteria. He called such cells phagocytes
(from the Greek phago - I devour, cytos - a cell), and the open phenomenon -
phagocytosis. The doctrine of phagocytosis was the basis for the study of
inflammation, which, as Mechnikov showed, is an active reaction of the body to the
introduction of pathogenic microbes.
 I. I. Mechnikov (1845-1916)

Mechnikov's scientific activity is multifaceted and wide. He investigated the causes of

aging and looked for ways to prolong human life. Mechnikov believed that poisonous
products formed as a result of the vital activity of putrefactive microorganisms living
in the large intestine poison the human body. To avoid this harmful effect, he
proposed to populate the intestines with lactic acid bacteria - putrefactive
antagonists.
With such a change in the intestinal microflora, Mechnikov believed, putrefactive
poisons would not adversely affect the human body and his life would last. At
present, it has been proven that it is impossible to completely change the natural
flora of the intestine, but the idea of using one type of microorganisms in the fight
against others (pathogenic), expressed by Mechnikov, has now been put into
practice with the use of antibiotics for the treatment of many infectious diseases,
bacterial preparations for the treatment of intestinal diseases.
Mechnikov studied cholera, typhoid and tuberculosis. He developed a method for
the experimental reproduction of syphilis (together with the French scientist E.
Roux), explored many other issues of biology and medicine. In 1886, he organized
the first bacteriological station in the country in Odessa and created a school of
microbiologists. However, his progressive scientific and social activities displeased
the tsarist government and he was forced to leave his homeland. From 1887 until the
end of his life he lived in Paris and worked at the Pasteur Institute. In our country
and abroad, many scientific institutes and laboratories are named after him.
One of the closest assistants and students of Mechnikov was A.M. Bezredka (1868-
1940). His works on the problems of immunity and anaphylaxis have found wide
application in practice.
L. A. Tarasevich (1868-1927) is also a student of Mechnikov. He conducted research
in the field of studying the mechanism of immunity and anaphylaxis, was a brilliant
organizer of the fight against epidemics in Russia, carried out vaccinations against
tuberculosis and intestinal infections. In 1918, he organized the Institute for the
Control of Serums and Vaccines, which bears his name (GISK named after L. A.
Tarasevich).
P. V. Tsiklinskaya (1859-1923), the first female professor of bacteriology, head of the
department of bacteriology at the Moscow Higher Women's Courses, is among the
outstanding microbiologists and students of Mechnikov. The main works of
Tsiklinskaya are devoted to the study of the intestinal flora and its significance for
human health. The results of Tsiklinskaya's research on the nature of childhood
diarrhea are still of theoretical and practical importance.
Mechnikov's closest assistant during his work at the bacteriological station in Odessa
was N. F. Gamaleya (1859-1949). He was a prominent immunologist who pioneered
the use of so-called chemical vaccines. He studied rabies, causative agents of
tuberculosis, cholera. In 1898, Gamaleya first observed bacteriophagy - the
dissolution of bacteria. The name of N. F. Gamaleya was given to several
microbiological institutes of our country.
The name of G. N. Gabrichevsky (1860-1907), head of the Moscow School of
Microbiology, is associated with the study and production of vaccines and sera in
Russia. He showed the importance of hemolytic streptococcus as the causative agent
of scarlet fever and proposed a vaccine that reduced mortality from this disease. The
introduction of serum by Gabrichevsky into the practice of treating diphtheria was
also an undoubted success. Gabrichevsky was the chairman of the Pirogov Society of
Russian Doctors, the author of the first Russian textbook of medical
microbiology. The Moscow Institute of Microbiology and Epidemiology is named
after him.
D. K. Zabolotny (1866-1929) - an outstanding microbiologist and epidemiologist. He
can rightly be considered the founder of Russian epidemiology. Zabolotny's works
are devoted to the fight against plague, cholera, and syphilis. He infected himself
with a suspension of Vibrio cholerae in order to find out the possibility of vaccination
against this disease by mouth with a killed vaccine. I. G. Savchenko did the same
experiment on himself.
Soil microbiology occupies a special place in general microbiology. The founder of
this science, S. N. Vinogradsky (1856-1953), established the role of microorganisms in
the circulation of nitrogen, carbon, phosphorus, sulfur and iron. He studied nitrifying
and nitrogen-fixing soil bacteria.
His student V. L. Omelyansky (1867-1928) devoted his research to elucidating the
role of bacteria in the nitrogen and carbon cycles, and to the anaerobic
decomposition of fiber.
Many more names of domestic microbiologists can be named, who have the honor
of discovering the causative agents of various infectious diseases, creating medicinal
preparations, developing vaccines and sera to prevent infections. We must not
forget those who risked their lives in the study of pathogens of infectious diseases or
in the fight against them: I. A. Deminsky, V. Khavkin, M. A. Lebedeva, A. L. Berlin and
others.
The Great October Socialist Revolution opened up broad opportunities for the
fruitful work of microbiologists.
The first period in the development of Soviet microbiology was associated with the
elimination of epidemics of typhus and relapsing fever, cholera; preventing the
introduction of plague and smallpox. The solution of this problem required the
organization of the production of vaccines and sera for preventive vaccinations, the
organization of sanitary and educational work, and the training of highly qualified
personnel. A brilliant constellation of Soviet microbiologists helped to solve the set
tasks.
L. A. Zilber (1894-1966) - microbiologist, immunologist, epidemiologist. His work on
the study of spring-summer encephalitis led to the discovery of the causative agent
and carriers of this disease. For a long time, Zilber studied the etiology and
immunology of malignant tumors and created a virogenetic theory of their origin.

L. A. Zilber (1894-1966)

The studies of ZV Ermolyeva (1898-1974) were devoted to the study of cholera and
the fight against this infection. For the first time in the USSR, she received penicillin,
which saved thousands of lives during the Great Patriotic War.
 Z. V. Ermolyeva (1898-1974)

The author of classical works on the study of brucellosis and rickettsiosis P. F.

Zdrodovsky (1890-1976) created and introduced into practice a number of
preventive and therapeutic drugs. He also worked on the immunology of malaria and
intestinal diseases caused by protozoa.
1950-1970 were a period of unprecedented flourishing of Soviet microbiology. At
this time, major scientists launched their work: M. P. Chumakov, A. A. Smorodintsev,
V. M. Zhdanov, V. D. Timakov and others. Their research is related to the
development and implementation of vaccines against various infections. Vaccines
from weakened pathogens of plague, tularemia, and brucellosis have been created
and are being used. A polio vaccine has been developed and is routinely used. The
introduction of this vaccine has almost eliminated polio (infantile paralysis) in our
country.
The further development of microbiology, virology and immunology is closely
connected with the successes of molecular biology and genetics. These disciplines
raise microbiological research to a new, higher level, allowing a broad insight into the
essence of pathogenicity, cell structure, patterns of the body's immune response,
etc.
In this very brief outline of the history of microbiology, only a small part of the
problems and questions on the solution of which Soviet microbiologists are
constantly working is elucidated. Their devotion to their work, high qualification and
creative burning are the key to the future success of domestic medicine.

Part I. General microbiology

Chapter 1. Microbiological laboratory - L. B.
Bogoyavlenskaya
Microbiological laboratories are organized at hospitals, polyclinics and sanitary and
epidemiological stations (SES).
The task of the medical microbiological laboratory is the diagnosis of infectious
diseases. To do this, the pathogen is isolated and the body's immune response to the
introduction of microorganisms (serological diagnostics) is determined. In addition,
carry out the identification of carriers of pathogenic (pathogenic)
microorganisms. There are laboratories in which virological studies are carried out. In
special sanitary and bacteriological laboratories, studies are carried out in order to
identify the degree of microbial contamination of the external environment and
various objects.
The material for microbiological research is most often human excretions (feces,
urine, vomit, sputum, wound discharge), as well as blood, bile, cerebrospinal fluid,
washings of the stomach, bronchi, cadaveric (sectional) material, etc.
Working in a microbiological laboratory with infectious material makes it mandatory
to place it in an isolated room. To comply with all the rules for working with
infectious material and conducting microbiological studies, the laboratory must have
several rooms:
1. Laboratory 4. Washing.
rooms. 5. Preparatory.
2. Boxing with a pre-box. 6. Sterilization.
3. Cooking room 7. Reception.
nutrient media. 8. Vivarium.
The laboratory room is designed for microbiological research. It should be spacious
and bright. The walls are painted with light oil paint, the floor is covered with
linoleum, laboratory tables are covered with plastic or glass, which is convenient for
wet cleaning and disinfection. The laboratory room is equipped with: work tables for
a doctor and a laboratory assistant, a place for staining preparations, a thermostat, a
refrigerator, a centrifuge, a microscope, cabinets, a sink with hot and cold water
supply, gas burners (in the absence of gas they work with alcohol burners).
The number of laboratory rooms is determined by the volume of work of the
laboratory. In large laboratories, separate rooms are allocated for working with
various types of pathogens.
The desktop is installed by the window so that the light falls from the side or
directly. A burner, bacteriological loops, jars with a disinfectant solution and cotton
wool are placed on the table.
Attention! Before starting work, everything necessary for the study is placed on the
table. The burner is installed at a distance equal to the forearm of the worker, that is,
in a position that excludes unnecessary movements during work. The size of the
flame in the burner and the correct glow are adjusted before starting work.
In the thermostat during routine research, the temperature should be 37 ° C. In large
laboratories, a special thermal room can be equipped. Temperature is recorded
daily.
Some nutrient media, diagnostic preparations, blood, bile, etc. are kept in the
refrigerator.
A centrifuge is used to separate solid particles from a liquid (for example,
erythrocytes from serum).
Racks, dishes, dry nutrient media, reagents, etc. are kept in cabinets.
Near the sink there should be a container with a disinfectant solution for cleaning
hands and a first aid kit with a set of items for first aid.
Boxing is a strictly isolated room for microbiological work in conditions requiring
special sterility. Deferrization of air is carried out using bactericidal lamps (BUV-15,
BUV-30, etc.) or a water bath (before work, the box is treated with steam of boiling
water with a disinfectant solution). The supply of disinfected air of a certain
temperature and humidity into the box through the supply and exhaust ventilation is
the best way to ensure the necessary conditions for work. Usually two people work
in boxing. They enter the box through the anteroom, in which they change clothes
(robe, slippers, cap, mask) and go to the box through the second door.
Attention! In boxing, they don’t talk and avoid unnecessary movements.
The room for the preparation of culture media should be located next to the
washing and sterilization room. This room should have a sink with hot and cold water
supply, a distiller, a stove (gas or electric), cabinets or racks for storing dry nutrient
media, chemicals, and sterile dishes.
Washing room - a room for washing and processing dishes, which should have a sink
(with cold and hot water) and a stove. The washing room is equipped with tables,
racks, equipped with dishes for washing dishes: detergents, ruffs, rags.
In the sterilization room there are devices for sterilizing clean dishes, nutrient media
and decontaminating waste material: autoclaves, a drying cabinet, etc. (see Chapter
5).
If there is a separate preparation room, it is used for preparing, packing dishes and
other ancillary work.
In the registry , or part of the premises replacing it, they receive and register the
material received for research, and issue conclusions of microbiological research.
Vivarium - a room for keeping experimental animals, available only in large
laboratories (see chapter 11).

Rules of conduct and work in a microbiological

laboratory

1. Employees are allowed to work only after they are familiar with the rules of
conduct and working hours.
2. All employees are subject to prophylactic vaccinations, mainly against intestinal
infections.
3. Each employee has a bathrobe and cap; in the laboratory wear changeable shoes.
4. Each employee is obliged to strictly observe personal hygiene, keep the workplace
clean.
5. The material entering the laboratory is registered in a special journal and marked.
6. All incoming material for research is considered infected (contagious). It is placed
on a special tray, and the container with the material is wiped with a disinfectant
solution from the outside.
7. Pour the test material from one container to another should be over a disinfectant
solution. The liquid material is sucked off using a rubber balloon, put on a pipette.
8. If the test material gets on hands, a table or other objects, they are treated with a
disinfectant solution.
9. At the end of the work, hands, tools, workplace are treated with a disinfectant
solution. Cultures are rendered harmless or, if necessary, stored in a refrigerator,
which is sealed. The material that requires further research is placed in a thermostat,
which is also sealed. When storing pathogenic cultures in the laboratory, they are
recorded in a special journal. Indicate the number of cultures, the dates of their
receipt, reseeding, destruction.
10. It is strictly forbidden to eat and smoke in the laboratory.
11. In the laboratory, wet cleaning of the premises is carried out daily using
disinfectant solutions. Walls, floors, inventory are washed weekly with hot water and
soap. Boxing is removed at the end of the working day, and before work it is
irradiated with bactericidal lamps.
The mode of work in laboratories depends on the degree of risk of infection for
persons working with pathogens or material containing them.
Microorganisms are divided into four groups according to the degree of danger of
infection:
I. The causative agents of the plague.
II. Causative agents of highly contagious epidemic diseases (cholera, brucellosis,
tularemia, anthrax, glanders, melioidosis, leptospirosis).
III. Causative agents of epidemic bacterial infections: intestinal (typhoid fever,
paratyphoid A and B, dysentery), tuberculosis, diphtheria, whooping cough,
meningitis, gonorrhea, listeriosis, trachoma, leprosy; pathogenic anaerobes,
spirochetes (causative agents of epidemic relapsing fever and syphilis), etc.
IV. Salmonella, Proteus, Escherichia, Klebsiella, hemoglobinophilic bacteria,
staphylococci, streptococci, pathogens of gas gangrene, etc.
With material possibly infected with pathogens of especially dangerous infections
(EOI) of groups I and II, and with cultures of microorganisms of these groups, they
work in special laboratories with the permission of the health authorities of the USSR
or the Union republics. With pathogens belonging to group III - in the laboratories of
SES, hospitals, etc., to group IV - in all microbiological laboratories.
Of great importance for microbiological research is the technique of taking the test
material and the method of delivering it to the laboratory. Any material must be
collected in a sterile container under conditions that protect it from contamination
by foreign microflora.
Excrements are taken with a special rectal loop, which is inserted into the rectum by
8-15 cm. The loop is placed in a test tube with a preservative (glycerin mixture,
phosphate buffer mixture, etc.). You can also use sterile cardboard plates or a vessel
treated with a disinfectant solution (10% bleach) and rinsed thoroughly with hot
water to remove traces of bleach.
Urine is taken with a sterile catheter into sterile vials or test tubes.
Sputum is collected in sterile jars. Blood from a vein is taken sterile into a vial with a
special nutrient medium, for serological reactions - into a dry test tube.
Purulent discharge from the wound, smears from the throat and nose are taken with
sterile cotton swabs and placed in sterile test tubes. The vomit is collected in a sterile
wide-mouth jar covered with wax paper.
Cadaveric (sectional) material should be taken in the first hours after the death of
the patient, since the intestinal microflora spreads very quickly throughout the
body. Blood is taken from the heart with a sterile syringe, pieces of the liver, spleen
and other organs are cut out with sterile scissors. All samples for microbiological
examination are placed in sterile vessels.
A label is attached to the test tube, jar, vials with material for research, on which the
last name, first name, patronymic, age of the patient and the date of taking the
material are indicated. In the direction, the information given on the label is
repeated, and additionally reported: the nature of the material, the institution that
sent the material, the clinical diagnosis, the purpose of the study and the name of
the doctor sending the material.
Delivery of the test material to the laboratory is carried out as soon as possible in
special metal bins, containers, and cases. Material containing microorganisms that
are unstable in the external environment is transferred in special vessels in which the
temperature is maintained at 37 ° C; when delivering viral material, thermoses with
ice are used to create a low temperature.
Attention! Proper collection and transportation of test material ensures the
effectiveness of microbiological studies.

Methods of microbiological research

The microscopic method is used to study stained smears and smears from native
material in a microscope and allows you to characterize the morphology (shape) of
the pathogen, its relationship to various dyes, and mobility. Using this method, it is
possible to confirm the clinical diagnosis of gonorrhea, diphtheria, relapsing fever,
syphilis and some other diseases.
The microbiological method is used to isolate and study a pure culture of the
pathogen, i.e., to establish the etiology of the disease. Laboratory diagnosis of most
infectious diseases (typhoid fever, dysentery, cholera, whooping cough, etc.) is based
on the use of this method.
The serological method (from Latin serum - serum) detects in the blood serum
substances formed in response to the introduction of a pathogen into the human
body (antibodies). With its help, the diagnosis of brucellosis, tularemia, typhoid
fever, etc. is confirmed.
Biological (experimental) method - introduction to experimental animals of a pure
culture of microorganisms, poisons they secrete (toxins) or test material in order to
obtain changes characteristic of a given infection. This method makes it possible to
reproduce an infectious disease. It is used to diagnose botulism, tetanus, toxic
infections, etc.

Control questions

1. What are the tasks of a microbiological laboratory?

2. What premises does the microbiological laboratory have?
3. How should one behave when working in a microbiological laboratory?
4. How is material collected and sent to the laboratory for microbiological testing?

Chapter 2. Microscope and microscopic methods of

research - M. Ya. Korn
Microscopes are used to detect and study microorganisms. Light microscopes are
designed to study microorganisms that are at least 0.2 microns in size (bacteria,
protozoa, etc.), and electronic microscopes are designed to study smaller
microorganisms (viruses).
Distinguish between simple and complex light microscopes. The optics of simple
microscopes is represented by a single lens with high magnification. In compound
microscopes, the optical system consists of a lens for obtaining an enlarged image of
an object and an eyepiece for further magnification of the obtained image and its
examination.
Modern light microscopes, which allow not only to see microorganisms, but also to
study their structure, are complex optical instruments, the handling of which
requires certain knowledge, skills and great accuracy.
Biological light microscopes, depending on the field of application, the complexity of
the device and the configuration with various optics, are divided into working,
laboratory and research. Now our industry produces a series of microscopes
"Biolam". Microscopes of this series have designations indicating which group they
belong to (P - workers, L - laboratory, I - research), the equipment is indicated by a
number, for example, workers from P11 to P17 (Fig. 1).
 Rice. 1. Microscopes working series 'Biolam'. a - 'Biolam P16'; b - 'Biolam R11'

A microscope is divided into mechanical and optical parts.

The mechanical part includes a tripod (consisting of a base and a tube holder) and a
tube mounted on it with a revolver for mounting and changing lenses, an object
table for the preparation, devices for attaching a condenser and light filters,
mechanisms built into the tripod for coarse (macromechanism, macroscrew) and fine
(micromechanism, microscrew) for moving the object stage or tube holder.
The optical part of the microscope is represented by objectives, eyepieces, and an
illumination system, which, in turn, consists of an Abbe condenser located under the
object stage, a mirror with a flat and concave side, and a separate or built-in
illuminator with a low-voltage incandescent lamp and a transformer. The objectives
are screwed into the revolver, and the corresponding eyepiece, through which the
image is observed, is installed on the opposite side of the tube.
There are monocular (having one eyepiece) and binocular (having two identical
eyepieces and making it possible to observe with two eyes) tubes. In addition, the
microscope tube can be straight vertical (mainly for photographing) and inclined.
The main role in obtaining a clear image is played by the lens. It builds an enlarged,
real and inverted image of the object. Then this image is additionally enlarged when
viewed through an eyepiece, which, similarly to a conventional magnifier, gives an
enlarged virtual image.
On fig. 2 shows a diagram of the path of rays in a microscope.
 Rice. Fig. 2. Scheme of the path of rays in a microscope with illumination adjustment
according to Köhler. a - schematic diagram of the microscope and lighting system: 1 -
light source; 2 - collector; 3 - field aperture of the illuminator; 4 - mirror; 5 - aperture
 diaphragm of the condenser; 6 - condenser; 7 - preparation; 7' - enlarged actual
intermediate image formed by the lens; 7" - enlarged imaginary final image
observed through the eyepiece; 8 - lens; 9 - exit pupil of the lens; 10 - field aperture of
the eyepiece; 11 - eyepiece; 12 - eye; b - principle of illumination according to Köhler:
1 - light source; 2 - collector; 3 - field diaphragm; 4 - light filter; 5 - aperture
diaphragm; 6 - condenser; 7 - object; 8 - lens

The magnification of a microscope can be determined by multiplying the

magnification of the objective by the magnification of the eyepiece (usually the
magnification of the objective and the eyepiece is indicated on the frame: the
objective is up to 100x, the eyepiece is 4x, 5x, 7x, 10x, 12.5x, 15x and 20x ).
However, magnification does not determine image quality. The quality of the image,
its clarity is determined by the resolution of the microscope, that is, the ability to
distinguish separately two closely spaced points. The resolution limit - the minimum
distance at which these points can still be seen separately - depends on the
wavelength of the light that illuminates the object and the numerical aperture of the
lens. The numerical aperture, in turn, depends on the angular aperture of the
objective and the refractive index of the medium between the front lens of the
objective and the preparation. Angular aperture is the maximum angle at which rays
passing through an object can enter the lens. The larger the aperture and the closer
the refractive index of the medium between the lens and the preparation is to the
refractive index of glass, the higher the resolution of the lens.
Distinguish between useful and useless magnification. Useful magnification is usually
equal to the numerical aperture of the objective magnified by 500-1000
times. Higher ocular magnification does not bring out new details and is useless.
Depending on the medium that is between the objective and the preparation, there
are "dry" lenses of small and medium magnification (up to 40×) and immersion
lenses with a maximum aperture and magnification (90-100×).
A feature of immersion lenses is that an immersion liquid is placed between the front
lens of such an objective and the preparation, which has a refractive index the same
as glass (or close to it), which ensures an increase in the numerical aperture and
resolution of the lens.
Distilled water is used as an immersion liquid for water immersion lenses, and cedar
oil or a special synthetic immersion oil is used for oil immersion lenses. The
disadvantage of cedar oil is its rapid thickening. For lenses operating in the
ultraviolet region of the spectrum, glycerol is used as an immersion liquid. An image
obtained with lenses has various disadvantages: spherical and chromatic aberrations,
curvature of the image field, etc. In lenses consisting of several lenses, these
disadvantages are to some extent corrected. Depending on the degree of correction
of these shortcomings, achromatic lenses and more complex apochromatic lenses
are distinguished. Accordingly, lenses in which the curvature of the image field is
corrected are called plan achromats and plan apochromats. The use of these lenses
produces a sharp image across the entire field, while the image obtained with
conventional lenses does not have the same sharpness in the center and at the
edges of the field of view. All characteristics of the lens are usually engraved on its
frame: own magnification, aperture, lens type (APO - apochromat, etc.); water
immersion lenses have the designation VI and a white ring around the frame in its
lower part, oil immersion lenses have the designation MI and a black ring.
All objectives are designed to work with a 0.17 mm cover slip. The thickness of the
coverslip especially affects image quality when working with strong dry systems
(40×). When working with immersion objectives, cover slips thicker than 0.17 mm
should not be used because the thickness of the cover slip may be greater than the
working distance of the objective, and in this case, when trying to focus the objective
on the specimen, the front lens of the objective may be damaged.
Eyepieces consist of two lenses and also come in several types, each of which is used
with a specific type of lens, further eliminating image imperfections. The type of
eyepiece and its magnification are marked on its frame.
The condenser is designed to focus the light from the illuminator on the
preparation. It consists of several lenses that convert parallel beams from the
illuminator into converging ones. One of the details of the condenser is the aperture
diaphragm, which is essential for proper illumination of the preparation. The
illuminator consists of a low-voltage incandescent lamp with a thick filament, the
incandescence of which can be adjusted, a collector lens and a field diaphragm (the
opening of which determines the diameter of the illuminated field on the
specimen). The mirror directs the light from the illuminator into the condenser. In
order to maintain the parallelism of the beams coming from the illuminator to the
condenser, it is necessary to use only the flat side of the mirror. Image quality also
depends to a large extent on proper lighting.
Setting the illumination and focusing of the microscope. There are several different
ways of illuminating a specimen under microscopy. The most common is the Köhler
light installation method, which is as follows:
1) set the illuminator against the microscope mirror;
2) turn on the illuminator lamp and direct the light onto a flat (!) microscope mirror;
3) place the preparation on the microscope stage;
4) cover the microscope mirror with a sheet of white paper and focus the image of
the lamp filament on it;
5) remove a sheet of paper from the mirror;
6) close the aperture diaphragm of the condenser. By moving the mirror and slightly
moving the lamp socket, the image of the filament is focused on the aperture
diaphragm.
Attention! The distance of the illuminator from the microscope should be such that
the image of the lamp filament is equal to the diameter of the aperture diaphragm of
the condenser.
7) open the aperture diaphragm of the condenser, cover the field diaphragm of the
illuminator and significantly reduce the lamp incandescence;
8) at low magnification (10×), looking into the eyepiece, a sharp image of the
preparation is obtained;
9) by slightly turning the mirror, the image of the field diaphragm, which looks like a
bright spot, is transferred to the center of the field of view. By lowering and raising
the condenser, a sharp image of the edges of the field diaphragm is obtained (a
colored border may be visible around them);
10) open the field diaphragm of the illuminator to the edges of the field of view,
increase the incandescence of the lamp filament and slightly (by 1/3 ) reduce the
opening of the aperture diaphragm of the condenser ;
11) When changing the lens, you need to check the light setting.
Attention! After finishing the adjustment of the light according to Köhler, in no case
should you change the position of the condenser, the opening of the field and
aperture diaphragms.
The illumination of the preparation can only be adjusted with neutral light filters or
by changing the incandescence of the lamp using a rheostat.
For correct illumination of the preparation, when working with low magnification
objectives (up to 10×), it is necessary to unscrew and remove the upper lens of the
condenser.
Attention! When working with lenses that give high magnification - with strong dry
(40 ×) and immersion (90 ×) systems, in order not to damage the front lens, the
following technique is used when focusing: observing from the side, lower the lens
with a macro screw almost until it comes into contact with the preparation, then,
looking into the eyepiece, the lens is very slowly raised with a macro screw until an
image appears, and with the help of a micro screw, the final focusing of the
microscope is performed.
Microscope care . A microscope is a precision optical instrument that requires
careful handling. When working with a microscope, do not use great effort. In no
case should you touch the surface of lenses, mirrors and filters with your fingers.
To protect the internal surfaces of the objectives, as well as the prisms of the tube
from dust, you must always leave the eyepiece in the tube.
When cleaning the outer surfaces of the lenses, remove dust from them with a soft
(squirrel) brush washed in ether. If necessary, gently wipe the lens surfaces with a
well-washed, soap-free linen or cambric cloth lightly moistened with clean gasoline,
ether, or a special mixture for cleaning optics. It is not recommended to wipe the
lens optics with xylene, as this may cause them to stick.
From mirrors with external silvering, you can only remove dust by blowing it off with
a rubber bulb. You cannot wipe them.
It is also impossible to unscrew and disassemble the lenses yourself - this will
inevitably lead to their damage.
Upon completion of work on the microscope, it is necessary first of all to carefully
remove the remnants of immersion oil from the front lens of the objective in the
manner described above. Then lower the stage (or condenser in microscopes with a
fixed stage) and cover the microscope with a cover.
To preserve the appearance of the microscope, it is necessary to periodically wipe it
with a soft cloth slightly soaked in acid-free Vaseline and then with a dry, soft, clean
cloth.

Phase contrast microscopy

Light waves are characterized by wavelength, amplitude and phase. The human eye
is able to distinguish wavelength (color) and amplitude (intensity, brightness of light),
but cannot detect differences in phase.
Microscopy of colored objects shows a change in amplitude (a decrease in the
brightness of light) and selective absorption of light of a certain wavelength (a
change in color).
When observing unstained microorganisms that differ from the environment only in
refractive index, there is no change in intensity, but only the phase of the
transmitted light waves changes. Therefore, the eye cannot notice changes and these
objects look low-contrast, transparent.
To observe such objects, phase-contrast microscopy is used, based on the
transformation of phase changes introduced by the object into amplitude changes
that are visible to the eye.
The phase-contrast device can be installed on any biological microscope and consists
of: 1) a set of objectives with special phase plates; 2) a condenser with a rotating
disk. It has annular diaphragms corresponding to the phase plates in each of the
lenses; 3) auxiliary microscope.
The phase contrast adjustment is basically as follows:
1) replace the lenses and the microscope condenser with phase-contrast ones;
2) install a low magnification lens and a hole in the condenser disk without an
annular diaphragm (indicated by the number "0");
3) adjust the light according to Koehler;
4) choose a phase lens of the appropriate magnification and focus it on the
preparation;
5) turn the condenser disk and set the annular diaphragm corresponding to the lens;
6) remove the eyepiece from the tube and insert an auxiliary microscope in its
place. Adjust it so that the phase plate (in the form of a dark ring) and the annular
diaphragm (in the form of a light ring of the same diameter) are clearly
visible. Adjusting screws on the condenser align these rings precisely. Remove the
auxiliary microscope and reinstall the eyepiece.
Through the use of this microscopy method, the contrast of living unstained
microorganisms is sharply increased and they appear dark against a light background
(positive phase contrast) or light against a dark background (negative phase
contrast). Our industry produces the KF-4 device for positive phase contrast.
Phase-contrast microscopy is also widely used to study tissue culture cells, to
observe the effect of various viruses on cells, etc. In these cases, biological
microscopes with reverse optics, the so-called inverted microscopes, are often
used. In such microscopes, the objectives are located at the bottom, and the
condenser is at the top. Sometimes they are enclosed in a thermostat to monitor the
dynamics of changes in tissue culture cells and are equipped with a movie camera.
The morphology of some microorganisms cannot be studied using the microscopy
methods described above. These include various spirochetes and, in particular,
leptospira, some large viruses. Dark-field microscopy is used to observe these
microorganisms.

Dark field microscopy

Dark-field microscopy is based on the ability of microorganisms to strongly scatter

light. For dark-field microscopy, ordinary objectives and special dark-field condensers
are used. There are several types of such capacitors, differing in design.
The main feature of dark-field condensers is that their central part is darkened and
direct rays from the illuminator do not fall into the microscope objective. The object
is illuminated by oblique side beams, and only beams scattered by particles in the
preparation enter the microscope objective. Dark-field microscopy is based on the
Tyndall effect, a well-known example of which is the detection of dust particles in the
air when illuminated by a narrow beam of sunlight.
To prevent direct rays from the illuminator from entering the lens, its aperture must
be smaller than the aperture of the condenser. To reduce the aperture, a diaphragm
is placed in a conventional lens or special lenses equipped with an iris diaphragm are
used.
Under dark-field microscopy, microorganisms appear brightly glowing against a black
background. With this method of microscopy, the smallest microorganisms can be
detected, the dimensions of which lie outside the resolution of the
microscope. However, dark-field microscopy allows you to see only the contours of
the object, but does not make it possible to study the internal structure.
Usually, using dark-field microscopy, preparations of the "crushed drop" type are
studied. At the same time, very strict requirements are imposed on the quality of
slides and coverslips and preparation of the preparation. Slides should be no thicker
than 1.1-1.2 mm, coverslips - 0.17 mm, without scratches and dirt. When preparing
the preparation, special attention should be paid to the absence of bubbles and large
particles (all these defects will be visible brightly luminous and will not allow
observing the preparation).
Dark-field microscopy requires bright light sources, so more powerful illuminators
and maximum lamp incandescence should be used.
The darkfield lighting setup is basically as follows:
1) set the light according to Kohler; 2) replace the bright-field condenser with a dark-
field one; 3) immersion oil or, in extreme cases, distilled water is applied to the
upper lens of the condenser; 4) raise the condenser until it comes into contact with
the lower surface of the glass slide; 5) a low magnification lens is focused on the
preparation; 6) with the help of centering screws, a bright spot is transferred to the
center of the field of view (sometimes having a darkened central area); 7) by raising
and lowering the condenser, the darkened central area disappears and a uniformly
illuminated bright spot is obtained. If this fails, then it is necessary to check the
thickness of the slide (usually this phenomenon occurs when using too thick slides -
the light cone is focused in the thickness of the glass).
After the correct setting of the light, a lens of the desired magnification is installed
and the preparation is examined.

Luminescent (fluorescent) microscopy

Luminescent (fluorescent) microscopy is based on the ability of certain substances to

luminesce, i.e., glow when illuminated with invisible ultraviolet or blue light. An
example of such a glow is the well-known fluorescent lamps, in which, as a result of
irradiation with ultraviolet rays, a special composition glows - a phosphor that covers
the inside of the lamp bulb.
The color of luminescence is usually shifted to a longer wavelength part of the
spectrum compared to the light that excites it. So, if luminescence is excited by blue
light, then its color can be from green to red, if luminescence is excited by invisible
ultraviolet radiation, then its color can be in any part of the visible spectrum. This
feature of luminescence makes it possible, using special light filters that absorb
exciting light, to observe a relatively weak luminescent glow.
The device of a luminescent microscope and the rules for working with it differ from
a conventional light microscope mainly in the following:
1. The presence of a powerful light source in the illuminator, emitting mainly in the
short-wave (ultraviolet, blue) part of the spectrum (mercury-quartz lamp of ultrahigh
pressure). Quartz halogen lamps (KGM) are used in special luminescent illuminators
that are mounted on a conventional microscope.
2. The presence of a system of light filters: a) exciting filters pass only that part of the
spectrum that excites luminescence;
b) a heat-shielding light filter protects other light filters, preparation and optics of a
luminescent microscope from overheating. In domestic luminescent microscopes,
the heat-shielding function is also performed by a cell with plane-parallel glasses
filled with distilled water. This cuvette is installed directly after the collector.
When working with a fluorescent microscope, special attention should be paid to
ensure that this cuvette is completely filled with water and that the water is
absolutely clean and transparent, since microorganisms can multiply in the water
during prolonged use of the microscope and it becomes cloudy;
c) "blocking" light filters are located between the preparation and the
eyepiece. These light filters absorb exciting radiation and transmit luminescence light
from the specimen to the observer's eye.
In our country, a very effective method of illuminating preparations for excitation of
luminescence has been developed, which is used in all domestic luminescent
microscopes. This method consists in the fact that the preparation is illuminated by
light falling on it through the lens. Due to this, the illumination is increased when
using objects with a large numerical aperture, i.e. those used to study
microorganisms. A very important role in this method of illumination is played by a
special interference beam-splitting plate that directs light into the lens and is a
translucent mirror that selectively reflects and directs into the lens only that part of
the spectrum that excites luminescence, and transmits only luminescence light into
the eyepiece.
The optics of the objectives of a luminescent microscope are made of non-
luminescent grades of optical glass and are glued together with a special non-
luminescent adhesive. The letter "L" is engraved on the frame of such lenses. When
working with oil immersion objectives for fluorescent microscopy, a special non-
luminescent immersion oil is used.
The rules for setting up a luminescent microscope are detailed in the instructions for
the microscope.
On fig. 3 shows the Lumam luminescent microscope manufactured by the Leningrad
Optical and Mechanical Association (LOMO).
 Rice. 3. Microscope luminescent research series 'Lumam IZ'

To study microorganisms in a fluorescent microscope, they are preliminarily stained

(fluorochromized) with highly diluted solutions of special luminescent dyes
(fluorochromes), which selectively bind to certain cell structures. Fluorochromes
differ from conventional dyes in that they are used in very low concentrations (up to
a few µg/ml); in addition, they can stain not only fixed, but also living
cells. Fluorescence microscopy is also used to record the results of an
immunofluorescence test (IF) (see Chapter 12).

electron microscopy

Various methods of light microscopy make it possible to study relatively large

microorganisms (bacteria, protozoa), but do not make it possible to observe objects
whose size is less than 0.2 microns, since the resolution of the microscope depends
on the wavelength of visible light. Therefore, the structure of viruses cannot be
studied in a light microscope.
Fundamentally new opportunities for studying the fine structure of bacteria and
viruses appeared after the invention of the electron microscope.
In an electron microscope, instead of light waves, a stream of electrons in a deep
vacuum is used to build an image.
The "lenses" that focus the electrons are the electromagnetic field created by the
electromagnetic coils. The image in an electron microscope is observed on a
fluorescent screen and photographed. Objects in electron microscopy are also in a
deep vacuum, so they are subjected to fixation and special processing. In addition,
they must be very thin, since the flow of electrons is strongly absorbed. In this
regard, ultrathin sections with a thickness of 20–50 nm are used as objects, which is
much less than the thickness of viral particles. The resolution of modern electron
microscopes is 0.15 nm, which makes it possible to obtain a useful magnification of
millions of times.
An electron microscope is very different from a light microscope in its size and
complexity of the device. The column of the microscope, in which the object is
located and the electrons are accelerated, exceeds the height of a person, and a
separate room is needed to accommodate the microscope.

Control questions

1. What are the main parts of a microscope?

2. How to properly adjust the light in a microscope?
3. What is the principle of phase contrast microscopy?
4. What is dark-field microscopy based on and when is it used?
5. What is fluorescent microscopy based on?
6. What is an electron microscope and what is its resolution (magnification)?

Chapter 3. Fundamentals of classification and

morphology of microorganisms - O. B. Orleanskaya
Microorganisms (from Latin micros - small) - organisms invisible to the naked
eye. These include protozoa, spirochetes, fungi, bacteria, viruses, which are studied
by microbiology. The size of microorganisms is measured in micrometers (µm). In the
microcosm there is a wide variety of forms that are divided into groups based on the
general principles of biological classification.
The first general biological classification was created in the 18th century by the
Swedish scientist K. Linnaeus, based on morphological features and including flora
and fauna. With the development of science, classification began to take into
account not only morphological, but also physiological, biochemical and genetic
characteristics of microorganisms. At present, it is impossible to talk about a unified
classification of all living organisms: while maintaining the same principles, the
classifications of macro- and microorganisms have their own characteristics.
The main steps of all classifications are: kingdom - department - class (group) - order
- family - genus - species. The main classification category is the species - a set of
organisms that have a common origin, similar morphological and physiological
characteristics and metabolism.
Microorganisms belong to the kingdom of prokaryotes, whose representatives,
unlike eukaryotes, do not have a formalized nucleus. Hereditary information in
prokaryotes is enclosed in a DNA molecule located in the cytoplasm of the cell.
For microorganisms, a unified international classification was adopted in 1980, based
on the system proposed by the American scientist Bergi.
In order to determine which species a microorganism belongs to, it is necessary to
study its features (cell shape, sporulation, mobility, enzymatic properties) using
various methods and, using the determinant, find its systematic position - identify it.
Variants exist within a species: morphovariants differ in morphology, biovariants in
biological properties, chemovariants in enzymatic activity, serovarians in antigenic
structure, phage variants in sensitivity to phages.
To designate microorganisms, the general biological binary or binomial (double)
nomenclature introduced by K. Linnaeus is adopted. The first name denotes the
genus and is written with a capital letter. The second name denotes the species and
is written with a lowercase letter. For example, Staphylococcus aureus is
Staphylococcus aureus. The names may reflect the names of the researchers who
discovered the microorganisms: brucella - in honor of Bruce, Escherichia - in honor of
Escherich, etc. A number of names include organs that affect this microorganism:
pneumococci - lungs, meningococci - meninges, etc. .
 bacteria

Bacteria are single-celled organisms that lack chlorophyll. The average size of a

bacterial cell is 2-6 microns. The size and shape of bacterial cells inherent in
microorganisms of a certain type can change under the influence of various factors
(depending on the age of the bacterial culture, habitat, etc.). This phenomenon is
called polymorphism.
According to the cell shape, bacteria are divided into three groups: spherical, rod-
shaped, and convoluted (Fig. 4).
 Rice. 4. Forms of microorganisms and methods of their coloring. a - spherical
bacteria: 1 - staphylococci (Gram stain); 2 - streptococci (Gram stain); 3 -
pneumococci (Burri-Ginsu stain); 4 - meningococci and gonococci (Gram stain); b -
 rod-shaped bacteria: 1 - Escherichia coli (Gram stain); 2 - the causative agent of
diphtheria (stained with methylene blue); 3 - the causative agent of tuberculosis
(coloring according to Ziehl - Nielsen); 4 - the causative agent of tetanus (Ozheshko
stain); 5 - cholera vibrio (Gram stain); c - spirochete: 1 - pale treponema; 2 -
relapsing fever spirochete; 3 - leptospira

globular bacteriaare called cocci (from lat. coccus - berry) and have a cell diameter
of 0.5 to 1 micron. The shape of cocci is diverse: spherical, lanceolate, bean-
shaped. According to the mutual arrangement of cells after division, among cocci,
the following are distinguished: micrococci (from Latin micros - small) - cells divide in
different planes and are arranged singly; diplococci (from lat. diploos - double) - cells
divide in one plane and then are arranged in pairs; these include lanceolate
pneumococci and bean-shaped gonococci and meningococci; streptococci (from lat.
streptos - chain) - cells divide in one plane and do not diverge, forming a
chain; staphylococci (from lat. staphyle - bunch) - cells divide in different planes,
forming clusters in the form of a bunch of grapes; tetracocci (from lat. tetra - four) -
cells divide in two mutually perpendicular planes and are arranged in four; sarcins
(from lat. sarcio - connect) - the cells are divided in three mutually perpendicular
planes and are arranged in the form of bales or packages of 8 or 16 cells each.
Cocci are widely distributed in the external environment, as well as in humans and
animals. Almost all groups of cocci, excluding micrococci, tetracocci and sarcins,
include pathogens of infectious diseases.
The rod-shaped forms are called bacteria. Their average dimensions are from 1 to 6
microns in length and from 0.5 to 2 microns in thickness.
Bacteria differ in appearance: their ends can be rounded (E. coli), chopped off
(anthrax), pointed (causative agent of plague) or thickened (causative agent of
diphtheria). After division, the bacteria can be arranged in pairs - diplobacteria
(Klebsiella), in a chain (the causative agent of anthrax), sometimes at an angle to
each other or crosswise (the causative agent of diphtheria). Most bacteria are
randomly distributed.
Among the bacteria there are curved forms - vibrios (the causative agent of cholera).
The convoluted forms include spirilla and spirochetes. The shape of their cell
resembles a spiral. Most spirillae are not disease-causing.
 The structure of a bacterial cell
To study the structure of a bacterial cell, along with a light microscope, electron
microscopic and microchemical studies are used to determine the ultrastructure of a
bacterial cell.
A bacterial cell (Fig. 5) consists of the following parts: a three-layer membrane,
cytoplasm with various inclusions, and a nuclear substance (nucleoid). Additional
structural formations are capsules, spores, flagella, pili.

Rice. 5. Schematic representation of the structure of a bacterial cell. 1 - shell; 2 -

mucous layer; 3 - cell wall; 4 - cytoplasmic membrane; 5 - cytoplasm; 6 - ribosome; 7
- polysome; 8 - inclusions; 9 - nucleoid; 10 - flagellum; 11 - drinking

The cell membrane consists of an outer mucosal layer, a cell wall, and a cytoplasmic
membrane.
The mucous capsular layer is outside the cell and performs a protective function.
The cell wall is one of the main structural elements of the cell, maintaining its shape
and separating the cell from the environment. An important property of the cell wall
is selective permeability, which ensures the penetration of essential nutrients (amino
acids, carbohydrates, etc.) into the cell and the removal of metabolic products from
the cell. The cell wall maintains a constant osmotic pressure inside the cell. The
strength of the wall is provided by murein, a substance of a polysaccharide
nature. Some substances destroy the cell wall, such as lysozyme.
Bacteria that are completely devoid of a cell wall are called protoplasts. They retain
the ability to breathe, divide, synthesize enzymes; to the influence of external
factors: mechanical damage, osmotic pressure, aeration, etc. Protoplasts can only be
preserved in hypertonic solutions.
Bacteria with partially destroyed cell walls are called spheroplasts. If you suppress
the process of cell wall synthesis with penicillin, then L-forms are formed, which in all
types of bacteria are spherical large and small cells with vacuoles.
The cytoplasmic membrane adheres tightly to the cell wall from the inside. It is very
thin (8-10 nm) and consists of proteins and phospholipids. This is a semi-permeable
boundary layer through which the cell is nourished. The membrane contains
permease enzymes that carry out active transport of substances, and respiratory
enzymes. The cytoplasmic membrane forms mesosomes that take part in cell
division. When a cell is placed in a hypertonic solution, the membrane can separate
from the cell wall.
The cytoplasm is the interior of a bacterial cell. It is a colloidal system consisting of
water, proteins, carbohydrates, lipids, various mineral salts. The chemical
composition and consistency of the cytoplasm change depending on the age of the
cell and environmental conditions. The cytoplasm contains the nuclear substance,
ribosomes and various inclusions.
Nucleoid, the nuclear substance of a cell, its hereditary apparatus. The nuclear
substance of prokaryotes, unlike eukaryotes, does not have its own membrane. The
nucleoid of a mature cell is a double strand of DNA coiled into a ring. The DNA
molecule encodes the genetic information of the cell. According to genetic
terminology, the nuclear substance is called genophore or genome.
Ribosomes are located in the cytoplasm of the cell and perform the function of
protein synthesis. The ribosome contains 60% RNA and 40% protein. The number of
ribosomes in a cell reaches 10,000. Combining together, ribosomes form polysomes.
Inclusions - granules containing various reserve nutrients: starch, glycogen, fat,
volutin. They are located in the cytoplasm.
Bacterial cells in the process of life form protective organelles - capsules and spores.
Capsule - the outer compacted mucous layer adjacent to the cell wall. This is a
protective organ that appears in some bacteria when they enter the body of humans
and animals. The capsule protects the microorganism from the protective factors of
the body (causative agents of pneumonia and anthrax). Some microorganisms have a
permanent capsule (Klebsiella).
controversyfound only in rod-shaped bacteria. They are formed when a
microorganism enters unfavorable environmental conditions (high temperatures,
drying, pH changes, a decrease in the amount of nutrients in the environment,
etc.). Spores are located inside the bacterial cell and represent a compacted area of
the cytoplasm with a nucleoid, dressed in its own dense shell. In chemical
composition, they differ from vegetative cells in a small amount of water, an
increased content of lipids and calcium salts, which contributes to the high resistance
of spores. Sporulation occurs within 18-20 hours; when a microorganism enters
favorable conditions, the spore germinates into a vegetative form within 4-5
hours. Only one spore is formed in a bacterial cell, therefore, spores are not
reproductive organs,
The spore-forming aerobic bacteria are called bacilli, and the anaerobic bacteria are
called clostridia.
Spores differ in shape, size and location in the cell. They can be located centrally,
subterminally and terminally (Fig. 6). In the causative agent of anthrax, the spore is
located centrally, its size does not exceed the diameter of the cell. The spore of the
causative agent of botulism is located closer to the end of the cell - subterminally
and exceeds the width of the cell. In the causative agent of tetanus, a rounded spore
is located at the end of the cell - terminally and significantly exceeds the width of the
cell.
Flagella- organs of movement, characteristic of rod-shaped bacteria. These are thin
filamentous fibrils, consisting of a protein - flagellin. Their length significantly
exceeds the length of a bacterial cell. Flagella extend from the basal body located in
the cytoplasm and exit to the cell surface. Their presence can be detected by
determining the mobility of cells under a microscope, in a semi-liquid nutrient
medium, or by staining with special methods. The ultrastructure of the flagella was
studied using an electron microscope. According to the location of the flagella,
bacteria are divided into groups (see Fig. 6): monotrichous - with one flagellum (the
causative agent of cholera); amphitrichous - with bundles or single flagella at both
ends of the cell (spirilla); lophotrichous - with a bundle of flagella at one end of the
cell (fecal alkaline former); peritrichous - flagella are located over the entire surface
of the cell (intestinal bacteria).
 Rice. 6. Variants of the location of spores and flagella in bacteria. I - disputes: 1 -
central; 2 - subterminal; 3 - terminal; II - flagella: 1 - monotrichous; 2 -
amphitriches; 3 - lophotrichous; 4 - peritrichous

Pili or fimbria are villi located on the surface of bacterial cells. They are shorter and
thinner than flagella and also have a spiral structure. Consist of drinking from protein
- pilin. Some pili (there are several hundred of them) serve to attach bacteria to
animal and human cells, while others (single ones) are associated with the transfer of
genetic material from cell to cell.

Mycoplasmas

Mycoplasmas are cells that do not have a cell wall, but are surrounded by a three-
layer lipoprotein cytoplasmic membrane. Mycoplasmas can be spherical, oval, in the
form of threads and stars. Mycoplasmas according to Bergi's classification are
separated into a separate group. Currently, these microorganisms are receiving
increasing attention as causative agents of inflammatory diseases. Their sizes are
different: from a few micrometers to 125-150 nm. Small mycoplasmas pass through
bacterial filters and are called filterable forms.

Spirochetes

Spirochetes (see Fig. 52) (from Latin speira - bend, chaite - hair) - thin, convoluted,
mobile unicellular organisms, measuring from 5 to 500 microns in length and 0.3-
0.75 microns in width. With the simplest, they are related by the method of
movement by shortening the internal axial thread, consisting of a bundle of
fibrils. The nature of the movement of spirochetes is different: translational,
rotational, flexion, wavy. The rest of the cell structure is typical of bacteria. Some
spirochetes stain weakly with aniline dyes. Spirochetes are divided into genera
according to the number and shape of thread curls and its end. In addition to
saprophytic forms, common in nature and the human body, among spirochetes there
are pathogens - the causative agents of syphilis and other diseases.

Rickettsia

Rickettsia are microorganisms ranging in size from 0.2 to 30 microns. They have the
usual cell structure for bacteria: a two-layer membrane, cytoplasm, nucleoid. The
shape of rickettsia can be rod-shaped, filiform and cocci. All rickettsia are
intracellular parasites, that is, they can develop only in the cells of a living
organism. They cause infectious diseases such as typhus and various fevers. Carriers
of rickettsia are arthropods: ticks, lice and fleas, in the body of which rickettsia
multiply.

Viruses

Viruses (see Fig. 53) are the smallest non-cellular organisms. The virus particle is
called a virion. Virions are 15 to 400 nm in size. Most viruses can only be seen with
an electron microscope. The virion envelope, the capsid, consists of protein
molecules. Inside is only one type of nucleic acid - DNA or RNA. According to the type
of nucleic acid, viruses are divided into two groups - DNA and RNA viruses. All viruses
are obligate (mandatory) parasites and are cultivated in laboratories in chicken
embryos, animals, or tissue culture. The shape of the virions is diverse: spherical,
rod-shaped, cuboidal and spermatozoic. Reproduction of viruses is carried out by
separate synthesis of the shell and nucleic acid in the host cell, followed by assembly
of virions. This process is called reproduction. In the host organism, some viruses
form intracellular inclusions and elementary bodies, which are visible in a
conventional light microscope, since their sizes are several micrometers. These
formations are of diagnostic value. Viruses cause disease in bacteria, plants, and
animals. The most important human infectious diseases of a viral nature are
influenza, measles, poliomyelitis, hepatitis and rabies.
Among viruses, a group of phages is distinguished (from Latin phagos - devouring),
causing lysis (destruction) of microorganism cells. While retaining the properties and
composition inherent in viruses, phages differ in the structure of the virion (see
Chapter 8). They do not cause disease in humans and animals.
Control questions
1. Tell us about the classification of microorganisms.
2. What are the main properties of representatives of the kingdom of prokaryotes.
3. List and characterize the main forms of bacteria.
4. Name the main organelles of the cell and their purpose.
5. Give a brief description of the main groups of bacteria and viruses.

Study of the morphology of microorganisms

To study the morphology of microorganisms, a microscopic research method is

used. An important condition for the successful use of this method is the correct
preparation of a smear from the test material or bacterial culture. Culture refers to
microorganisms grown on nutrient media in the laboratory.
Smear preparation technique
For work it is necessary to have clean and fat-free glass slides and coverslips. New
glasses are boiled for 15-20 minutes in a 2-5% soda solution or soapy water, rinsed
with water and placed in weak hydrochloric acid, then washed thoroughly with
water.
Glasses that were in use and contaminated with dyes or immersion oil can be treated
in two ways: 1) immersed for 2 hours in concentrated sulfuric acid or a chromium
mixture, and then rinsed thoroughly; 2) boil for 30-40 minutes in a 5% solution of
soda or alkali. Raw glass can be degreased by rubbing it with soap and then cleaning
it with a dry cloth.
Attention! If the glass is well degreased, then a drop of water spreads on it evenly,
without breaking up into small drops.
Glasses are stored in vessels with ground stoppers in Nikiforov's mixture (equal
volumes of alcohol and ether) or in 96% alcohol. Glass solutions are removed with
tweezers.
Attention! During operation, the glass is held by the edges with your fingers.
The material for research is applied to a glass slide with a bacterial loop, a needle or
a Pasteur pipette. Most often, a bacterial loop is used (Fig. 7), made of a platinum or
nichrome thread 5-6 cm long. The loop is fixed in a loop holder or soldered into a
glass rod. The end of the wire is bent in the form of a ring 1×1.5 or 2×3 µm in size.

Rice. 7. Needle and loops for sowing. 1 - needle; bacterial loops: 2, 3 - loops are
cooked incorrectly; 4 - the loop is cooked correctly

Attention! A properly prepared loop, when immersed in water and removed from
there, retains a water film.
Before preparing a smear, the working part of the loop is burned in the flame of a
burner in a vertical position: first the loop itself, and then the metal rod. This
manipulation is carried out after the end of sowing.
Preparation of a smear from a culture grown on a liquid nutrient medium. A fat-
free glass slide is burned in a burner flame and cooled. A culture is applied to a glass
slide placed on a stand (Petri dish, tripod). The culture tube is held with the thumb
and forefinger of the left hand. The loop is held in the right hand. Without releasing
the loop, with the little finger of the right hand, press the cork to the palm of your
hand and carefully remove it from the test tube. Movements should be smooth and
calm. The throat of the tube is burned in the flame of a burner. Insert the loop into
the test tube. Cool the loop against the wall of the tube and then immerse it in the
culture. Remove the loop without touching the walls of the tube. Close the plug,
after passing it through the flame of the burner. Put the test tube in a tripod. The
culture is applied to a glass slide with a loop, evenly distributing it in a circular
motion. Then the loop is burned in the flame of the burner. The smear is left to dry.
Attention! The smear should be evenly spread, thin and small (about the size of a
two-kopeck coin).
Preparation of a smear from a culture grown on solid nutrient medium . A drop of
isotonic sodium chloride solution (0.9%) is applied to the prepared glass slide with a
Pasteur pipette or loop. The culture is carefully removed by a loop from the agar in a
test tube or Petri dish and emulsified in a drop on glass. The prepared smear should
be uniform and not thick. When it dries, a slight coating remains on the glass slide.
Preparation of a smear from pus or sputum . The material is taken with a sterile
pipette or loop and applied to the middle of the glass slide. Cover the first slide with
the second slide so that a third of the first and second slides remain free. Glasses are
pushed apart with effort. Get two large swabs.
Preparing a blood smear . A drop of blood is applied to a glass slide at a distance of
one third from the left edge. Then the edge of a specially polished glass, tilted at an
angle of 45°, is touched to a drop of blood. By pressing the polished glass to the
object, they move it forward. A properly prepared smear is yellowish and
translucent.
Preparation of smears-imprints from the internal organs of corpses and foodstuffs
of a solid consistency . The surface of an organ or food product is cauterized with a
hot scalpel, and a piece of material is cut out of this area. This piece is carefully
grasped with tweezers and the cut surface is touched to the glass slide in two or
three places, making a series of strokes-imprints.
Drying the smear
The smear is dried in air at room temperature. If necessary, it can be dried near the
flame of the burner, holding the glass in a horizontal position by the edges with the
thumb and forefinger, stroke up.
Attention! At high temperatures, damage to the structure of cells can occur.
 Smear fixation
Smears are fixed after complete drying in order to: 1) fix microorganisms on the
glass; 2) neutralize the material; 3) killed microorganisms perceive color better. A
fixed swab is called a preparation.
fixation methods. 1. Physical - in the burner flame: the glass is taken with tweezers or
thumb and forefinger and passed through the top of the burner flame three times
for 6 s.
2. Chemical - in liquid: cellular elements in blood smears and smears-imprints are
destroyed under the action of high temperatures, so they are treated with one of the
fixative liquids: a) methyl alcohol - 5 min; b) ethyl alcohol - 10 min; c) Nikiforov's
mixture - 10-15 minutes; d) acetone - 5 min; e) acid and formalin vapors - a few
seconds.
Staining preparations
After fixation, the staining of the preparation is started.
Preparations are stained on a specially equipped table covered with linoleum, plastic,
glass, etc. A vessel with distilled water is needed on the table; a stand of two tubes
or sticks connected by rubber tubes on both sides (for placing
preparations); tweezers, cylinders, pipettes, filter paper, a set of dyes, a container for
draining them. The painting table should be near the water tap.
The ratio of microorganisms to dyes is called their tinctorial properties. Aniline dyes
are widely used in microbiology. Most microorganisms perceive basic dyes better.
The following dyes are most commonly used: red (basic magenta, sour magenta,
Congo red, neutral red); blue (methylene and toluidine); violet (gentian, methyl,
crystalline); brown-yellow (vesuvin, chrysoidin); green (diamond, malachite).
All dyes are produced in the form of amorphous or crystalline powders. Saturated
alcohol and phenol solutions are prepared from them, and then water-alcohol or
water-phenol solutions of dyes are used for work. If concentrated solutions of dyes
are used for staining, then the preparation is preliminarily covered with filter paper,
on which the dye is applied. In this case, pieces of dye remain on the paper.
Attention! A drop of dye is applied with a pipette so that it covers the entire
preparation.
 Dye Recipes
1. Saturated alcohol solutions (initial):
dye - 1 g
alcohol 96% - 10 ml
The mixture is placed in a thermostat until completely dissolved for several
days. Shake daily. Stored in bottles with ground stoppers.
2. Carbol fuchsin Ziel (for staining acid-resistant microorganisms, spores and
capsules):
saturated alcohol solution
basic fuchsin - 10 ml
carbolic acid solution 5% - 90 ml
Attention! Carbolic acid is poured into the dye, and not vice versa.
The mixture is shaken vigorously for several minutes, filtered and poured into a vial
for storage.
3. Pfeiffer magenta (for Gram stain and for the simple stain method):
Magenta Tsilya - 1 ml
distilled water - 9 ml
The dye is prepared immediately before use.
4. Carbolic gentian violet (for Gram stain):
saturated alcohol solution
gentian violet - 10 ml
carbolic acid 5% - 100 ml
The solutions are mixed and filtered through filter paper.
5. Lugol's solution (for Gram stain and starch reagent):
potassium iodide - 2 g
crystalline iodine - 1 g
distilled water - 10 ml
The mixture is placed in a frosted glass bottle, corked well and placed in a thermostat
for a day, then 300 ml of distilled water are added.
6. Alkaline Methylene Blue Loeffler Solution:
saturated alcohol solution
methylene blue - 30 ml
potassium hydroxide solution 1% - 1 ml
distilled water - 100 ml
7. Papers according to Sinev (for Gram staining):
1% alcohol solution of crystal violet
Filter paper strips are soaked in the solution and dried.
Staining methods are divided into indicative (simple) and differential (complex),
revealing the chemical and structural features of the bacterial cell.
Easy coloring method
The drug is placed on a stand for coloring, the test material up. A dye solution is
applied to it with a pipette. After the specified time, the dye is carefully drained, the
preparation is washed with water and dried with filter paper. With a simple method,
one dye is used. Methylene blue and alkaline blue Leffler stain the preparation for 3-
5 minutes, Pfeiffer fuchsin - 1-2 minutes (see Fig. 4).
A drop of immersion oil is applied to the stained and dried preparation and
microscoped using an immersion system.
Sophisticated staining methods
Gram stain (universal method) . The most common method of differential staining is
the Gram stain.
Depending on the results of staining, all microorganisms are divided into two groups
- gram-positive and gram-negative.
Gram-positive bacteria contain a magnesium salt of RNA in the cell wall, which forms
a complex compound with iodine and a basic dye (gentian, methyl, or crystal
violet). This complex is not destroyed by the action of alcohol, and the bacteria retain
their purple color.
Gram-negative bacteria are not able to retain the basic dye, as they do not contain
the magnesium salt of RNA. Under the action of alcohol, the dye is washed out, the
cells become discolored and stained with an additional dye (magenta) red.
1. A piece of paper is applied to the preparation according to Sinev and a few drops
of water or a solution of gentian violet are applied. Color 1-2 min. Remove the paper
or drain the dye.
2. Without washing with water, Lugol's solution is applied until it turns black (1 min),
then the dye is drained.
3. Without rinsing with water, 96% alcohol is applied until the dye leaves (30-60
s). You can lower the drug into a glass of alcohol for 1-2 seconds.
4. Wash the preparation with water.
5. Stained with Pfeiffer magenta for 3 minutes, washed with water and dried.
Microscopically using an immersion system.
Ziehl-Nielsen stain (for acid-fast bacteria) . This method is used to detect
tuberculosis and leprosy bacteria, which have a large amount of lipids, wax and
hydroxy acids in the cell membrane. Bacteria are acid-, alkali- and alcohol-
resistant. To increase the permeability of the cell wall, the first stage of staining is
carried out with heating.
1. The fixed preparation is covered with filter paper and Ziel's fuchsin is
applied. Holding the glass with tweezers, the preparation is heated over the flame of
the burner until the vapors escape. Add a new portion of the dye and heat 2 more
times. After cooling, the paper is removed and the preparation is washed with water.
2. The drug is decolorized with a 5% solution of sulfuric acid, immersed 2-3 times in a
solution or pouring acid on glass, then washed several times with water.
3. Stained with a water-alcohol solution of methylene blue for 3-5 minutes, washed
with water and dried.
Microscopically using an immersion system.
Acid-resistant bacteria are stained red, the rest - blue (see Fig. 4).
Staining according to Ozheshko (identification of spores) . 1. A few drops of a 0.5%
solution of hydrochloric acid are poured onto an air-dried smear and heated until
vapors form. The drug is dried and fixed over a flame.
2. Stained according to the Ziehl-Nielsen method. Acid-resistant spores are stained
pink-red, and the bacterial cell is stained blue (see Fig. 4).
Staining according to Burri - Guinsu (capsule detection) . This method is called
negative, since the background of the drug and the bacterial cell are stained, while
the capsule remains unstained.
1. A drop of black ink, diluted 10 times, is applied to a glass slide. They add a bit of
culture to it. A smear is made with the edge of a grinding glass, just like a smear of
blood, and dried.
2. Fix chemically with alcohol or sublimate. Rinse carefully with water.
3. Stain with Pfeiffer magenta for 3-5 minutes. Wash carefully and air dry.
Attention! Do not use filter paper, so as not to damage the preparation.
Microscopically using an immersion system. The background of the drug is black, the
cells are red, the capsules are unstained (see Fig. 4).
Vital staining of microorganisms
To study a living culture, methylene blue and other dyes are most often used in large
dilutions (1: 10,000). A drop of the test material is mixed on a glass slide with a drop
of dye and covered with a cover slip. Microscopically with a 40x objective.
The study of the mobility of microorganisms
For the study, a culture of bacteria grown in a liquid nutrient medium or a
suspension of bacteria in an isotonic sodium chloride solution is used.
crushed drop method . Place a drop of culture on a glass slide and cover it with a
coverslip. In order to avoid the formation of air bubbles, the coverslip is brought with
an edge to the edge of the drop and sharply lowered. To protect the drug from
drying out, it is placed in a humid chamber.
The wet chamber is a Petri dish with wet filter paper at the bottom. Two matches are
placed on the paper and the drug is placed on them. The cup is closed with a lid.
Microscopic at 40x objective magnification in a dark field (see Chapter 2).
Hanging drop method (Fig. 8). To prepare the preparation, you need a glass with a
hole, a coverslip and petroleum jelly. The edges of the hole are covered with a thin
layer of Vaseline.

Rice. 8. Hanging drop chamber

A drop of culture is applied to the coverslip. Then carefully cover the coverslip with a
glass with a hole so that the drop is in the center. The sticky slides are quickly turned
over with the coverslip up. The drop is in a hermetic chamber and remains for a long
time. Under microscopy, first, at low magnification (8×), the edge of the drop is
found, and then the preparation is studied at high magnification.

Control questions

1. How to prepare a bacterial loop?

2. Name the goals and methods of fixing smears.
3. Name the main dyes.
4. What methods study the mobility of microorganisms?

Exercise

1. Take finished preparations, study them and draw the main forms of
microorganisms.
2. Prepare smears from various materials (culture, pus, blood, imprint smears).
3. Stain preparations with complex methods (according to Gram, Ziehl - Nielsen,
Ozheshko, Burri - Gins).

Chapter 4
Physiology studies the vital functions of microorganisms: nutrition, respiration,
growth and reproduction. Physiological functions are based on continuous
metabolism (metabolism).
The essence of metabolism consists of two opposite and at the same time
interconnected processes: assimilation (anabolism) and dissimilation (catabolism).
In the process of assimilation, nutrients are assimilated and used for the synthesis of
cellular structures. During the processes of dissimilation, the nutrients decompose
and oxidize, while the energy necessary for the life of the microbial cell is
released. As a result of the breakdown of nutrients, complex organic compounds are
broken down into simpler, low molecular weight ones. Some of them are removed
from the cell, while others are again used by the cell for biosynthetic reactions and
are included in the processes of assimilation. All processes of synthesis and
breakdown of nutrients are performed with the participation of enzymes.
A feature of microorganisms is an intensive metabolism. In a day, under favorable
conditions, one microbial cell can process such an amount of nutrients that is 30-40
times its mass.

The chemical composition of bacteria

To understand the processes of metabolism, it is necessary to know the chemical

composition of microorganisms. Microorganisms contain the same chemicals as the
cells of all living organisms.
The most important elements are organogens (carbon, hydrogen, oxygen, nitrogen),
which are used to build complex organic substances: proteins, carbohydrates and
lipids. Microorganisms also contain ash or mineral elements. Most of them are
chemically bound to organic substances, the rest are present in the cell in the form
of salts.
In quantitative terms, the most significant component of the cell is water, which
makes up 75-85%; the share of dry matter, which consists of organic (proteins,
nucleic acids, carbohydrates, lipids) and mineral compounds, accounts for 15-25%.
Water . The importance of water in the life of the cell is great. All substances enter
the cell with water, and metabolic products are removed with it. Water in a
microbial cell is in a free state as an independent compound, but most of it is
associated with various chemical components of the cell (proteins, carbohydrates,
lipids) and is part of cellular structures.
Free water takes part in chemical reactions occurring in the cell, is a solvent for
various chemical compounds, and also serves as a dispersion medium for
colloids. The content of free water in a cell can vary depending on environmental
conditions, the physiological state of the cell, and its age. Thus, spore forms of
bacteria have significantly less water than vegetative cells. The largest amount of
water is observed in capsular bacteria.
Proteins (50-80% of dry matter) determine the most important biological properties
of microorganisms. These are simple proteins - proteins and complex - proteins. Of
great importance in the life of the cell are nucleoproteins - the combination of
protein with nucleic acids (DNA and RNA). In addition to nucleoproteins, microbial
cells contain small amounts of lipoproteins, glycoproteins, and chromoproteins.
Proteins are distributed in the cytoplasm, nucleoid, they are part of the structure of
the cell wall. Enzymes, many toxins (poisons of microorganisms) belong to proteins.
The species specificity of microorganisms depends on the quantitative and
qualitative composition of protein substances.
Nucleic acids in a microbial cell perform the same functions as in cells of animal
origin. DNA is contained in the nucleus (nucleoid) and determines the genetic
properties of microorganisms. RNA is involved in the biosynthesis of cellular proteins
and is found in the nucleus and cytoplasm. The total amount of nucleic acids ranges
from 10 to 30% of the dry matter of a microbial cell and depends on its type and age.
Carbohydrates (12-18% dry matter) are used by the microbial cell as a source of
energy and carbon. They consist of many structural components of the cell (cell
membrane, capsule, and others). Carbohydrates are also part of teichoic acid, which
is characteristic of gram-positive bacteria.
Cells of microorganisms contain simple (mono- and disaccharides) and high-
molecular (polysaccharides) carbohydrates. A number of bacteria may have
inclusions that chemically resemble glycogen and starch; they play the role of
reserve nutrients in the cell. The carbohydrate composition is different in different
types of microorganisms and depends on their age and development conditions.
Lipids (0.2-40% dry matter) are essential components of the cytoplasmic membrane
and cell wall, they are involved in energy metabolism. In some microbial cells, lipids
play the role of storage substances.
Lipids consist mainly of neutral fats, fatty acids, phospholipids. Their total number
depends on the age and type of microorganism. For example, in Mycobacterium
tuberculosis, the amount of lipids reaches 40%, which makes these bacteria resistant
to environmental factors.
In the cells of microorganisms, lipids can be associated with carbohydrates and
proteins, forming a complex complex that determines the toxic properties of
microorganisms.
Minerals - phosphorus, sodium, potassium, magnesium, sulfur, iron, chlorine and
others - average 2-14% of dry matter.
Phosphorus is part of nucleic acids, phospholipids, many enzymes, as well as ATP
(adenosine triphosphoric acid), which is an energy accumulator in the cell. Sodium is
involved in maintaining the osmotic pressure in the cell. Iron is found in respiratory
enzymes. Magnesium is part of magnesium ribonucleate, which is localized on the
surface of gram-positive bacteria.
For the development of microorganisms, trace elements are needed, which are
contained in the cell in very small quantities. These include cobalt, manganese,
copper, chromium, zinc, molybdenum and many others. Trace elements are involved
in the synthesis of certain enzymes and activate them. The ratio of individual
chemical elements in a microbial cell may vary depending on the type of
microorganism, the composition of the nutrient medium, the nature of the exchange
and the conditions of existence in the external environment.

Bacteria nutrition

All microorganisms need nutrients to carry out the processes of nutrition,

respiration, reproduction.
Microorganisms use various organic and inorganic compounds as nutrients and
energy sources; for normal life, they also require microelements and growth factors.
The process of nutrition of microorganisms has a number of features: firstly, the
supply of nutrients occurs through the entire surface of the cell; secondly, the
microbial cell has an exceptional speed of metabolic reactions; thirdly,
microorganisms are able to quickly adapt to changing environmental conditions. A
variety of conditions for the existence of microorganisms causes different types of
nutrition.
Types of nutrition are determined by the nature of the assimilation of carbon and
nitrogen. Water serves as a source of other organogens - hydrogen and
oxygen. Microorganisms also need water to dissolve nutrients, since they can only
enter the cell in dissolved form.
According to the assimilation of carbon, microorganisms are divided into two types:
autotrophs and heterotrophs.
Autotrophs (from the Greek autos - itself, trophe - nutrition) are able to synthesize
complex organic substances from simple inorganic compounds. They can use carbon
dioxide and other inorganic carbon compounds as a carbon source. Many soil
bacteria (nitrifying bacteria, sulfur bacteria, etc.) are autotrophs.
Heterotrophs (from the Greek heteros - another, trophe - nutrition) need ready-
made organic compounds for their growth and development. They can absorb
carbon from carbohydrates (most often glucose), polyhydric alcohols, organic acids,
amino acids, and other organic substances.
Heterotrophs represent an extensive group of microorganisms, among which
saprophytes and parasites are distinguished.
Saprophytes (from the Greek sapros - rotten, phyton - plant) receive ready-made
organic compounds from dead organisms. They play an important role in the
decomposition of dead organic residues, such as decay bacteria, etc.
Parasites (from the Greek parasites - parasite) live and multiply at the expense of
organic substances of living cells of plants, animals or humans. Such microorganisms
include rickettsiae, viruses, and some protozoa (see Chapter 11).
According to the ability to assimilate nitrogen, microorganisms are also divided into
two groups: aminoautotrophs and aminoheterotrophs. Aminoautotrophs for cell
protein synthesis use molecular nitrogen from the air (nodule bacteria, Azotobacter)
or assimilate it from ammonium salts. Aminoheterotrophs obtain nitrogen from
organic compounds - amino acids, complex proteins. These include all pathogenic
microorganisms and most saprophytes.
According to energy sources, among microorganisms, phototrophs are distinguished,
which use the energy of sunlight for biosynthetic reactions (purple sulfur bacteria)
and chemotrophs, which receive energy due to the oxidation of inorganic substances
(nitrifying bacteria, etc.) and organic compounds (most bacteria, including those
pathogenic for human species).
However, a sharp boundary between the types of nutrition of microbes cannot be
drawn, since there are types of microorganisms that can switch from a heterotrophic
type of nutrition to an autotrophic one, and vice versa.
At present, a new terminology has been introduced to characterize the types of
nutrition: heterotrophs are called organotrophs, and autotrophs are called
lithotrophs (from the Greek litos - stone), since such microorganisms are able to
grow in a purely mineral environment.
growth factors . Microorganisms for their growth and reproduction need special
substances that they cannot synthesize themselves and must receive them ready-
made. These substances are called growth factors, and they are needed by microbial
cells in small quantities. These include various vitamins, some amino acids (necessary
for protein synthesis), purine and pyrimidine bases (used for the construction of
nucleic acids), etc. Many growth factors are part of various enzymes and play the
role of catalysts in biochemical processes.
Knowledge of the needs of microorganisms for nutrients and growth factors is very
important, in particular, for the creation of nutrient media used for their cultivation.
Transport of nutrients . Nutrients can penetrate into the cytoplasm of microbial cells
only in the form of small molecules and in dissolved form.
Complex organic substances (proteins, polysaccharides, etc.) are previously exposed
to enzymes secreted by the microbial cell, and after that they become available for
use. The transport of nutrients into the cell and the release of metabolic products
from it is carried out mainly through the cytoplasmic membrane.
Nutrients enter the cell in several ways:
1. Passive diffusion, i.e., the movement of substances through the thickness of the
membrane, as a result of which the concentration of substances and osmotic
pressure are equalized on both sides of the membrane. In this way, nutrients can
penetrate when the concentration in the environment significantly exceeds the
concentration of substances in the cell.
2. Facilitated diffusion - the penetration of nutrients into the cell with the help of
their active transfer by special carrier molecules called permeases. These are
substances of an enzymatic nature that are localized on the cytoplasmic membrane
and have specificity. Each permease adsorbs the corresponding nutrient on the outer
side of the cytoplasmic membrane, enters into a temporary relationship with it and
diffuses in a complex through the membrane, giving away its transported substance
to the cytoplasm on the inner side. This process takes place without the use of
energy, since the movement of substances occurs from a higher concentration to a
lower one.
3. Active transport of nutrients is also carried out with the help of permeases, but
this process requires energy. In this case, the nutrient can penetrate into the cell if
its concentration in the cell significantly exceeds the concentration in the
environment.
4. In some cases, the transported substance can be subjected to chemical
modification, and this method of transporting substances is called the transfer of
radicals or the translocation of chemical groups. According to the mechanism of
transfer of the transported substance, this process is similar to active transport.
The release of substances from the microbial cell is carried out either in the form of
passive diffusion, or in the process of facilitated diffusion with the participation of
permeases.

Enzymes and their role in metabolism

Enzymes are protein substances produced by living cells. They are biological catalysts
and play an important role in the metabolism of microorganisms.
According to the chemical structure, properties and mechanism of action, microbial
enzymes are similar to enzymes that are formed in the cells and tissues of animals
and plants. Enzymes of a microbial cell are localized mainly in the cytoplasm, some
are contained in the nucleus and cell membrane. Microorganisms can synthesize a
wide variety of enzymes belonging to six known classes: oxidoreductases,
transferases, hydrolases, lyases, isomerases, ligases .
A characteristic property of enzymes is the specificity of action, i.e., each enzyme
reacts with a specific chemical compound or catalyzes one or more closely related
chemical reactions. For example, the enzyme lactase breaks down lactose, maltase
breaks down maltose.
The activity of enzymes depends on the temperature of the medium, pH and other
factors. For many pathogenic microorganisms, the optimal pH value is 7.2-7.4, and
the optimal temperature is in the range of 37-50 ° C.
Enzymes of microorganisms are classified into exoenzymes and
endoenzymes. Exoenzymes, released into the external environment, break down
nutrient macromolecules into simpler compounds that can be absorbed by the
microbial cell. So, exoenzymes include hydrolases that cause the hydrolysis of
proteins, fats, carbohydrates. As a result of these reactions, proteins are broken
down into amino acids and peptones, fats into fatty acids and glycerol,
carbohydrates (polysaccharides) into disaccharides and monosaccharides. The
breakdown of proteins is caused by protease enzymes, fats by lipases, and
carbohydrates by carbohydrases. Endoenzymes are involved in metabolic reactions
that occur inside the cell.
Microorganisms also distinguish between constitutive and inductive
enzymes. Constitutive enzymes are constantly present in the microbial cell,
regardless of the conditions of existence. These are mainly enzymes of cellular
metabolism: proteases, lipases, carbohydrases, etc. Inductive (adaptive) enzymes are
synthesized in the cell only under the influence of the appropriate substrate in the
nutrient medium, and when the microorganism is forced to absorb it. For example, if
bacteria that do not produce the enzyme amylase, which breaks down starch under
normal conditions, are planted on a nutrient medium where starch is the only source
of carbon, then they begin to synthesize this enzyme. Thus, inductive enzymes allow
the microbial cell to adapt to the changed conditions of existence.
Along with metabolic enzymes, many pathogenic bacteria also produce aggression
enzymes that serve to overcome the natural protective barriers of the
macroorganism and are pathogenicity factors. These enzymes include hyaluronidase,
deoxyribonuclease, lecitovitellase, etc. For example, hyaluronidase breaks down the
intercellular substance of the connective tissue (hyaluronic acid) and thereby
contributes to the spread of the pathogen in the macroorganism.
The secretion of various enzymes by microorganisms determines their biochemical
properties. The enzyme composition of any microorganism is a fairly constant
feature, and different types of microorganisms are quite clearly distinguished by the
set of enzymes. Therefore, the study of the enzymatic composition is important for
the differentiation and identification of various microorganisms.
Practical use of microbial enzymes . Since ancient times, man has used the
enzymatic activity of yeast in brewing and winemaking. The use of enzymes in the
food industry can significantly intensify the technological process, increase the yield
and improve the quality of the finished product. Enzymes isolated from certain types
of microscopic fungi are used in the process of making wheat dough, which makes it
possible to increase the volume and porosity of baked bread, improve its freshness,
aroma, and taste. Enzyme preparations of some microorganisms are used to
accelerate the processes of extracting juices from fruits and berries.
In order to obtain high-quality feed for farm animals, microbial synthesis processes
are used in the ensiling of green grasses; thanks to the enzymatic activity of yeast
that breeds on oil waste (paraffins), protein-vitamin concentrates are obtained,
which are a valuable nutrient - they are added to roughage for animals.
Enzymes allow certain microorganisms to digest methane, and these types of
bacteria are used to fight methane in mines. It is known that bacterial enzymes (in
particular, hay bacillus) are widely used as bioadditives to Oka washing powder and
Bio washing paste. These preparations remove protein contamination, as enzymes
break down proteins into water-soluble substances that are easily washed off during
washing.
In the medical industry, with the help of enzymes of microorganisms, vitamins,
hormones, and alkaloids are obtained.

Breath bacteria

Respiration (or biological oxidation) of microorganisms is a set of biochemical

processes, as a result of which the energy necessary for the vital activity of microbial
cells is released.
All physiological processes, such as movement, growth and reproduction, the
formation of spores and capsules, the production of toxins, can be carried out with a
constant supply of energy. Microorganisms obtain energy by oxidizing various
chemical compounds: carbohydrates (usually glucose), alcohols, organic acids, fats,
etc. The essence of oxidation is that the oxidized substance gives up electrons, and
the recovered one receives them.
According to the type of respiration, all microorganisms are divided into obligate
(strict) aerobes, obligate anaerobes and facultative (optional) anaerobes.
Obligate aerobes (mycobacterium tuberculosis, etc.) live and develop with free
access to oxygen, i.e., oxidation reactions are carried out in them with the
participation of molecular oxygen with the release of a large amount of energy. An
example is the oxidation of glucose under aerobic conditions:
C 6 H 12 O 6 + 6O 2 → 6CO 2 + 6H 2 O + 2882.6 kD (688.5 kcal)
There are also microaerophiles that need small amounts of oxygen (some leptospira,
brucella).
Obligate anaerobes (clostridia of tetanus, botulism, etc.) are able to live and
reproduce only in the absence of free oxygen in the air. Respiration in anaerobes
occurs by fermentation of the substrate with the formation of a small amount of
energy. So, with anaerobic decomposition of 1 mol of glucose, much less energy is
released than with aerobic respiration:
C 6 H 12 O 6 → 2C 2 H 5 OH + 2CO 2 + 130.6 kD (31.2 kcal)
The presence of free oxygen for obligate anaerobes is detrimental. This is due to the
fact that in the presence of oxygen, the end product of the oxidation of organic
compounds is hydrogen peroxide. And since anaerobes do not have the ability to
produce the enzyme catalase, which breaks down hydrogen peroxide, it accumulates
and has a toxic effect on bacteria.
Facultative anaerobes can reproduce both in the presence of molecular oxygen and
in its absence. These include most pathogenic and saprophytic bacteria.
The processes of decomposition of organic substances in oxygen-free conditions,
accompanied by the release of energy, are also called fermentation. Depending on
the participation of certain microorganisms and the end products of the breakdown
of carbohydrates, several types of fermentation are distinguished: alcoholic, carried
out by yeast; lactic acid, caused by lactic acid bacteria; butyric acid, caused by butyric
acid bacteria, etc.
The release of heat during the respiration of microorganisms can be observed when
growing cultures in vessels protected from heat loss - the temperature of the
nutrient medium will gradually increase. With the release of excess heat during the
respiration of some microorganisms, the processes of spontaneous combustion of
peat, manure, wet hay and cotton are associated.
The biochemical mechanisms of respiration are described in more detail in textbooks
of biological chemistry.

Pigments of microorganisms

Some microorganisms (bacteria, fungi) in the process of metabolism form coloring

substances - pigments. Pigments are heterogeneous in chemical composition and
properties. They are divided into water-soluble (blue pigment - pyocyanin, secreted
by Pseudomonas aeruginosa); soluble in alcohol and insoluble in water (red pigment
- prodigiosan, secreted by a wonderful wand); insoluble neither in water nor in
alcohol (black and brown pigments of yeasts and molds).
Water-insoluble pigments (lipochromes) usually stain bacterial colonies (for example,
yellow, golden, fawn pigments of staphylococci), and soluble pigments stain the
nutrient medium (Pseudomonas aeruginosa).
The formation of pigments in microbial cells occurs in the light with sufficient access
to oxygen and a certain composition of the nutrient medium.
Pigmentation in some cases is a persistent sign of microorganisms, which allows it to
be used as a test for the identification of certain bacteria (for example, staphylococci,
Pseudomonas aeruginosa).
Pigment formation in microorganisms has a certain physiological
significance. Pigments protect the microbial cell from natural ultraviolet radiation,
take part in the processes of respiration, some have an antibiotic effect
(prodigiosan).
Of particular interest is the story of the miraculous bacterium Serratia marcescens,
which forms red colonies on bread, potatoes and other starchy foods that look like
drops of fresh blood. The ancient Roman historian Quintus Curtius Rufus in his book
"History of Alexander the Great" described one of his victories in the conquest of
Asia Minor, associated with this amazing microbe. In 332 BC. e. during the siege of
the city of Tyros, an unpleasant event occurred in the army of Alexander the Great -
large red spots appeared in the bread, resembling blood stains, and the soldier was
seized with fear. They considered it a bad omen. However, the cunning court sage
Alexander interpreted this "sign" as follows: "Blood spots are indeed a sign of the
gods, but since they are inside baked bread, this means the death of the
troops, within the besieged walls of the city. The gods indicate their favor to the
troops of Alexander and make it clear that his victory is assured. "The sage's
interpretation raised the spirit of the army so much that the soldiers enthusiastically
attacked the walls of the city and soon captured it.
The appearance of such red spots on products during the time of religious prejudice
and obscurantism of the Middle Ages was widely used by churchmen to propagate
"God's punishment" for unbelief and served as the basis for cruel reprisals against
freethinkers.

Luminous and aroma-producing microorganisms

Among microorganisms (bacteria, fungi) there are those that have the ability to glow
(luminesce). The glow of bacteria arises as a result of intense oxidation processes,
accompanied by the release of energy. The glow of sea water, fish scales, the body of
small crustaceans, rotten wood is explained by the presence of luminous bacteria or
photobacteria on them.
All luminous bacteria are aerobes. Most of their species live in sea water, as they
reproduce better at high salt concentrations (halophilic microbes). Spiders, ants,
termites living in symbiosis with photobacteria can glow. Luminous bacteria emit a
green or bluish light that is clearly visible in the dark. Mushrooms, such as autumn
mushrooms, also glow at night.
Luminous bacteria do not cause decay processes; for most species, the optimum
temperature for life is 15-18 ° C. They grow well on fish and meat substrates, which
causes the glow of meat and fish.
At the beginning of the 20th century, they tried to use luminous bacteria for practical
purposes, they were proposed to be used for "safe lamps" in powder magazines.
Microorganisms capable of producing aromatic substances, such as acetic-ethyl,
acetic-amyl esters, have been identified. The smells of some microbes determine the
aromatic properties of wines, milk, butter, cream, cheeses, etc. Aroma-forming
bacteria are widely used in the preparation of various foods.
Some microorganisms in the process of life form substances with an unpleasant odor
(indole, skatole, hydrogen sulfide), which is associated with the decomposition of
organic substances.

Growth and reproduction of bacteria

One of the most important manifestations of the vital activity of microorganisms is

their growth and reproduction.
Growth is defined as an increase in the size of an individual and the orderly
reproduction of all cellular components and structures.
Reproduction is understood as the ability of microorganisms to reproduce
themselves, as a result of which the number of individuals in the population
increases. The main mode of reproduction in bacteria is transverse fission. Before
dividing in bacterial cells that have reached a certain age, doubling of DNA molecules
occurs. Each daughter cell receives a copy of the mother's DNA. The division process
is considered complete when the cytoplasm of the daughter cells is separated by a
septum (Fig. 9).

Rice. 9. Ultrathin section of a dividing E. coli cell

The cytoplasmic membrane and the cell wall take part in the formation of the
partition. If a septum is formed in the middle of a dividing cell, then daughter cells of
the same size appear (isomorphic division). Sometimes a septum is formed closer to
one of the ends, then the daughter cells have an unequal size (heteromorphic
division).
The division of bacteria (cocci) can occur in different planes with the formation of
diverse combinations of cells: chains of streptococci, paired compounds (diplococci),
tetrads of cocci, bales (sarcina), clusters (staphylococci). Rod-shaped and convoluted
forms are divided transversely and only in one plane.
In some bacteria, reproduction occurs by the formation of a kidney (Mycobacterium
tuberculosis, nodule bacteria), which is smaller in size than the original cell.
The rate of reproduction of bacteria is high, due to the intensity of their
metabolism. In most bacteria, each cell divides within 15-30 minutes. It has been
calculated that bacteria go through as many generations in 24 hours as a human
does in 5,000 years. There are types of bacteria that divide slowly, once a day, for
example, Mycobacterium tuberculosis.
For each type of bacteria, the rate of reproduction can be different and depends on
the age of the culture, nutrient medium, temperature, pH value, and many other
factors.
The reproduction of bacteria in a liquid nutrient medium has a number of features
and occurs in several successive phases (Fig. 10).

Rice. 10. Phases of reproduction of bacteria. ab - initial stationary; bd - logarithmic

growth phase; de - stationary phase; eg - dying stage
Phase 1 - initial stationary (latent): microbial cells adapt to the nutrient medium,
while the intensity of metabolic processes increases, the cell size increases. Bacteria
begin to multiply only towards the end of the first phase.
Phase 2 - logarithmic growth: bacteria multiply vigorously, as a result of which the
number of cells increases exponentially. In this phase, bacteria have the highest
biochemical and biological activity.
Phase 3 - stationary: the concentration of bacterial cells in the medium remains
constant. This is due to the fact that the number of newly emerging bacteria is
almost equal to the number of dying cells. The duration of this phase is different for
different bacteria.
Phase 4 - dying off: there are fewer and fewer viable bacterial cells, and they
gradually die. The causes of cell death may be the depletion of the nutrient medium,
the accumulation of harmful metabolic products in it. In this phase, bacteria can
change their morphological, biochemical, and other properties. The phase of dying in
different types of bacteria is not the same. The complete death of the culture can
occur in a few days, weeks or months.
An increase in the number of bacteria multiplied in liquid nutrient media can be
observed after 18-24 hours - either cloudiness of the medium appears, or the
formation of a film or sediment. At the same time, the nature of the change in the
medium depends both on the type and age of the bacterial culture, and on the
composition of the nutrient medium itself.
When multiplying on dense nutrient media, bacteria form colonies typical for each
microbial species on the surface of the medium and inside it. Each colony is a
population of microorganisms that has developed from a single cell of a particular
type of bacterium. Colonies of bacteria vary in size, shape, structure, consistency and
color. The appearance of colonies in some bacteria is so characteristic that it can
serve as a differential sign for the identification of microorganisms. For example,
colonies of the causative agent of anthrax can be compared to curls or a lion's mane
(see Fig. 47).
Spirochetes and rickettsia also reproduce by transverse fission. The process of
reproduction (reproduction) of viruses (see "Viruses").

Control questions
1. What is the chemical composition of a microbial cell?
2. What types of nutrition are distinguished in microorganisms?
3. How is the transport of nutrients into the microbial cell?
4. How do microorganisms differ in the type of respiration?
5. What are the methods of reproduction of bacteria?

Chapter 5. The influence of environmental factors on

microorganisms - N. A. Belskaya
The life of microorganisms is closely dependent on environmental conditions. All
environmental factors affecting microorganisms can be divided into three groups:
physical, chemical and biological, the beneficial or destructive effect of which
depends both on the nature of the factor itself and on the properties of the
microorganism.

Physical factors

Of the physical factors, temperature, drying, radiant energy, and ultrasound have the
greatest influence on the development of microorganisms.
temperature . The vital activity of each microorganism is limited by certain
temperature limits. This temperature dependence is usually expressed by three main
points: minimum - the temperature below which the reproduction of microbial cells
stops; optimum - the best temperature for the growth and development of
microorganisms; maximum - the temperature above which the vital activity of cells
weakens or stops. The optimal temperature usually corresponds to the temperature
conditions of the natural habitat.
All microorganisms in relation to temperature are divided into psychrophiles,
mesophiles and thermophiles.
Psychrophiles (from the Greek psychros - cold, phileo - love), or cold-loving
microorganisms, grow at relatively low temperatures: the minimum temperature is 0
° C, the optimum is 10-20 ° C, the maximum is 30 ° C. This group includes
microorganisms, living in the northern seas and oceans, soil, sewage. This also
includes luminous and iron bacteria, as well as microbes that cause food spoilage in
the cold (below 0 ° C).
Mesophiles (from Greek mesos - middle) - the most extensive group, including most
saprophytes and all pathogenic microorganisms. The optimum temperature for them
is 28-37°C, the minimum is 10°C, the maximum is 45°C.
Thermophiles (from the Greek termos - heat, heat), or heat-loving microorganisms,
develop at temperatures above 55 ° C, the temperature minimum for them is 30 ° C,
the optimum is 50-60 ° C, and the maximum is 70-75 ° C. They are found in hot
mineral springs, the surface layer of the soil, self-heating substrates (dung, hay,
grain), the intestines of humans and animals. There are many spore forms among
thermophiles.
High and low temperatures have different effects on microorganisms. Some are
more sensitive to high temperatures. Moreover, the higher the temperature beyond
the maximum, the faster the death of microbial cells occurs, which is due to the
denaturation (coagulation) of cell proteins.
Vegetative forms of mesophilic bacteria die at a temperature of 60°C within 30-60
minutes, and at 80-100°C - after 1-2 minutes. Bacterial spores are much more
resistant to high temperatures. For example, spores of anthrax bacilli withstand
boiling for 10-20 minutes, and spores of Clostridium botulism - 6 hours. pressure 1
atm (in autoclave) for 30 min.
The action of high temperatures on microorganisms is the basis for sterilization - the
complete release of various objects from microorganisms and their spores (see
below).
Many microorganisms are extremely resistant to low temperatures. Salmonella
typhoid and Vibrio cholerae survive for a long time in ice. Some microorganisms
remain viable at liquid air temperatures (-190°C), while bacterial spores can
withstand temperatures down to -250°C.
Only certain types of pathogenic bacteria are sensitive to low temperatures (for
example, bordetella pertussis and parapertussis, Neisseria meningococcus,
etc.). These properties of microorganisms are taken into account in laboratory
diagnostics and during transportation of the test material - it is delivered to the
laboratory protected from cooling.
The effect of low temperatures stops putrefactive and fermentation processes,
which is widely used to preserve food in refrigeration units, cellars, and glaciers. At
temperatures below 0 ° C, microbes fall into a state of suspended animation - a
slowdown in metabolic processes occurs and reproduction stops. However, in the
presence of appropriate temperature conditions and a nutrient medium, the vital
functions of microbial cells are restored. This property of microorganisms is used in
laboratory practice to preserve cultures of microbes at low temperatures. A rapid
change in high and low temperatures (freezing and thawing) also has a detrimental
effect on microorganisms - this leads to rupture of cell membranes.
Drying . Water is essential for the normal functioning of microorganisms. Drying
leads to dehydration of the cytoplasm, disruption of the integrity of the cytoplasmic
membrane, as a result of which the nutrition of microbial cells is disturbed and their
death occurs.
The timing of the death of different types of microorganisms under the influence of
drying is significantly different. So, for example, pathogenic Neisseria (meningococci,
gonococci), leptospira, pale treponema and others die when dried after a few
minutes. Vibrio cholerae withstands drying for 2 days, salmonella typhoid - 70 days,
and Mycobacterium tuberculosis - 90 days. But the dried sputum of tuberculosis
patients, in which pathogens are protected by a dry protein sheath, remains
infectious for 10 months.
Spores are particularly resistant to drying, as well as to other environmental
influences. Spores of anthrax bacilli retain the ability to germinate for 10 years, and
mold spores - up to 20 years.
The adverse effect of drying on microorganisms has long been used to preserve
vegetables, fruits, meat, fish and medicinal herbs. At the same time, once in
conditions of high humidity, such products quickly deteriorate due to the restoration
of the vital activity of microbes.
For the storage of cultures of microorganisms, vaccines and other biological
preparations, the freeze-drying method is widely used. The essence of the method is
that the microorganisms or preparations are first subjected to freezing, and then
they are dried under vacuum. At the same time, microbial cells enter a state of
suspended animation and retain their biological properties for several months or
years.
Radiant Energy . In nature, microorganisms are constantly exposed to solar
radiation. Direct sunlight causes the death of many microorganisms within a few
hours, with the exception of photosynthetic bacteria (green and purple sulfur
bacteria). The destructive effect of sunlight is due to the activity of ultraviolet rays
(UV rays). They inactivate cell enzymes and damage DNA. Pathogenic bacteria are
more sensitive to UV rays than saprophytes. Therefore, it is better to store microbial
cultures in the laboratory in the dark. Buchner's experience is demonstrative in this
respect.
In a Petri dish with a thin layer of agar, a plentiful inoculation of any bacterial culture
is carried out. On the outer surface of the seeded cup, letters cut out of black paper
are glued, forming, for example, the word "typhus". The cup, turned upside down, is
exposed to direct sunlight for 1 hour. Then the papers are removed, and the cup is
placed for a day in a thermostat at 37 ° C. Bacterial growth is observed only in those
places of the agar that were protected from UV rays by glued letters. The rest of the
agar remains clear, i.e., there is no growth of microorganisms (Fig. 11).

Rice. 11. Effect of light on bacteria

The importance of sunlight as a natural factor in the improvement of the external

environment is great. It frees air, water from natural reservoirs, upper layers of soil
from pathogenic bacteria.
The bactericidal (bacteria-destroying) effect of UV rays is used to sterilize indoor air
(operating rooms, dressing rooms, boxes, etc.), as well as water and milk. The source
of these rays are ultraviolet radiation lamps, bactericidal lamps.
Other types of radiant energy - X-rays, α-, β-, γ-rays have a detrimental effect on
microorganisms only in large doses, about 440-280 J / kg. The death of microbes is
due to the destruction of nuclear structures and cellular DNA. Small doses of
radiation stimulate the growth of microbial cells. Microorganisms are much more
resistant to radioactive radiation than higher organisms. Known thionic bacteria
living in deposits of uranium ores. Bacteria were found in the water of nuclear
reactors at a concentration of ionizing radiation of 20–30 kJ/kg.
The bactericidal effect of ionizing radiation is used to preserve certain food products,
sterilize biological preparations (sera, vaccines, etc.), while the properties of the
material to be sterilized do not change.
In recent years, disposable products have been sterilized by the radiation method -
polystyrene pipettes, Petri dishes, wells for serological reactions, syringes, as well as
suture material - catgut, etc.
Ultrasound causes significant damage to microbial cells. Under the action of
ultrasound, the gases in the liquid medium of the cytoplasm are activated, and high
pressure arises inside the cell (up to 10,000 atm). This leads to rupture of the cell
membrane and cell death. Ultrasound is used to sterilize food products (milk, fruit
juices), drinking water.
High pressure . Bacteria and especially their spores are resistant to mechanical
pressure. In nature, there are bacteria living in the seas and oceans at a depth of
1000-10000 m under pressure from 100 to 900 atm. Some types of bacteria can
withstand pressure up to 3000-5000 atm, and bacterial spores - even 20000 atm.

Chemical Factors

The effect of chemicals on microorganisms varies depending on the nature of the

chemical compound, its concentration, and the duration of exposure to microbial
cells. Depending on the concentration, a chemical substance can be a source of
nutrition or have a depressing effect on the vital activity of microorganisms. For
example, 0.5-2% glucose solution stimulates the growth of microbes, and 20-40%
glucose solutions delay the reproduction of microbial cells.
Many chemical compounds that have a detrimental effect on microorganisms are
used in medical practice as disinfectants and antiseptics.
Chemicals used for disinfection are called disinfectants. Disinfection is understood as
measures aimed at the destruction of pathogenic microorganisms in various
environmental objects. Disinfectants include halogen compounds, phenols and their
derivatives, salts of heavy metals, some acids, alkalis, alcohols, etc. They cause the
death of microbial cells, acting at optimal concentrations for a certain time. Many
disinfectants have a harmful effect on the tissues of the macroorganism.
Antiseptics are chemical substances that can cause the death of microorganisms or
delay their growth and reproduction. They are used for therapeutic purposes
(chemotherapy), as well as for the disinfection of wounds, skin, and mucous
membranes of a person. Hydrogen peroxide, alcohol solutions of iodine, brilliant
green, solutions of potassium permanganate, etc., have antiseptic properties. Some
antiseptic substances (acetic, sulfurous, benzoic acids, etc.) in doses that are
harmless to humans are used for food preservation.
According to the mechanism of action, chemicals with antimicrobial activity can be
divided into several groups.
1. Surfactants (fatty acids, soaps and other detergents) cause a decrease in surface
tension, which leads to disruption of the functioning of the cell wall and cytoplasmic
membrane of microorganisms.
2. Phenol, cresol and their derivatives cause coagulation of microbial proteins. They
are used for the disinfection of infectious material in microbiological practices and
infectious diseases hospitals.
3. Oxidizing agents, interacting with microbial proteins, disrupt the activity of
enzymes, cause protein denaturation. Active oxidizing agents are chlorine, ozone,
which are used to disinfect drinking water. Chlorine derivatives (chlorine,
chloramine) are widely used for disinfection purposes. Oxidizing properties are
hydrogen peroxide, potassium permanganate, iodine, etc.
4. Formaldehyde is used as a 40% solution (formalin) for disinfection. It kills
vegetative and spore forms of microorganisms. Formalin blocks the amino groups of
microbial cell proteins and causes their denaturation.
5. Heavy metal salts (mercury, lead, zinc, gold, etc.) coagulate microbial cell proteins,
causing their death. A number of metals (silver, gold, mercury, etc.) have a
bactericidal effect on microorganisms in negligible concentrations. This property is
called oligodynamic action (from Latin oligos - small, dinamys - strength). It has been
proven that water in silver vessels does not rot due to the bactericidal action of silver
ions. For the prevention of blennorrhea * newborns have long been used 1% solution
of silver nitrate. Colloidal solutions of organic silver compounds (protargol, collargol)
are also used as local antiseptics.
*
 ( Blennorrhea - inflammation of the conjunctiva of the eye caused by gonococci. )
Mercury preparations have a strong antimicrobial effect. Since ancient times,
mercury bichloride, or sublimate (at a dilution of 1:1000), has been used for
disinfection. However, it has a toxic effect on the tissues of the macroorganism and
its use is limited.
6. Dyes (brilliant green, rivanol, etc.) have the property of inhibiting the growth of
bacteria. Solutions of a number of dyes are used as antiseptics, and are also
introduced into the composition of some nutrient media to inhibit the growth of
associated microflora.
The destructive effect of a number of physical and chemical factors on
microorganisms forms the basis of aseptic and antiseptic methods widely used in
medical and sanitary practice.
Asepsis is a system of preventive measures that prevent microbial contamination of
an object (wound, surgical field, cultures of microorganisms, etc.), based on physical
methods.
Antiseptics - a set of measures aimed at the destruction of microorganisms in the
wound, the whole organism or on environmental objects, using various disinfecting
chemicals.

Biological factors

Under natural habitat conditions, microorganisms do not exist in isolation, but are in
complex relationships, which are reduced mainly to symbiosis, metabiosis and
antagonism.
Symbiosis is the cohabitation of organisms of different species, bringing them mutual
benefit. At the same time, together they develop better than each of them
separately.
Symbiotic relationships exist between rhizobia and leguminous plants, between
filamentous fungi and blue-green algae (lichens): Symbiosis of lactic acid bacteria and
alcohol yeast is used to prepare some lactic acid products (kefir, koumiss).
Metabiosis is a type of relationship in which the metabolic products of one type of
microorganism create the necessary conditions for the development of others. For
example, putrefactive microorganisms that break down protein substances
contribute to the accumulation of ammonium compounds in the environment and
create favorable conditions for the growth and development of nitrifying
bacteria. And the development of anaerobes in well-aerated soil would be
impossible without aerobes that absorb free oxygen.
Metabiotic relationships are widespread among soil microorganisms and underlie
the cycle of substances in nature.
Antagonism is a form of relationship in which one microorganism inhibits the
development of another or can cause its complete death. Antagonistic relationships
have developed among microorganisms in the struggle for existence. Wherever they
live, there is a continuous struggle between them for food sources, air oxygen, and
habitat. So, most pathogenic bacteria, having got into the external environment (soil,
water) with the secretions of patients, do not withstand long-term competition with
numerous saprophytes and die relatively quickly.
Antagonism may be due to the direct action of microorganisms on each other or the
action of their metabolic products. For example, protozoa devour bacteria, while
phages lyse them. The intestines of newborns are inhabited by lactic acid bacteria
Bifidobacterium bifidum. By secreting lactic acid, they suppress the growth of
putrefactive bacteria and thus protect the still fragile organism of infants from
intestinal disorders. Some microorganisms in the process of life produce various
substances that have a detrimental effect on bacteria and other microbes. These
substances include antibiotics (see "Antibiotics").

Control questions

1. What physical factors affect the vital activity of microorganisms?

2. What substances are classified as disinfectants and how do they differ in the
mechanism of action on microorganisms?
3. List the relationships that exist between microorganisms?
 Sterilization

Sterilization is despawning, i.e., the complete release of environmental objects from

microorganisms and their spores.
Sterilization is carried out in various ways:
1) physical (exposure to high temperature, UV rays, use of bacterial filters);
2) chemical (use of various disinfectants, antiseptics);
3) biological (use of antibiotics).
In laboratory practice, physical methods of sterilization are usually used.
The possibility and expediency of using one or another method of sterilization is due
to the characteristics of the material to be sterilized, its physical and chemical
properties.
Physical methods
Ignition in the flame of a burner or flaming is a method of sterilization in which the
object is completely decomposed, since both vegetative cells and spores of
microorganisms die. Usually calcined bacteriological loops, spatulas, pipettes, glass
slides and coverslips, small instruments. Scissors and scalpels should not be sterilized
by calcination, as the cutting surface becomes dull under the action of fire.
Dry heat sterilization
Sterilization by dry heat or hot air is carried out in Pasteur ovens (drying ovens). The
Pasteur oven is a double-walled cabinet made of heat-resistant materials - metal and
asbestos. Heat the cabinet with gas burners or electric heaters. Cabinets with electric
heating are equipped with regulators that provide the required temperature. To
control the temperature, there is a thermometer inserted into the hole in the top
wall of the cabinet.
Dry heat sterilizes mainly laboratory glassware. The dishes prepared for sterilization
are loosely loaded into the oven to ensure uniform and reliable heating of the
material to be sterilized. The cabinet door is tightly closed, the heating device is
turned on, the temperature is brought to 160-165 ° C and sterilized at this
temperature for 1 hour. At the end of sterilization, the heating is turned off, but the
cabinet door is not opened until the oven has cooled down; Otherwise, the cold air
entering the inside of the cabinet may cause cracks on hot dishes.
Sterilization in the Pasteur oven can be carried out at different temperatures and
exposures (sterilization time) (Table 1).

Table 1. Sterilization mode

Liquids (nutrient media, isotonic sodium chloride solution, etc.), items made of
rubber and synthetic materials cannot be sterilized by dry heat, since liquids boil and
pour out, and rubber and synthetic materials melt.
To control sterilization in the Pasteur oven, silk threads are moistened in a culture of
spore-forming bacteria, dried, placed in a sterile Petri dish and placed in the Pasteur
oven. Sterilization is carried out at a temperature of 165 ° C for 1 hour (for control,
some of the threads are left at room temperature). Then the sterilized and control
threads are placed on the surface of the agar in a Petri dish or placed in test tubes
with broth and incubated in a thermostat at a temperature of 37°C for 2 days. With
the correct operation of the Pasteur oven, in test tubes or cups with nutrient media
where the sterilized threads were placed, there will be no growth, since bacterial
spores will die, while bacterial spores on threads that have not been sterilized
(control) will also germinate on nutrient media growth will be noted.
To determine the temperature inside the Pasteur oven, you can use sucrose or
edible granulated sugar, caramelized at a temperature of 165-170 ° C.
Preparation of laboratory glassware for sterilization in the Pasteur oven . Before
sterilization, laboratory glassware (Petri dishes, graduated and Pasteur pipettes,
vials, flasks, test tubes) must be thoroughly washed, dried and wrapped in paper,
otherwise after sterilization it may again become contaminated with air bacteria.
Petri dishes are wrapped in paper one or more pieces or placed in special metal
cases.
Cotton swabs are inserted into the upper ends of the pipettes to prevent the test
material from entering the mouth. Graduated pipettes are wrapped in long strips of
paper 4-5 cm wide. The volume of the wrapped pipette is noted on the paper. In
cases, graduated pipettes are sterilized without additional paper wrapping.
Note . If the graduation on the pipettes is poorly visible, it is restored before
sterilization. Oil paint is applied to the pipette and, not allowing the paint to dry,
barium sulfate powder is rubbed into it with a cloth. After that, excess paint is
removed with a rag, which remains only in the graduation notches. Pipettes treated
in this way should be rinsed.
The sharp ends of the Pasteur pipettes are sealed in a burner flame and wrapped in
paper, 3-5 pieces each. Wrap the Pasteur pipettes carefully so as not to break off the
sealed ends of the capillaries.
Vials, flasks, test tubes are closed with cotton-gauze stoppers. The cork should fit
into the neck of the vessel for 2/3 of its length , not too tight, but not too loose. A paper
cap is put on top of the corks for each vessel (except for test tubes). The test tubes
are tied up in 5-50 pieces and wrapped over with paper.
Note . At high temperatures, the paper in which cups and pipettes are wrapped, and
cotton wool, turn yellow and may even char, so each new grade of paper obtained by
the laboratory should be tested at the accepted temperature regime.
Control questions
1. What is meant by the term sterilization?
2. What are the methods of sterilization?
3. What is sterilized by roasting on fire?
4. Describe the device and mode of operation of the Pasteur furnace.
5. What is sterilized in the Pasteur oven?
6. How is glassware prepared for sterilization?
7. Why is it impossible to sterilize culture media and rubber objects in the Pasteur
oven?
Exercise
Prepare Petri dishes, graduated pipettes, Pasteur pipettes, test tubes, flasks and vials
for sterilization.
Sterilization by boiling
Boiling is a sterilization method that guarantees sterilization provided that there are
no spores in the sterilized material. They are used for processing syringes of
instruments, glass and metal utensils, rubber tubes, etc.
Boiling sterilization is usually carried out in a sterilizer - a rectangular metal box with
a tight-fitting lid. The material to be sterilized is placed on the mesh in the sterilizer
and filled with water. To increase the boiling point and eliminate water hardness,
add 1-2% sodium bicarbonate (it is better to use distilled water). The sterilizer is
closed with a lid and warmed up. The beginning of sterilization is considered the
moment of boiling water, boiling time is 15-30 minutes. At the end of sterilization,
the mesh with tools is removed by the side handles with special hooks, and the tools
in it are taken with sterile tweezers or forceps, which are boiled along with the rest
of the tools.
Steam sterilization is carried out in two ways: 1) steam under pressure; 2) flowing
steam.
Steam sterilization under pressure is carried out in an autoclave. This method of
sterilization is based on the effect of saturated water vapor on the sterilized
materials at a pressure above atmospheric. As a result of such sterilization, both
vegetative and spore forms of microorganisms die during a single treatment.
Autoclave (Fig. 12) - a massive boiler, covered with a metal casing on the outside,
hermetically sealed with a lid, which is tightly screwed to the boiler with hinged
bolts. Another, smaller diameter, which is called the sterilization chamber, is inserted
into the outer boiler. Items to be sterilized are placed in this chamber. Between both
boilers there is a free space called a water-steam chamber. Water is poured into this
chamber through a funnel fixed on the outside to a certain level, marked on a special
water-metering tube. When water is boiled in a steam chamber, steam is
produced. The sterilization chamber is equipped with an outlet cock with a safety
valve for steam to escape when the pressure rises above the required level. A
manometer is used to determine the pressure created in the sterilization chamber.
 Rice. 12. Scheme of the autoclave. M - pressure gauge; PC - safety valve; B - funnel
for water; K  2 - tap for water release; K  3 - tap for steam release

Normal atmospheric pressure (760 mm Hg) is taken as zero. There is a certain

relationship between the pressure gauge readings and temperature (Table 2).

Table 2. Autoclave operation mode

Autoclaves with automatic mode control are now available. In addition to the usual
pressure gauge, they are equipped with an electrocontact pressure gauge, which
prevents the pressure from increasing above the set value and thus ensures the
constancy of the desired temperature in the autoclave.
Various nutrient media (except those containing native proteins), liquids (isotonic
sodium chloride solution, water, etc.) are sterilized with steam under
pressure; appliances, especially those with rubber parts.
The temperature and duration of autoclaving of nutrient media is determined by
their composition, specified in the recipe for the preparation of the nutrient
medium. For example, simple media (meat-peptone agar, meat-peptone broth) are
sterilized for 20 minutes at 120 ° C (1 atm). However, at this temperature it is
impossible to sterilize media containing native proteins, carbohydrates and other
substances easily changed by heating. Media with carbohydrates are sterilized
fractionally at 100°C or in an autoclave at 112°C (0.5 atm) for 10-15 minutes. Various
liquids, devices with rubber hoses, plugs, bacterial candles and filters are sterilized
for 20 minutes at 120 ° C (1 atm).
Attention! In autoclaves, the infected material is also neutralized. Cups and test
tubes containing cultures of microorganisms are placed in special metal buckets or
tanks with holes in the lid for steam penetration and sterilized in an autoclave at 126
° C (1.5 atm) for 1 hour. Instruments are sterilized in the same way after working
with bacteria that generate controversy.
Only specially trained persons are allowed to work with the autoclave, who must
strictly and accurately follow the rules specified in the instructions attached to the
device.
Autoclaving technique . 1. Before work, check the serviceability of all parts and the
lapping of taps.
2. Through a funnel fixed on the outside of the boiler, water (distilled or boiled) is
poured up to the upper mark of the water gauge glass so that scale does not
form. The faucet under the funnel is closed.
3. The material to be sterilized is placed on a special mesh in the sterilization
chamber. Items should not be loaded too tightly, as steam must pass freely between
them, otherwise they will not heat up to the correct temperature and may remain
unsterile.
4. The rubber gasket on the lid is rubbed with chalk for better sealing.
5. The lid is closed and bolted to the body of the autoclave, and the bolts are twisted
in pairs crosswise.
6. Fully open the exhaust cock connecting the sterilization chamber to the outside
air, and begin to heat the autoclave. The autoclave is usually heated by gas or
electricity.
When the autoclave is heated, the water boils, the resulting steam rises between the
walls of the boilers and through special holes in the wall of the inner boiler (see Fig.
12), enters the sterilization chamber and exits through the open outlet cock. First,
the steam escapes along with the air that was in the autoclave. It is essential that all
air is expelled from the autoclave, otherwise the pressure gauge reading will not
correspond to the temperature in the autoclave.
The appearance of a continuous strong jet of steam indicates the complete removal
of air from the autoclave; after that, the outlet cock is closed and the pressure inside
the autoclave begins to rise gradually.
7. The beginning of sterilization is considered the moment when the pressure gauge
readings reach the specified value. Heating is regulated so that the pressure in the
autoclave does not change for a certain time.
8. After the sterilization time has elapsed, the heating of the autoclave is stopped,
the steam is released through the outlet cock. When the gauge needle drops to zero,
open the lid. To avoid burns from the steam remaining in the autoclave, open the lid
towards you.
The temperature level in the autoclave, i.e. the correctness of the pressure gauge,
can be checked. For this, various substances are used that have a certain melting
point: antipyrine (113 ° C), resorcinol and sulfur (119 ° C), benzoic acid (120 ° C). One
of these substances is mixed with a negligible amount of dye (magenta or methylene
blue) and poured into a glass tube, which is sealed and placed in a vertical position
between the material to be sterilized. If the temperature is sufficient, the substance
will melt and turn into the color of the corresponding dye.
To check the effectiveness of sterilization, a test tube with a known spore culture is
placed in the autoclave. After autoclaving, the tube is transferred to a thermostat for
24-48 hours, the absence or presence of growth is noted. The absence of growth
indicates the correct operation of the device.
Sterilization with flowing steam is carried out in the Koch apparatus. This method is
used in cases where the object to be sterilized changes at a temperature above 100 °
C. Fluid steam sterilizes nutrient media containing urea, carbohydrates, milk,
potatoes, gelatin, etc.
The Koch apparatus (boiler) is a metal cylinder sheathed on the outside (to reduce
heat transfer) with felt or asbestos. The cylinder is closed with a conical lid with a
hole for steam to escape. Inside the cylinder there is a stand, up to the level of which
water is poured. A bucket with a hole is placed on the stand, into which the material
to be sterilized is placed. The Koch apparatus is heated with gas or electricity. The
sterilization time is counted from the moment of vigorous release of steam at the
edges of the lid and from the steam outlet. Sterilize for 30-60 minutes. At the end of
sterilization, the heating is stopped. The bucket with the material is removed from
the apparatus and left at room temperature until the next day. Warming up is carried
out for 3 days in a row at a temperature of 100 ° C for 30-60 minutes. This method is
called fractional sterilization. During the first heating, the vegetative forms of
microbes die, while the spore ones remain. During the day, the spores have time to
germinate and turn into vegetative forms that die on the second day of
sterilization. Since it is possible that some of the spores did not have time to
germinate, the material is kept for another 24 hours, and then a third sterilization is
carried out. Sterilization with flowing steam in the Koch apparatus does not require
special control, since the sterility of the prepared nutrient media serves as an
indicator of the correct operation of the device. Steam sterilization can also be done
in an autoclave with the lid unscrewed and the outlet cock open. the material is kept
for another 24 hours, and then a third sterilization is carried out. Sterilization with
flowing steam in the Koch apparatus does not require special control, since the
sterility of the prepared nutrient media serves as an indicator of the correct
operation of the device. Steam sterilization can also be done in an autoclave with the
lid unscrewed and the outlet cock open. the material is kept for another 24 hours,
and then a third sterilization is carried out. Sterilization with flowing steam in the
Koch apparatus does not require special control, since the sterility of the prepared
nutrient media serves as an indicator of the correct operation of the device. Steam
sterilization can also be done in an autoclave with the lid unscrewed and the outlet
cock open.
Control questions
1. What culture media can be steam sterilized?
2. What is a sterilizer and how does it work?
3. Why should distilled water be used for sterilization by boiling?
4. Describe the device and mode of operation of the autoclave.
5. What is sterilized in an autoclave?
6. What controls the correct sterilization in autoclaving?
7. What is steam sterilization?
8. Describe the device of the Koch apparatus.
9. What is the purpose of fractional sterilization?
Exercise
Fill the form.

Fractional sterilization can also be carried out in a Koch coiler.

The Koch Coiler is used to coagulate whey and egg nutrient media, and
simultaneously with the compaction of the medium, it is sterilized.
The Koch coiler is a double-walled flat metal box, covered on the outside with a
heat-insulating material. Water is poured into the space between the walls through a
special hole located in the upper part of the outer wall. The hole is closed with a
stopper into which a thermometer is inserted. Close the device with two covers:
glass and metal. Through the glass lid, you can observe the clotting process. Test
tubes with media are placed on the bottom of the coiler in an inclined position.
The coiler is heated by gas or electricity. The media are sterilized once at a
temperature of 90 ° C for 1 hour or fractionally - 3 days in a row at 80 ° C for 1 hour.
Tyndallization * - fractional sterilization at low temperatures - is used for substances
that are easily destroyed and denatured at a temperature of 60 ° C (for example,
protein liquids). The material to be sterilized is heated in a water bath or in special
devices with thermostats at a temperature of 56-58 ° C for an hour for 5 days in a
row.
*
 ( Method of sterilization, named after Tyndall, who proposed it. )
Pasteurization - sterilization at 65-70 ° C for 1 hour, proposed by Pasteur for the
destruction of non-spore forms of microbes. Pasteurize milk, wine, beer, fruit juices
and other products. Milk is pasteurized in order to get rid of lactic acid and
pathogenic bacteria (Brucella, Mycobacterium tuberculosis, Shigella, Salmonella,
Staphylococcus, etc.). Pasteurization of beer, fruit juices, wine kills microorganisms
that cause various types of fermentation. Pasteurized foods are best kept
refrigerated.
Control questions
1. What is the purpose and device of the Koch coiler?
2. What are the methods of sterilization in the coiler?
3. What is tyndalization?
4. What is pasteurization?
Sterilization by ultraviolet irradiation
Sterilization with UV rays is carried out using special installations - bactericidal
lamps. UV rays have a high antimicrobial activity and can cause the death of not only
vegetative cells, but also spores. UV irradiation is used to sterilize air in hospitals,
operating rooms, children's institutions, etc. In a microbiological laboratory, a box is
treated with UV rays before work.
Control questions
1. What are the properties of ultraviolet rays?
2. In what cases is sterilization by ultraviolet radiation used?
Mechanical sterilization with bacterial filters
Filtration sterilization is used in cases where the objects to be sterilized change when
heated. Filtration is carried out using bacterial filters made of various finely porous
materials. The pores of the filters must be small enough (up to 1 µm) to provide
mechanical retention of bacteria, so some authors consider filtration to be a
mechanical sterilization method.
The filtration method sterilizes nutrient media containing protein, serum, some
antibiotics, and also separates bacteria from viruses, phages and exotoxins.
In microbiological practice, Seitz asbestos filters, membrane filters and Chamberlan
and Berkefeld filters (candles) are used.
Seitz filters are discs made from a mixture of asbestos and cellulose. Their thickness
is 3-5 mm, diameter is 35-140 mm. The domestic industry produces filters of two
grades: "F" (filtering) - retaining suspended particles, but passing bacteria; "SF"
(sterilizing) - with smaller pores, trapping bacteria, but passing viruses. Crumpled
asbestos plates, as well as plates with breaks and cracks, are unsuitable for work.
Membrane filters are made from nitrocellulose. They are white discs 0.1 mm thick
and 35 mm in diameter. Depending on the pore size, they are designated nos. 1, 2, 3,
4, and 5 (Table 3).

Table 3. Characteristics of membrane filters

Filter No. 1 is most suitable for sterilization. In addition to those listed, they also
produce the so-called pre-filter, designed to free the filtered liquid from large
particles contained in it.
Chamberlant and Berkefeld filters (candles) are hollow cylinders closed at one
end. Chamberlain candles are made from kaolin mixed with sand and quartz. They
are standardized by pore size and denoted L 1 , L 2 , L 3 ... L 13 . Berkefeld filters
(candles) are prepared from infusor earth, they are designated V, N, W by the size of
the pores, which corresponds to a pore diameter of 3-4, 4-7, 8-12 microns.
Work with bacterial filters is carried out as follows. The filter must be fixed in a
special holder, which is inserted into the filter receiver. The receiver is usually a
Bunsen flask. The holders, in most cases made of stainless steel, consist of two parts:
the upper one, which has the shape of a cylinder without a bottom, and the lower
one, the supporting part, ending with a tube. The Seitz filters, with the rough surface
up, are placed on the metal grid and screwed tightly between the top and bottom of
the holder. The mounted filter is fixed in a rubber stopper inserted into the neck of
the Bunsen flask. A cotton swab is inserted into the outlet tube of the flask, which is
connected to a vacuum pump. The prepared installation is wrapped in paper and
sterilized in an autoclave at a pressure of 1 atm for 20-30 minutes.

Rice. 13. Seitz filter

Immediately prior to filtration, the outlet end of the Bunsen flask is connected by a
rubber tube to an oil or water jet pump. The joints of the various parts are filled with
paraffin to create tightness. The liquid to be filtered is poured into the cylinder of the
apparatus and the pump is turned on to create a vacuum in the receiver. As a result
of the resulting pressure difference, the filtered liquid passes through the filter pores
into the receiver, and the microbes remain on the filter surface.
Membrane filters are sterilized by boiling in distilled water before use. To prevent
the filters from twisting, they are first placed in distilled water, heated to a
temperature of 50-60 ° C, and boiled over low heat for 30 minutes, changing the
water 2-3 times. The filter holder and receiver are sterilized in advance, the device is
mounted under aseptic conditions. In order not to tear the membrane filter on the
metal mesh, circles of sterile filter paper are placed under it. Then, with sterile
tweezers with smooth tips, take the membrane filter from the sterilizer and place it
on the support grid with a shiny surface down.
Candles (Chamberlant) sterilized in an autoclave are connected by means of a rubber
tube to a receiver and lowered into a vessel (usually a cylinder) with a filtered
liquid. Filtration takes place using a vacuum pump. A sterile filtrate enters the
receiver, and the bacteria are retained by the pores of the candle.
Membrane and asbestos filters are designed for single use. Candles after use are
boiled in tap water, then calcined in a muffle furnace.
Before subsequent use, the candles are checked for integrity. The candle is lowered
into a vessel with water and air is passed through. If air bubbles appear on the
surface of the candle, then cracks have formed in the candle and it is unusable.
Control questions
1. What is the filter sterilization method? What is sterilized by this method?
2. What bacterial filters do you know? How is the filtering device mounted, what
conditions must be observed?
Chemical methods
This type of sterilization is used to a limited extent, and it serves mainly to prevent
bacterial contamination of nutrient media and immunobiological preparations
(vaccines and sera).
Substances such as chloroform, toluene, and ether are most often added to nutrient
media. If it is necessary to free the medium from these preservatives, it is heated in a
water bath at 56 ° C (the preservatives evaporate).
For the conservation of vaccines, sera, merthiolate, boric acid, formalin, etc. are
used.
biological sterilization
Biological sterilization is based on the use of antibiotics. This method is used in the
cultivation of viruses.
Control questions
1. What is chemical sterilization and when is it used?
2. What is biological sterilization?
The main methods of sterilization are presented in table. 4.

Table 4. Main methods of sterilization

1
 ( Sterilization incomplete: spores remain in the sterilized material. )
2
 ( Sterilization incomplete: viruses remain in the sterilized material. )

Disinfection

Various disinfectants are used in microbiological practice: 3-5% phenol solutions, 5-

10% Lysol solutions, 1-5% chloramine solutions, 3-6% hydrogen peroxide solutions,
1-5% formalin solutions, sublimate solutions in dilution 1: 1000 (0.1%), 70° alcohol,
etc.
The spent pathological material (pus, feces, urine, sputum, blood, cerebrospinal
fluid) is disinfected before it is drained into the sewer. Disinfection is carried out with
dry bleach or 3-5% chloramine solution.
Contaminated with pathological material or cultures of microorganisms, pipettes
(graded and Pasteur), glass spatulas, slides and coverslips are dipped for a day in
glass jars with a 3% solution of phenol or hydrogen peroxide.
Upon completion of work with infectious material, the laboratory assistant must
treat the workplace and hands with a disinfectant solution. The surface of the
desktop is wiped with a piece of cotton wool moistened with a 3% phenol
solution. Hands are disinfected with 1% chloramine solution. To do this, moisten a
cotton ball or gauze with a disinfectant solution and wipe the left hand, then the
right, and then wash their hands with warm water and soap.
The choice of disinfectant, its concentration and duration of exposure (exposure)
depend on the biological properties of the microbe and on the environment in which
the contact of the disinfectant with pathogenic microorganisms will occur. For
example, sublimate, phenol, alcohols are unsuitable for the disinfection of protein
substrates (pus, blood, sputum), since under their influence protein coagulation
occurs, and the coagulated protein protects microorganisms from exposure to
disinfectants.
When disinfecting material infected with spore forms of microorganisms, a 5%
solution of chloramine, 1-2.5% solutions of activated chloramine, 5-10% solutions of
formalin and other substances are used.
Disinfection, which is carried out throughout the day in the course of work, is called
current, and at the end of work - final.
Disinfectants and prescriptions for the preparation of working solutions from
them . Chlorine lime is a white lumpy powder with a strong smell of chlorine, it does
not completely dissolve in water. The bactericidal effect depends on the content of
active chlorine, the amount of which ranges from 28 to 36%. Bleach containing less
than 25% active chlorine is unsuitable for disinfection.
If stored improperly, bleach decomposes and loses some of the active
chlorine. Decomposition is facilitated by heat, moisture, sunlight, so bleach should
be stored in a dry, dark place, in a tightly closed container.
Dry bleach is used to disinfect excretions of humans and animals (at the rate of 200 g
per 1 liter of feces and 10 g per 1 liter of urine).
Preparation of the initial 10% clarified bleach solution. Take 1 kg of dry bleach, place
in an enameled bucket and grind. Then pour cold water up to a volume of 10 liters,
mix well, cover with a lid and leave for a day in a cool place. After that, the resulting
10% clarified solution is carefully drained and filtered through several layers of gauze
or filtered through a dense cloth. Store in dark glass bottles, closed with a wooden
stopper, in a cool place, no more than 10 days. Working solutions of the required
concentration are prepared from the main solution immediately before their
use. The amount of basic solution required for the preparation of 0.2-10% clarified
solutions of bleach is given in table. 5.
 Table 5. Scheme for the preparation of bleach solutions

The concentration of clarified bleach solutions from 0.2 to 10% is chosen depending
on the nature of the object to be disinfected and the resistance of the pathogen.
Chloramine - a crystalline substance of white or yellowish color, contains 24-28%
active chlorine. It dissolves well in water at room temperature, so its solutions are
prepared immediately before disinfection. Use 0.2-10% solutions of chloramine. The
ratio between the percentage concentration of the solution and the amount of
chloramine in grams per 1 and 10 liters is given in table. 6.

Table 6. Scheme for the preparation of various solutions of chloramine

Dissolve chloramine in a glass or enamel bowl. When storing chloramine solutions in

a dark glass container with a ground stopper, their activity persists for up to 15 days.
activated chloramine. The disinfecting properties of chloramine are enhanced by
adding an activator to it in a ratio of 1:1 or 1:2. Ammonium compounds - chloride,
sulfate, ammonium nitrate - are used as an activator. Activated chloramine is used at
a concentration of 0.5, 1 and 2.5%. Prepare them immediately before
use. Chloramine and ammonium salt are weighed separately. First, chloramine is
dissolved in water, and then the activator is added.
The advantage of activated chloramine solutions over conventional ones is that
when an activator is added, the release of active chlorine is accelerated. Therefore,
the drug has a detrimental effect not only on the vegetative forms of
microorganisms, but also on their spores. Activated chloramine is used at lower
concentrations and at lower exposure.
Phenol (carbolic acid) is a colorless needle-shaped crystals with a sharp characteristic
odor. Under the action of light, air and moisture, the crystals acquire a crimson-red
color. Store in closed dark glass jars and in a place protected from light.
Phenol is soluble in water, alcohol, ether, fatty oils. Possessing high hygroscopicity, it
absorbs moisture from the environment and becomes liquid. Liquid carbolic acid
contains 90% crystalline phenol and 10% water.
Apply 3-5% aqueous solutions of carbolic acid, prepared from crystalline phenol and
liquid carbolic acid according to the scheme shown in table. 7. The activity of phenol
increases when it is dissolved in hot water (40-50 ° C).

Table 7. Scheme for the preparation of various solutions of carbolic acid

Attention! Crystalline phenol or liquid carbolic acid, getting on the skin, can cause
skin irritation, and in high concentrations - severe burns. Therefore, carbolic acid
must be handled with great care. When making solutions, wear rubber gloves or, in
extreme cases, lubricate your hands with petroleum jelly.
If carbolic acid comes into contact with the skin, immediately wash it off with warm
water and soap or 40 ° ethyl alcohol.
Note. For the preparation of disinfectant solutions of phenol, it is more convenient
and safer to use liquid carbolic acid.
 Control questions
1. What disinfectants are used in microbiological practice?
2. Describe the appearance and basic properties of bleach, chloramine, phenol.
3. What solutions of disinfectants are used to disinfect material infected with spore
forms of microorganisms?
Exercise
Prepare 2 liters of a 5% working solution of clarified bleach; 500 ml of 3% chloramine
solution, 300 ml of 1% activated chloramine solution.
Attention! Before you start preparing solutions, do the calculations.

Chapter 6. The spread of microorganisms in nature - L.

B. Bogoyavlenskaya
Microorganisms are ubiquitous in the environment. They are found in soil, water, air,
human and animal bodies. Microorganisms are involved in the processes of
transformation of substances, their assimilation by plants and animals.
Microorganisms have the ability to adapt (adapt) to a variety of environmental
conditions. They are found in various combinations (associations) and
quantities. Each object has its own characteristic microflora. Our knowledge of the
characteristics of the spread of microorganisms helps to prevent infectious diseases
and even eliminate some of them.

Soil microflora

In the soil, microorganisms find the most favorable conditions for their
development. Organic matter, mineral compounds, sufficient soil moisture create
conditions for the accumulation of a huge number of microorganisms in it.
The richest in microorganisms is cultivated, cultivated soil (up to 5 billion per 1 g of
soil), the least is desert soil, poor in moisture and organic matter (200 million per 1
g).
The number of microorganisms in the soil is also not the same in different climatic
conditions: in the southern regions it is much higher. Their distribution is uneven in
different layers of the soil. So, in the surface layer of the soil, due to the destructive
effect of sunlight and drying, there are relatively few microorganisms, at a depth of
10-20 cm their number reaches a maximum and then, as they deepen, their number
rapidly falls.
Soil microflora is very diverse; it consists of nitrifying, nitrogen-fixing, denitrifying,
cellulose-decomposing bacteria; gray and iron bacteria, fungi, algae, protozoa. Most
microorganisms living in the soil take part in the cycle of substances in nature: the
decomposition of organic substances to inorganic ones, the assimilation of mineral
elements and the fixation of atmospheric nitrogen by plants. Microorganisms change
the structure and chemical composition of the soil.
Soil can serve as a pathway for the transmission of infectious agents. Pathogenic
bacteria enter the soil with excretions of humans and animals, corpses and
waste. Most of them, due to a lack of nutrients, the influence of sunlight and the
action of antagonist microbes, quickly die. However, some microorganisms persist
for a time sufficient for the spread of infection (from several hours to several
months). There are also microorganisms that persist for a long time (many years) in
the soil, through which the infection of animals and humans occurs. These include
spore-forming bacteria: pathogens of anthrax, tetanus, gas gangrene. And, finally, for
some microorganisms, the soil is a permanent habitat: pathogens of botulism,
actinomycetes, etc.

Water microflora

The water of open reservoirs is a natural habitat for many microorganisms. They

enter the water from the soil, with excretions of humans and animals, waste,
sewage.
The usual microflora of the soil is saprophytes. Pseudomonas, micrococci, vibrios live
in water. In addition, pathogens of infectious diseases can get into the water, persist
and even multiply. For example, Escherichia coli and typhoid pathogens survive in
water for a long time, while cholera pathogens multiply.
The intensity of water contamination by microorganisms and the composition of
microflora depend on the degree of pollution of the reservoir, especially with organic
compounds. Close to populated areas where water bodies are polluted with sewage,
household and industrial waters, the number of microorganisms in the water is
especially high, and the microflora is more diverse.
Self-purification processes are constantly taking place in water - microorganisms die
from the action of sunlight and chemicals, precipitation, exposure to antibiotic
substances produced by other microorganisms, algae, fungi.
The water of the seas and oceans is also rich in microorganisms, but there are much
fewer of them than in freshwater open reservoirs. There are especially many
microorganisms in the layer of bottom silt, on which they form a thin film. The
cleanest are soil waters that come to the surface through artesian wells and springs.
Water plays a big role in the transmission of infectious diseases. The causative agents
of intestinal infections, poliomyelitis, tularemia, leptospirosis often cause "water"
epidemics, and for cholera, water is the main route of infection transmission.
Determining the purity of water and preventing its pollution is one of the mandatory
measures in the fight against infectious diseases.

Air microflora

The air does not contain nutrient substrates necessary for the development of
microorganisms. In addition, solar radiation, temperature changes and other factors
have an adverse effect on microorganisms. Despite this, a significant number of
microorganisms are constantly in the air, which enter the air with dust from the soil
surface. Most often in the air there are spores of fungi and bacteria, pigment
saprophytic bacteria, mold and yeast fungi, and various cocci.
The number of microorganisms in the air varies widely.
The most polluted air in large industrial cities. In rural areas, the air is much cleaner,
and the least amount of microorganisms is found in the air above the forest,
mountains, and seas.
There are fewer microorganisms in the upper layers of the atmosphere than in the
lower ones; less in winter than in summer; more indoors than outdoors. Especially a
lot of bacteria in poorly ventilated areas in the absence of wet cleaning.
Pathogenic microorganisms enter the air along with droplets of saliva and sputum,
coughing, sneezing, talking sick people, as well as dust from contaminated objects
and infected soil.
Microorganisms are in the air in the form of an aerosol (droplets of liquid or in the
smallest solid particles suspended in the air).
Inhaling air contaminated with pathogenic microorganisms can make a person
sick. This way of transmission of infection is called airborne (air-dust).
Low-resistant pathogenic microorganisms are usually transmitted only at a distance
close to the patient (the causative agent of measles, influenza, whooping
cough); dust particles carry cocci, spores and more resistant microorganisms. The
latter include pathogens of anthrax, tuberculosis, etc. Epidemics of diseases spread
through the air usually occur in winter when people gather in enclosed spaces that
are insufficiently ventilated and in the absence of daily wet cleaning.
To prevent these diseases, gauze masks are used, which are used by medical
personnel, patients, employees of children's institutions.

Microflora of the human body

Normal human microflora has developed as a result of the interaction of micro- and
macroorganism in the process of evolution. The totality of microbial species
characteristic of individual organs and cavities of the body - biocenosis - is a
necessary condition for the normal functioning of the body. Violation of the
biocenosis, the appearance of microorganisms unusual for it, especially pathogens,
causes the development of the disease.
The human fetus is sterile during pregnancy. Already during childbirth,
microorganisms enter the child's body from the birth canal of the mother. They also
come from the mother's skin, the hands of staff, surrounding objects and the air.
During a person's life, the nature of the microflora changes, but in general it is
constant and characteristic of individual organs. Human internal organs are usually
sterile (blood, brain, liver, etc.). Organs and tissues that communicate with the
environment contain microorganisms.
The microflora of the skin is quite constant. It is represented by staphylococci,
streptococci, diphtheroids, spore-forming bacteria, yeast-like fungi. The nutrient
substrate for them is the secretions of the sebaceous and sweat glands, dead cells
and decay products. Microorganisms that have fallen on clean healthy skin usually
die from the effects of secretions from various glands and bacteria that constantly
live on the skin.
Pollution of the skin promotes the development of pathogenic microorganisms, so it
is very important to keep the skin clean at all times.
The microflora of the oral cavity is abundant and varied. Constant temperature,
humidity, availability of nutrients, alkaline reaction of saliva create favorable
conditions for the development of microorganisms. Various types of cocci, lactic acid
bacteria, diphtheroids, spirochetes predominate; spindle-shaped sticks,
actinomycetes and yeast-like fungi are found.
Oral microorganisms play an important role in the development of dental caries,
stomatitis, inflammation of soft tissues. In the first stage of the inflammatory
process, streptococci, bacteroids, and actinomycetes predominate. As caries
develops, putrefactive bacteria join them: Proteus, Clostridia, etc. Oral hygiene is of
great importance in preventing these diseases.
Microflora of the gastrointestinal tract . Usually, the microflora of the stomach is
extremely poor due to the destructive effect of acidic gastric juice. In the small
intestine, despite the alkaline reaction, there are also few microorganisms due to the
unfavorable action of enzymes. In the large intestine, conditions for the reproduction
of microorganisms are more favorable. Throughout a person's life, the microflora of
the large intestine changes: lactic acid bacteria predominate in infants, in adults
bacteroids, bifidobacteria, E. coli, fecal streptococcus, etc. are usually found. About a
third of fecal masses are various microorganisms.
The microflora of the respiratory tract . A person breathes in a huge number of
microorganisms along with the air. However, most of them linger in the nasal cavity
or are brought out with the help of the ciliated epithelium of the upper respiratory
tract. In the nasopharynx and pharynx, staphylococci, streptococci, diphtheroids, etc.
are usually found. When the body is weakened (cooling, exhaustion, injuries),
microorganisms - permanent inhabitants of the upper respiratory tract - can cause
various diseases, affecting the lower respiratory tract (bronchitis, pneumonia ).
The microflora of the mucous membrane of the eyes is very poor due to the action
of lysozyme contained in tears. Nevertheless, staphylococci and diphtheroids are
found on the conjunctiva.
The microflora of the vagina changes throughout a woman's life. In girls, coccal flora
predominates, in adult women - Dederlein's stick.
Normal human microflora is a necessary condition for maintaining his
health. Violation of microbial biocenoses in various organs and systems of the body
leads to the development of pathological processes, a decrease in the body's
defenses, and the development of dysbacteriosis.

Control questions

1. What characterizes the microflora of soil, water, air?

2. What is the role of the normal microflora of the human body?

Chapter 7. Nutrient media and microbiological research

- G. I. Katz
Microbiological research is the isolation of pure cultures of microorganisms,
cultivation and study of their properties. Pure cultures are cultures that consist of
microorganisms of the same species. They are needed in the diagnosis of infectious
diseases, to determine the species and type of microbes, in research work, to obtain
microbial waste products (toxins, antibiotics, vaccines, etc.).
For the cultivation of microorganisms (cultivation under artificial conditions in vitro)
requires special substrates - nutrient media. On the media, microorganisms carry out
all life processes (eat, breathe, multiply, etc.), so they are also called culture media.

Nutrient media

Culture media are the basis of microbiological work, and their quality often
determines the results of the entire study. Environments should create optimal
(best) conditions for the life of microbes.
Environment Requirements
Environments must meet the following requirements:
1) be nutritious, i.e. contain in an easily digestible form all the substances necessary
to meet nutritional and energy needs. They are sources of organogens and mineral
(inorganic) substances, including trace elements. Mineral substances not only enter
the cell structure and activate enzymes, but also determine the physicochemical
properties of media (osmotic pressure, pH, etc.). When cultivating a number of
microorganisms, growth factors are introduced into the media - vitamins, some
amino acids that the cell cannot synthesize;
Attention! Microorganisms, like all living things, need a lot of water.
2) have an optimal concentration of hydrogen ions - pH, since only with an optimal
reaction of the environment that affects the permeability of the shell,
microorganisms can absorb nutrients.
For most pathogenic bacteria, a weakly alkaline environment (pH 7.2-7.4) is
optimal. The exception is Vibrio cholerae - its optimum is in the alkaline zone (pH 8.5-
9.0) and the causative agent of tuberculosis, which needs a slightly acidic reaction
(pH 6.2-6.8).
So that during the growth of microorganisms, acidic or alkaline products of their vital
activity do not change pH, the media must have buffering properties, i.e., contain
substances that neutralize products; exchange;
3) be isotonic for microbial cells; i.e., the osmotic pressure in the medium should be
the same as inside the cell. For most microorganisms, the optimal environment is
0.5% sodium chloride solution;
4) be sterile, since foreign microbes prevent the growth of the microbe under study,
the determination of its properties, and change the properties of the medium
(composition, pH, etc.);
5) dense media must be moist and have an optimal consistency for microorganisms;
6) have a certain redox potential, i.e., the ratio of substances that donate and accept
electrons, expressed by the RH 2 index . This potential indicates the saturation of the
medium with oxygen. Some microorganisms need a high potential, others need a low
one. For example, anaerobes reproduce at RH 2 not higher than 5, and aerobes - at
RH 2 not lower than 10. The redox potential of most environments satisfies the
requirements for it of aerobes and facultative anaerobes;
7) be as unified as possible, i.e. contain constant amounts of individual
ingredients. Thus, the media for the cultivation of most pathogenic bacteria should
contain 0.8-1.2 g/l of amino nitrogen NH 2 , i.e., the total nitrogen of amino groups of
amino acids and lower polypeptides; 2.5-3.0 g/l of total nitrogen N; 0.5% chlorides in
terms of sodium chloride; 1% peptone.
It is desirable that the media be transparent - it is more convenient to monitor the
growth of cultures, it is easier to notice the contamination of the environment by
foreign microorganisms.
 Media classification
The need for nutrients and the properties of the environment for different types of
microorganisms is not the same. This eliminates the possibility of creating a universal
environment. In addition, the choice of a particular environment is influenced by the
objectives of the study.
At present, a huge number of media* have been proposed, the classification of
which is based on the following features.
1. Initial components. According to the initial components, natural and synthetic
media are distinguished. Natural media are prepared from products of animal and
vegetable origin. to the present; Over time, media have been developed in which
valuable food products (meat, etc.) are replaced by non-food products: bone and fish
meal, fodder yeast, blood clots, etc. Despite the fact that the composition of nutrient
media from natural products is very complex and varies depending on the feedstock ,
these media have found wide application. Synthetic media are prepared from certain
chemically pure organic and inorganic compounds, taken in precisely specified
concentrations and dissolved in doubly distilled water. An important advantage of
these media is that their composition is constant (it is known how much and what
substances they contain), so these media are easily reproducible.
2. Consistency (degree of density). Media are liquid, solid and semi-liquid. Dense and
semi-liquid media are prepared from liquid media, to which agar-agar or gelatin is
usually added to obtain a medium of the desired consistency.
Agar-agar is a polysaccharide obtained from certain types of seaweed. It is not a
nutrient for microorganisms and serves only to compact the medium. Agar melts in
water at 80-100°C and solidifies at 40-45°C.
Gelatin is an animal protein. Gelatin media melt at 25-30°C, so cultures are usually
grown on them at room temperature. The density of these media at pH below 6.0
and above 7.0 decreases, and they harden poorly. Some microorganisms use gelatin
as a nutrient - as they grow, the medium liquefies.
In addition, clotted blood serum, clotted eggs, potatoes, and silica gel media are used
as solid media.
3. Composition . Environments are divided into simple and complex. The former
include meat-peptone broth (MPB), meat-peptone agar (MPA), Hottinger broth and
agar, nutritious gelatin and peptone water. Complex media are prepared by adding
to simple media blood, serum, carbohydrates and other substances necessary for the
reproduction of one or another microorganism.
4. Purpose : a) the main (generally used) media are used for the cultivation of most
pathogenic microbes. These are the aforementioned MPA, MPB, Hottinger broth and
agar, peptone water;
b) special media are used to isolate and grow microorganisms that do not grow on
simple media. For example, for the cultivation of streptococcus, sugar is added to the
media, for pneumo- and meningococci - blood serum, for the causative agent of
whooping cough - blood;
c) elective (selective) media serve to isolate a certain type of microbes, the growth of
which they favor, delaying or suppressing the growth of associated
microorganisms. So, bile salts, inhibiting the growth of Escherichia coli, make the
environment elective for the causative agent of typhoid fever. The media become
elective when certain antibiotics, salts are added to them, and the pH changes.
Liquid elective media are called accumulation media. An example of such a medium
is peptone water with a pH of 8.0. At this pH, Vibrio cholerae actively reproduces on
it, and other microorganisms do not grow;
d) differential diagnostic media make it possible to distinguish (differentiate) one
type of microbe from another by enzymatic activity, for example, Hiss media with
carbohydrates and an indicator. With the growth of microorganisms that break down
carbohydrates, the color of the medium changes;
e) preservative media are intended for primary inoculation and transportation of the
test material; they prevent the death of pathogenic microorganisms and suppress
the development of saprophytes. An example of such a medium is the glycerin
mixture used to collect feces in studies conducted to detect a number of intestinal
bacteria.
Recipes for some media are provided at the end of the next section and in the
second part of the tutorial.
Control questions
1. What requirements must the nutrient media meet?
2. How are media classified by source components?
3. What substances are used to seal media?
4. What media are simple or commonly used and what are they used for?
5. What environments are called complex, what serves as their basis?
6. What media allow one to obtain the preferential growth of some microbes while
suppressing others?
7. What media are used to study the enzymatic activity of microbes?
Exercise
Fill out the form, indicating which groups the environments are divided into.
Media preparation
Vessels for preparation of media should not contain foreign substances, such as
alkalis released by certain types of glass, or iron oxides, which can enter the medium
when it is boiled in rusty pans. It is best to use glass, enamel or aluminum
dishes. Large quantities of the medium (tens and hundreds of liters) are prepared in
special digesters or reactors (Fig. 14). Before use, the dishes must be thoroughly
washed, rinsed and dried. New glassware is preliminarily boiled for 30 minutes in a 1-
2% solution of hydrochloric acid or immersed in this solution overnight, after which it
is rinsed in running water for an hour.
 Rice. 14. General view of the reactor

Attention! Vessels intended for the preparation of media must not be used for other
purposes, such as storing chemicals or disinfectant solutions - even traces of these
substances can interfere with the growth of microorganisms.
The raw materials for the preparation of most media are products of animal or
vegetable origin: meat and its substitutes, milk, eggs, potatoes, soybeans, corn,
yeast, etc.
The main nutrient broths are prepared with meat water or with various digests
obtained by acid or enzymatic hydrolysis of the feedstock. Broths from digests are 5-
10 times more economical than from meat water. Digested media are richer in
amino acids, therefore, more nutritious; have a greater buffering capacity, i.e., have
a more stable pH value. In addition, digests can be prepared from meat substitutes
(blood clots, placenta, casein, etc.).
Currently, the supply of laboratories with meat water and digests is
centralized. Pancreatic Hottinger's digest, casein hydrolysates or fodder yeast are
more commonly used. The necessary media are prepared from these semi-finished
products according to certain recipes.
Stages of media preparation: 1) boiling; 2) establishing the optimal pH value; 3)
clarification; 4) filtration; 5) spill; 6) sterilization; 7) control.
The media are cooked on an open fire, in a water bath, in an autoclave or in steam-
heated digesters.
The establishment of the pH of the media is approximately carried out using
indicator papers. To accurately determine the pH, a potentiometer is used, using
glass electrodes in accordance with the instructions or a comparator (Michaelis
apparatus), consisting of a rack with test tube sockets (Fig. 15) and a set of standards
for a certain pH. In the preparation of media, the indicator metanitrophenol is
usually used, which changes its color in the range of 6.8-8.4.

Rice. 15. Comparator (apparatus macro-Michaelis). 1 - front view; 2 - rear view; 3 -

layout of test tubes in a rack

To determine the pH of the medium, 4 test tubes, the diameter and color of the glass
of which does not differ from the test tubes with standards, are placed in slots 1, 2,
3, and 5 (see Fig. 15). 5 ml of distilled water are poured into the 1st and 3rd tubes; in
the 5th - 7 ml; in the 2nd - 4 ml of water and 1 ml of the indicator. In nests 4 and 6
put the standards of the desired pH. Pour 2 ml of chilled medium into the 1st, 2nd
and 3rd tubes. The contents of the tubes are mixed.
The color of the liquids in the test tubes is compared in transmitted light by closing
the rear slot of the device with a filter (opaque or blue if the liquids are intensely
yellow). The pH of the test solution corresponds to the pH of the standard, the color
of which matches its color.
When preparing media with a given pH, standards are placed in nests 4 and 6, the pH
of which is close to the required one, and a certain amount of alkali solution is added
from the buret to the 2nd test tube with the test medium and indicator, if the liquid
in the 2nd test tube is lighter than the standards, or acid solution - if the standards
are lighter. Alkali (or acid) is added until the color of the liquid in the 2nd tube does
not match the color of the standards. The amount of alkali (or acid) added to 2 ml of
the medium in the 2nd test tube is calculated for the entire volume of the prepared
medium. For example, if to obtain the desired pH, 2 drops (0.1 ml) of 0.05 N were
used per 2 ml of the medium. alkali solution, then for alkalization of 1 liter you need
500 times more, i.e. 50 ml of 0.05 n. or 2.5 ml 1 N. alkali solution.
During sterilization, the pH of the media decreases by 0.2, therefore, to obtain a
medium with a pH of 7.2-7.4, it is first prepared with a pH of 7.4-7.6.
Clarification of media is carried out if they become cloudy or dark during
cooking. For clarification, egg white, beaten with double the amount of water, is
poured into a medium heated to 50 ° C, mixed and boiled. Coagulating, the protein
entrains particles suspended in the medium into the sediment. In the same way, you
can use blood serum instead of egg white (20-30 ml per 1 liter of medium).
Filtration of liquid and molten gelatinous media is carried out through wet paper or
cloth filters. Filtration of agar media is difficult - they solidify quickly. Usually they are
filtered through a cotton-gauze filter (a gauze napkin is placed in the funnel and a
lush lump of cotton wool is placed on it). Paper or cloth filters can be used if filtration
is carried out in a hot autoclave or in heated funnels.
Filtration of agar media can be replaced by settling. The medium is poured into a tall
cylindrical vessel and melted in an autoclave. When the medium slowly cools in the
switched off device, the particles suspended in it settle to the bottom. The next day,
the agar clot is removed from the vessel (for this, the vessel is briefly placed in hot
water) and the lower part with the accumulated sediment is cut off with a knife. The
upper part is melted and poured into appropriate containers.
The media are poured into test tubes (3-5 ml or 10 ml each), vials, flasks, mattresses
and bottles no more than 2/3 of the capacity , since during sterilization the stoppers may
get wet and the media will lose their sterility .
Media that are sterilized at temperatures above 100 ° C are poured into clean, dry
dishes. Media sterilized at a lower temperature must be poured into sterile
containers.
The media are poured using a funnel, at the end of which a rubber tube with a Mohr
clamp is put on. For measured spills, beakers, burettes, dispensers, syringes,
pipettes, etc. are used (Fig. 16).
 Rice. 16. Devices for volumetric pouring of media. a - laboratory installation; b -
automatic syringe-pipette; c - dispenser; g - semi-automatic; d - automatic dispenser

Dishes with the medium are usually closed with cotton-gauze stoppers, over which
paper caps are put on. It is important that when pouring, the medium does not wet
the edges of the dishes, otherwise corks may stick to them. A label must be attached
to each vessel with the name of the medium and the date of its preparation.
Sterilization . The sterilization mode depends on the composition of the medium and
is indicated in its recipe. An exemplary scheme of the mode of sterilization of media
is given in Table. 8.
 Table 8. Media sterilization mode
1
 ( Liquid media containing carbohydrates, proteins, or vitamins are best sterilized with bacteria filters.  )
Control of ready-made media: a) to control the sterility of the media, put in a
thermostat for 2 days, after which they are viewed. If no signs of growth appear on
the media, they are considered sterile and several samples of each series are
submitted for chemical control; b) chemical control: finally set the pH, the content of
total and amine nitrogen, peptone, chlorides (their amount must correspond to that
specified in the recipe).
Chemical control of media is carried out in a chemical laboratory; c) for biological
control, several samples of the medium are inoculated with specially selected
cultures of microorganisms, and their growth is used to judge the nutritional
(growth) properties of the medium. A label and a passport are attached to the
finished medium, in which the name and composition of the medium, control results,
etc. are indicated.
Media are stored at room temperature in cabinets, preferably specially designed for
them. Some media, such as blood and vitamin media, are kept refrigerated.
 Recipes for the preparation of simple (basic) media and isotonic
sodium chloride solution
Isotonic sodium chloride solution . To 1 liter of distilled water add 9 g of sodium
chloride. The solution is filtered, adjusted to the desired pH and, if necessary,
sterilized at 120°C for 30 minutes.
Meat peptone broth (MPB) . 1% peptone and 0.5% x are added to meat
water. including sodium chloride, boil over low heat for 10-15 minutes to dissolve
the substances, set the desired pH and boil again for 30-40 minutes until a
precipitate forms. Filtered, topped up to the original volume with water and
sterilized for 20 minutes at 120°C.
Hotinter broth . The Hottinger digest is diluted with water 5-6 times, depending on
how much amine nitrogen it contains and how much it should be in the broth
(indicated in the digest passport and the medium recipe). For example, to prepare a
medium with 1.2 g/l of amine nitrogen, a digest containing 9.0. g / l, it must be
diluted 7 5 times (9.0: 1.2). 0.5% sodium chloride is added to the diluted digest and
boiled over low heat until the salt dissolves. The pH is adjusted in the cooled
medium, filtered, poured and sterilized for 20 minutes at
Meat peptone agar (MPA) . To the finished broth (before or after sterilization) add 2-
3% of crushed agar-agar and boil, stirring, over low heat until the agar is completely
melted. MPA can be cooked in an autoclave or Koch apparatus. The prepared
medium, if necessary, is clarified, filtered and sterilized for 20 minutes at 120°C.
Semi-liquid agar contains 0.4-0.5% agar-agar .
Nutritious gelatin . 10-15% gelatin is added to the finished broth, heated BEFORE it
melts (do not boil!), poured into sterile dishes and sterilized with flowing steam.
Recipes for complex environments
Media with carbohydrates . The right amount (0.1-2%) of a certain carbohydrate (for
example, glucose) is added to the main broth or molten agar. After its dissolution, it
is poured into sterile dishes and sterilized with flowing steam. Since carbohydrates
are partially destroyed even with this sterilization mode, it is preferable to add a 25-
30% solution of carbohydrates sterilized through a bacterial filter in the required
volume with asepsis to sterile basic media - after sterility control, the medium is
ready for use.
Blood media are prepared from sterile plain media by adding aseptically (preferably
in a box) from 30% to 30% (usually 5%) of sterile defibrinated blood. Before this, agar
media are melted and cooled to 45 ° C. The temperature of the medium is
determined by bringing the vessel to the neck at the angle of the lower jaw. At the
right temperature, there should be a tolerable sensation of hot, but not
burning. After the addition of blood, until the medium has solidified, the contents of
the vessel are thoroughly mixed and poured into cups or test tubes.
Attention! Media with blood cannot be melted - the blood will change its properties.
Serum media are prepared in the same manner as blood media. To the main media
add 10-20% serum, not containing a preservative and pre-inactivated at 56 ° C for 30
minutes in a water bath or in an inactivator. During inactivation, a substance
(complement) that has a detrimental effect on microbes is destroyed.
Media with bile . Bile is added to simple media in an amount of 10-40% of the
volume of the medium, the desired pH is adjusted and sterilized for 20 minutes at
120 ° C. Sterile bile can be added to a sterile medium under aseptic conditions.
Pouring agar media into Petri dishes . Before pouring, the media are melted in a
water bath and cooled to 45-50 ° C. Usually, 15-20 ml of the medium is enough for a
cup with a diameter of 9 cm (layer height 0.25-0.3 cm). If the layer is higher, colonies
look less contrasting on it. With a very thin layer, the amount of nutrients and
moisture is sharply limited (the medium dries quickly) - the cultivation conditions
worsen.
Pour the media into sterile cups under aseptic conditions. Cups are placed lid up. The
vessel with the medium is taken in the right hand, holding it by the fire. The cork is
removed with the left hand, holding it with the little finger and palm. They burn the
neck of the vessel and slightly open the lid with two fingers of the left hand. The neck
of the vial is inserted under it, without touching it to the edge of the cup. When
pouring the medium, make sure that it is evenly distributed over the bottom of the
cup. If during spillage air bubbles form on the surface of the medium, they bring the
flame of a match or burner to them before the medium solidifies - the bubbles will
burst. The cup is then closed and the medium is allowed to solidify. If sowing is done
on the day of the spill, the medium must be dried. To do this, the cups in the
thermostat are carefully opened and the lids and cups are placed with the open side
down for 20-30 minutes. If sowing is done the day after the spill,
Preparation of slant agar . Tubes with 4-5 ml of sterile molten agar medium are
placed in an inclined position ( approximately at an angle of 20 °) so that the medium
does not go beyond 2/3 of the tubes, otherwise it may wet the stopper . After the
medium has solidified, the test tubes are placed vertically - the condensate is
allowed to drain. It is better to use freshly cut agar.
Attention! It is impossible to use an environment in which there is no condensate. It
should be melted again in a water bath and mowed.
Dry environments
Domestic industry produces dry media for various purposes: simple, elective,
differential diagnostic, special. These are powders in vials with screw caps. Store dry
media in a dark place tightly closed - they are hygroscopic. In the laboratory, the
media are prepared from powders according to the prescription on the label.
The advantage of dry media compared to laboratory-made media is standardization
(they are produced in large batches), ease of preparation, making them available in
any (even camping) conditions, stability, and economy. It is important that they can
be prepared from meat substitutes: casein hydrolyzate, fibrin, sprat, and even
protein fractions of microbial cells (sarcin).
Control questions
1. What should be the pH of the media for the cultivation of most pathogenic
microbes before sterilization and why?
2. At what temperature do agar media melt and solidify?
3. How should the dishes be prepared into which media with carbohydrates and
proteins are poured?
Exercise
1. Prepare MPB, MPA, broth and Hottinger agar with pH 7.2-7.4, pour into vials and
test tubes; sterilize.
2. Prepare Hiss medium from dry powders, pour into 4-5 ml tubes and sterilize.
3. Prepare blood agar and pour it into Petri dishes.
4. Prepare Endo, EMS, Ploskirev media from dry powders and pour them into Petri
dishes.
5. Prepare the agar slant.
 Seeding methods

An important stage of bacteriological research is sowing. Depending on the purpose

of the study, the nature of the inoculum and the medium, different methods of
sowing are used. All of them include the obligatory Purpose: to protect the crop from
foreign microbes. Therefore, you should work quickly, but without sudden
movements that increase air vibrations. You can't talk while planting. Crops are best
done in boxing.
Attention! Remember to follow personal safety rules when working with infectious
material.
Seeding from test tube to test tube . The tube with the inoculum and the tube with
the medium are held slightly obliquely in the left hand between the thumb and
forefinger so that the edges of the tubes are at the same level, and their bases are on
top of the brush. Usually, the inoculum tube is held close to you. In the right hand,
like a writing pen, they hold a bacterial loop, and sterilize it, holding it vertically in
the flame of the burner. With the little finger and the edge of the palm of the right
hand, both plugs are taken out at the same time. The plugs are removed not with a
jerk, but smoothly - with light screw movements. After removing the stoppers, the
edges of the test tubes are burned in the flame of a burner. The calcined loop is
introduced through the flame of a burner into a test tube with inoculum, cooled and,
having collected some material, is carefully transferred into a test tube with the
medium.
When sowing in a liquid medium, the inoculum is triturated on the wall of the test
tube over the liquid and washed off with the medium.
When sowing on liquid media with a swab, it is immersed in the medium and rinsed
in it for 3-5 s. When sowing on a solid medium, the material is rubbed into its surface
by rotating the swab, after which the swab is decontaminated (placed in the test
tube in which it was delivered to the laboratory and autoclaved).
Attention! Make sure that the medium does not spill out and wet the stopper.
When inoculating on agar slant, the material is usually triturated on the surface of
the medium in zigzag movements from the bottom up, starting from the condensate
boundary.
When sowing on solid media, poured into test tubes in a column, a column is pierced
with a loop with inoculum, producing the so-called "prick" seeding.
After sowing, the loop is removed from the test tube, the edges of the test tubes are
burned, and after passing the plugs through the flame of the burner, the test tubes
are closed, after which the loop is calcined.
Inoculation of liquid material can be done with sterile pipettes (Pasteur or
graduated). After sowing, the pipettes are immersed in a disinfectant liquid.
Inoculations in vials, mattresses and bottles are made in approximately the same way
as in test tubes, only the material is first collected (with a loop or in a pipette), and
then the vessel with the medium is opened.
Vessels with seeded culture are inscribed and placed in a thermostat.
Sowing on test tubes from Petri dishes . Having studied the nature of the growth of
the culture on the cup, from the side of the bottom, the area necessary for sowing is
marked with a wax pencil. Place the cup with the inoculum in front of you with the
lid up. The lid is slightly opened with the left hand and the burnt loop is inserted
under it. After cooling the loop, they collect the sowing material from the marked
area. The loop is taken out, the cup is closed and a test tube with the medium is
taken in the left hand. Sowing is carried out in the same way as from test tube to test
tube. After sowing, the cup is turned upside down.
Seeding on agar in Petri dishes . Sowing with a spatula. A spatula is a glass or metal
tube, the end of which is bent in the form of a triangle. A spatula can be made from a
Pasteur pipette by bending its thin end at an angle, previously heated in a burner
flame.
With the left hand, slightly open the lid, holding it with the thumb and
forefinger. The inoculum is applied to the surface of the medium with a loop, pipette
or glass rod, after which it is carefully rubbed with a circular motion of the spatula
until the spatula stops sliding freely over the surface of the medium, while holding
the lid with the left hand and simultaneously rotating the cup. At the end of sowing,
the spatula is removed from the cup and the lid is closed. A glass spatula is placed in
a disinfectant solution, and a metal spatula is calcined in a burner flame.
Loop sowing. A small amount of inoculum (sometimes it is pre-emulsified in a sterile
isotonic solution or broth) is rubbed with a loop into the surface of the medium at
the edge of the dish, passing the loop from side to side several times. Then, at the
place where the strokes ended, the agar is pierced with a loop, removing the excess
seed. The inoculum remaining on the loop is spread over the entire surface of the
medium in zigzag movements. At the end of sowing, close the cup and burn the loop.
Loop sowing into sectors. The cup from the bottom side is drawn into
sectors. Sowing is done in zigzag movements from the edge of the cup to the
center. It is necessary to ensure that the strokes do not enter the neighboring sector.
Sowing with a swab. A tampon with inoculum is introduced into a slightly ajar cup
and its contents are rubbed in a circular motion into the surface of the medium,
while rotating the tampon and cup.
Lawn sowing. Approximately 1 ml (20 drops) of the liquid culture (if the culture is
from a solid medium, emulsify it in sterile isotonic saline or broth) is applied to the
surface of the agar and carefully spread the liquid over the surface of the
medium. The cup is slightly tilted and the excess culture is sucked off with a pipette,
pouring it into the disinfectant solution. A pipette is placed there.
Sowing in the thickness of agar. A culture grown on a liquid medium or an emulsified
material is introduced into a vessel with agar melted and cooled to 45 ° C, mixed and
poured into a sterile Petri dish. You can add seed to an empty cup and pour 15-20 ml
of agar cooled to 45 ° C. To mix the contents of the cup, it is slightly shaken and
rotated. The cups are left on the table until the medium solidifies.
Seeded cups are signed from the bottom side and placed in a thermostat upside
down.
Control questions
1. Are aseptic conditions necessary during seeding? Justify your answer.
2. How should the workplace be processed after the end of crops?

Culture methods

For successful cultivation, in addition to properly selected media and properly

seeded, optimal conditions are necessary: temperature, humidity, aeration (air
supply). As a rule, suitable conditions can be created by carefully reproducing the
conditions of the natural environment.
temperature . The optimum temperature for the cultivation of most pathogenic
microorganisms (37 ° C) is created in a thermostat (Fig. 17). This is a device with
double walls, between which there is air or water heated by electricity. It is equipped
with a thermostat that automatically maintains the desired temperature, and a
thermometer to control the temperature.

Rice. 17. Thermostat

Test tubes with cultures in racks, wire meshes or jars are placed on thermostat
shelves. The cups in the thermostat must be upside down. In order for the air in the
thermostat to circulate freely and the heating to be uniform, the shelves in the
thermostat are made with slots and are not loaded tightly. In order not to cool the
cultures, the thermostat is not left open for a long time.
The laboratory assistant is obliged to register the temperature in the thermostat
daily and maintain cleanliness in the device, and in case of a malfunction, call the
master.
The overwhelming majority of microbes (all pathogenic ones belong to them) do not
need light - they are cultivated in the dark. However, to study pigment formation,
which occurs more actively in diffused light, the cultures after the thermostat are
kept for 2-3 days at room light.
Attention! Avoid exposure to direct sunlight, which is detrimental to crops.
Humidity . The life of microbes is impossible without moisture - nutrients penetrate
into the cell only in dissolved form. This must be taken into account when cultivating
on dense media: it is better to pour them into cups and mow in test tubes on the day
of sowing. When cultivating microbes that are especially sensitive to the lack of
moisture, such as gonococci, an open vessel with water is placed in a thermostat.
Terms of cultivation . Most pathogenic microbes are cultivated for 18-24 hours, but
there are species that grow slowly (up to 4-6 weeks). To keep moisture in them,
cotton plugs after sowing are replaced with sterile rubber ones or rubber caps are
put on them.
Attention! Rubber stoppers are sterilized in an autoclave wrapped in paper.
Aeration . According to the need of microbes for free oxygen, they are divided into
aerobes and anaerobes. Both groups require different culture conditions.
The supply of oxygen necessary for the cultivation of aerobes and facultative
anaerobes is carried out with passive and active aeration.
Passive aeration is cultivation on dense and liquid media in vessels closed with cotton
or cotton-gauze stoppers, or in Petri dishes. With such cultivation, microbes consume
oxygen dissolved in the medium, which is in the vessel above the medium and enters
through the stopper. Passively aerated crops can be grown on the surface or in a thin
layer of the environment, where atmospheric oxygen penetrates.
Active aeration is used in the deep cultivation of microbes, when they are grown in
large volumes of the medium. In order to sufficiently supply such cultures with
oxygen, they are placed in special rocking chairs - constant mixing of the culture
ensures its contact with air. During cultivation in liquid volumes reaching tens and
hundreds of liters, carried out in devices called reactors or fermenters, air is blown
through the culture using special devices.
The cultivation of anaerobes is more difficult than aerobes, since they must be
deprived of access to free oxygen in the air. To do this, air is removed from the
nutrient medium in various ways.
Cultivation of actinomycetes, fungi, mycoplasmas, L-forms, spirochetes and
protozoa . The cultivation of these microorganisms is fundamentally similar to the
cultivation of bacteria. Special environments have been developed for them and
modes have been selected that meet their needs.
Cultivation of rickettsiae and viruses . Rickettsia and viruses are obligate parasites,
that is, they can only develop in living cells. They are cultivated in tissue cultures,
experimental animals, developing chicken embryos.

Methods for isolating pure cultures of

microorganisms

A pure culture is the accumulation of microbes of the same species on a dense or

liquid nutrient medium.
There are a number of methods for isolating a pure culture, depending on the
properties of the material under study and the purpose of the study. Usually pure
cultures are obtained from isolated colonies - isolated accumulations of microbes on
a solid medium. It is believed that most often a colony develops from a single
microbial cell, i.e., it is a pure culture of this microorganism.
Steps for isolating a pure culture:
1st day - obtaining isolated colonies. A drop of the test material is applied with a
loop, pipette or glass rod to the agar surface in a Petri dish. With a spatula, rub the
material into the surface of the medium; without burning or turning over the
spatula, sowing is done on the 2nd and then on the 3rd cup. With such a sowing, the
1st cup has a lot of material and, accordingly, a lot of microbes, the 2nd one has less
and the 3rd even less.
It is possible to obtain isolated colonies by loop inoculation. To do this, the test
material is emulsified in broth or isotonic sodium chloride solution.
2nd day - study the growth of microbes on the cups. In the 1st dish, there is usually a
continuous growth - it is not possible to isolate an isolated colony. Isolated colonies
grow on the surface of the agar in the 2nd and 3rd plates. They are studied with the
naked eye, with a magnifying glass, at low magnification of a microscope, and
sometimes in a stereoscopic microscope (see Chapter 31). The desired colony is
marked from the side of the bottom of the plate and inoculated onto the agar
slant. Crops are placed in a thermostat.
Attention! Only isolated colonies can be reseeded.
3rd day - study the nature of growth on slant agar. They make a smear, stain it and,
making sure that the culture is clean, they begin to study it. This completes the
isolation of pure culture. A culture isolated from a specific source and studied is
called a strain.
When isolating a pure culture from blood (hemoculture), it is preliminarily "grown"
in a liquid medium: 10-15 ml of sterile blood is inoculated into 100-150 ml of a liquid
medium. This is done because there are usually few microbes in the blood. The ratio
of sown blood and nutrient medium 1:10 is not accidental - this is how blood dilution
is achieved (undiluted blood has a detrimental effect on microorganisms). The
culture flasks are placed in a thermostat. A day later (sometimes after a longer time,
depending on the isolated culture), seedings are made from the contents of the
flasks on dishes to obtain isolated colonies. If necessary, repeat sowing at intervals of
2-3 days.
When isolating a pure culture from urine, gastric lavage and other liquids, they are
preliminarily centrifuged under aseptic conditions and the sediment is
inoculated. Further isolation of pure culture is carried out in the usual way.
Elective media are widely used to isolate pure cultures.
In a number of methods for obtaining pure cultures, the biological characteristics of
the isolated microbe are used. For example, when spore-forming bacteria are
isolated, the crops are heated at 80 ° C for 10 minutes, thereby killing the vegetative
forms; when isolating the causative agent of tuberculosis, resistant to acids and
alkalis, with the help of these substances, the inoculum is freed from the
accompanying flora; to isolate pneumococcus and bacillus plague, the test material is
injected into white mice - in their body, which is highly sensitive to these pathogens,
these microbes multiply faster than others.
In research work, especially in genetic studies, it is necessary to obtain cultures from
a single cell. Such a culture is called a clone. To obtain it, they most often use a
micromanipulator - a device equipped with tools (needles, pipettes) of microscopic
dimensions. With the help of a holder under the control of a microscope, they are
introduced into the "hanging drop" preparation, the desired cell (one) is removed
and transferred to a nutrient medium.

Study of isolated cultures

The study of morphology, mobility, tinctorial properties (see Chapter 3), the nature
of growth on media (cultural properties), enzymatic activity, and a number of other
features of the isolated microbe makes it possible to establish its taxonomic position,
i.e., classify the microorganism: determine its genus, species, type, subtype,
variety. This is called identification. Identification of microorganisms is very
important in diagnosing infections, establishing the sources and routes of its
transmission, and in a number of other scientific and practical studies.
cultural properties
Different types of microorganisms grow differently on media. These differences serve
to differentiate them. Some grow well on simple media, others are demanding and
grow only on special ones. Microorganisms can produce abundant (lush) growth,
moderate or sparse. Cultures can be colorless, grayish, gray-blue. The cultures of
microorganisms that form the pigment have a variety of colors: white, yellow or
golden in staphylococcus, red in the miraculous stick, blue-green in the blue-green
stick, the pigment of which, soluble in water, colors not only colonies, but also the
environment.
On dense media, microorganisms, depending on the amount of inoculum, form
either a continuous coating ("lawn") or isolated colonies. Cultures are coarse and
delicate, transparent and opaque, with a matte, shiny, smooth, rough, dry, bumpy
surface.
Colonies can be large (4-5 mm in diameter and more), medium (2-4 mm), small (1-2
mm) and dwarf (less than 1 mm). They differ in shape, location on the surface of the
medium (convex, flat, dome-shaped, depressed, round, rosette-shaped), the shape
of the edges (smooth, wavy, indented).
In liquid media, microorganisms can form a uniform turbidity, give a precipitate
(granular, dusty, flaky) or a film (tender, rough, wrinkled).
On semi-liquid media, when inoculated with an injection, mobile microbes cause
clouding of the thickness of the medium, while immobile microbes grow only after
an “injection”, leaving the rest of the medium transparent.
Cultural properties are determined by studying the growth pattern of the culture
with the naked eye, with a magnifying glass, under a low magnification microscope,
or using a stereoscopic microscope. The size and shape of the colonies, the shape of
the edges and transparency are studied in transmitted light, looking at the cups from
the bottom. In reflected light (from the side of the lid) determine the nature of the
surface, color. The consistency is determined by touching the loop.
 Morphological properties

The study of the morphology of microbes also serves to differentiate

them. Morphology is studied in stained preparations. Determine the shape and size
of the cells, their location in the preparation, the presence of spores, capsules,
flagella. In stained preparations, the ratio of microbes to paints (tinctorial properties)
is determined - paints are perceived well or poorly, as it relates to differential colors
(what color is stained according to Gram, Ziehl - Nielsen, etc.). Vital (vital) staining
allows you to establish mobility, differentiate between living and dead cells, and
monitor their division. Division and motility can be studied in native (unstained)
preparations (see Chapter 3).

Enzymatic activity

The enzymatic activity of microorganisms is rich and varied. Using it, one can
establish not only the species and type of microbe, but also determine its variants
(the so-called biovars). Consider the main enzymatic properties and their qualitative
definition.
The breakdown of carbohydrates (saccharolytic activity), i.e., the ability to break
down sugars and polyhydric alcohols with the formation of acid or acid and gas, is
studied on Hiss media, which contain one or another carbohydrate and
indicator. Under the action of the acid formed during the breakdown of the
carbohydrate, the indicator changes the color of the medium. Therefore, these
media are called "variegated series". Microbes that do not ferment this carbohydrate
grow on the medium without changing it. The presence of gas is established by the
formation of bubbles in media with agar or by its accumulation in a "float" on liquid
media. "Float" - a narrow glass tube with a sealed end facing upwards, which is
placed in a test tube with the medium before sterilization (Fig. 18).
Rice. 18. Study of the saccharolytic activity of microorganisms. I - 'variegated row': a
- liquid medium with carbohydrates and Andrede's indicator; b - semi-liquid medium
with BP indicator: 1 - microorganisms do not ferment carbohydrate; 2 -
microorganisms ferment carbohydrate with the formation of acid; 3 -
microorganisms ferment carbohydrate with the formation of acid and gas; II -
colonies of microorganisms that do not decompose (colorless) and decompose
lactose (violet on the EMS medium - on the left, red on the Endo medium - on the
right)

In addition, saccharolytic activity is studied on Endo, EMS, Ploskirev

media. Microorganisms, fermenting milk sugar (lactose) in these media to acid, form
colored colonies - the acid changes the color of the indicator present in the
medium. Colonies of microbes that do not ferment lactose are colorless (see Fig. 18).
Milk with the growth of lactose fermenting microbes coagulates.
With the growth of microorganisms that form amylase, on media with soluble starch,
it is cleaved. This is known by adding a few drops of Lugol's solution to the culture -
the color of the medium does not change. Undigested starch gives a blue color with
this solution.
Proteolytic Properties(i.e., the ability to break down proteins, polypeptides, etc.) is
studied on media with gelatin, milk, whey, peptone. With the growth of gelatin-
fermenting microbes on the gelatin medium, the medium liquefies. The nature of the
liquefaction caused by different microbes is different (Fig. 19). Microbes that break
down casein (milk protein) cause peptonization of milk - it takes on the appearance
of whey. During the splitting of peptones, indole, hydrogen sulfide, ammonia can be
released. Their formation is established using indicator pieces of paper. The filter
paper is pre-impregnated with certain solutions, dried, cut into narrow strips 5-6 cm
long, and after sowing the culture on the BCH, placed under the cork between it and
the test tube wall. After incubation in a thermostat, the result is taken into
account. Ammonia causes litmus paper to turn blue; when hydrogen sulfide is
released on a paper soaked in a 20% solution of lead acetate and sodium
bicarbonate, lead sulfate is formed - the paper turns black; indole causes reddening
of a piece of paper soaked in a solution of oxalic acid (see Fig. 19).
 Rice. 19. Proteolytic properties of microorganisms. 1 - forms of liquefaction of
gelatin; II - determination of hydrogen sulfide; III - determination of indole: 1 -
negative result; 2 - positive result

In addition to these media, the ability of microorganisms to break down various

nutrient substrates is determined using paper discs impregnated with certain
reagents (paper indicator systems "SIB"). These disks are dipped into test tubes with
the culture under study, and after 3 hours of incubation in a thermostat at 37 ° C, the
decomposition of carbohydrates, amino acids, proteins, etc. is judged by the change
in the color of the disks.
Hemolytic properties (the ability to destroy red blood cells) are studied on media
with blood. In this case, liquid media become transparent, and a transparent zone
appears around the colony on dense media (Fig. 20). When methemoglobin is
formed, the medium turns green.
 Rice. 20. Hemolysis around colonies growing on blood agar

Preservation of crops

Isolated and studied cultures (strains) of value for science or production are stored in
museums of living cultures. The All-Union Museum is located in the State Research
Institute for Standardization and Control of Medical Biological Preparations named
after V.I. L. A. Tarasevich (GISK).
The task of storage is to maintain the viability of microorganisms and prevent their
variability. To do this, it is necessary to weaken or stop the exchange in the microbial
cell.
One of the most advanced methods of long-term preservation of cultures -
lyophilization - drying in a vacuum from a frozen state allows you to create a state of
suspended animation. Drying is carried out in special devices. Cultures are stored in
sealed ampoules at a temperature of 4 ° C, preferably at -30-70 ° C.
Recovery of dried crops. The tip of the ampoule is strongly heated in the flame of the
burner and touched with a cotton swab slightly moistened with cold water so that
microcracks form on the glass, through which air slowly seeps into the ampoule. At
the same time, passing through the heated edges of the cracks, the air is sterilized.
*
 ( If there is an excess of water on the swab, it can get into the ampoule and violate the sterility of the culture: it will be
sucked in through the formed microcracks, since there is a vacuum in the ampoule.  )

Attention! Do not forget that there is a vacuum in the sealed ampoule. If air enters it
immediately through a large hole, the culture in the ampoule can be sprayed and
ejected.
After allowing air to enter, quickly break with tweezers and remove the top of the
ampoule. The hole is lightly burned and a solvent (broth or isotonic solution) is
introduced into the ampoule with a sterile Pasteur pipette or syringe. Mix the
contents of the ampoule and inoculate on the media. The growth of regenerated
crops in the first crops may be slowed down.
It is also possible to preserve cultures for a long time in liquid nitrogen (-196 ° C) in
special devices.
The methods of short-term preservation of cultures are as follows: 1) subcultivation
(periodic transfers to fresh media) at intervals depending on the properties of the
microorganism, the medium and cultivation conditions. Cultures are stored at 4°C
between reseedings; 2) preservation under a layer of oil. The culture is grown in agar
in a column 5-6 cm high, poured with sterile vaseline oil (oil layer about 2 cm) and
stored vertically in the refrigerator. The shelf life of different microorganisms is
different, therefore, a culture is periodically sown from test tubes to check its
viability; 3) storage at -20-70°C; 4) storage in sealed tubes. As needed, the stored
material is sown on a fresh medium.

Control questions

1. What is included in the concept of "bacteriological research"?

2. What should be the culture for such research?
3. What is a microbial colony, culture, strain, clone?
4. What is included in the concept of "cultural properties of microbes"?

Exercise
1. Study and describe several colonies. Transfer them to the agar slant and to the
sector.
2. Study and describe the growth pattern - agar slant cultures. Determine the purity
and morphology of the culture in the stained preparation.
3. Transfer culture from agar slant to broth and differential diagnostic
media. Examine and record in the protocol the growth pattern of the culture on
these media and its enzymatic properties.

Chapter 8. Phages - G. I. Katz

Phages are viruses of bacteria and a number of other microorganisms. Under certain
conditions, they cause lysis (dissolution) of their hosts. The action of phages
manifests itself in nature and is used in practice.
History of the discovery and study of the phage . In 1898, N. F. Gamaleya showed
that the filtrate of anthrax bacilli causes lysis of fresh cultures of these
microorganisms. In 1915, F. Tuort discovered that white, opaque staphylococcal
colonies became transparent and disappeared, and that the staphylococcal lysing
agent passes through bacterial filters, retaining the ability to dissolve fresh cultures
of these microorganisms. The phenomenon of lysis of microorganisms has been
described, but its nature has not been studied. That is why the honor of discovering
the bacteriophage belongs to the Canadian scientist d'Herelle.
D'Herelle (1917) studied stool filtrates taken daily from a patient with dysentery and
introduced into test tubes with a freshly inoculated culture of the causative agent of
this disease. After incubation in a thermostat, the culture grew. But one day she did
not grow, but dissolved. This coincided with the beginning of the patient's recovery.
D'Herelle showed that the lysing ability of stool filtrates increased with successive
passages on fresh bacterial cultures. From this, the scientist concluded that it
dissolves their living agent passing through bacterial filters, i.e. the virus. At present,
his point of view is accepted by the majority of scientists.
d'Herelle called the discovered virus a bacteriophage * - a devourer of bacteria (from
the Greek phagos - devouring), and the phenomenon - bacteriophage.
*
 ( The term "phage" is now more commonly used, since similar "devourers" are also found in fungi and algae.  )
With the discovery of the electron microscope, the corpuscular nature of the phage
was confirmed and its morphology was studied.
D'Herelle's discovery attracted the attention of physicians who used phage to treat
and prevent a number of infectious diseases. Currently, phages are widely used in
medical practice and in various biological studies. Phages are studied by
bacteriologists, virologists, biochemists, geneticists, biophysicists, molecular
biologists, experimental oncologists, specialists in genetic engineering and
biotechnology, etc. The study of phage continues as one of the most interesting
chapters of biology.

Phage Properties

The nature of phages . Phages, according to most researchers, are organisms that,

like all living organisms, are able to reproduce, pass on their properties to offspring,
and change under the influence of various factors. They can infect and destroy only
young developing cells, being their parasites.
Phage morphology . Most phages consist of a head and a tail, so they are compared
to tadpoles or spermatozoa. The most studied are T-phages of E. coli (Fig. 21). Their
process is a hollow cylinder (rod) covered with a sheath and ending in a basal plate
with spines and fibrils. The size of phages, the shape and size of the head, the length
and structure of the process are different in different phages. For example, there are
phages with a long process, the sheath of which does not contract, phages with a
short process, without a process, and filamentous (Fig. 22).
 Rice. 21. The structure of the T-2 phage. 1 - head; 2 - DNA; 3 - rod; 4 - cover; 5 -
basal plate; 6 - spikes; 7 - tail fibrils

Rice. 22. Phage morphology. 1 - phages with a head, a process and a contracting

sheath; 2 - head and process, without contractility; 3 - head and short process; 4 -
tailless phages; 5 - filamentous phages

Chemical composition of phages . Like all viruses, phages are composed of a single

type of nucleic acid (DNA phages are more common) and a protein. The nucleic acid
molecule, twisted into a spiral, is located in the head of the phage. The shell of the
phage (capsid) and the process are of a protein nature. The free end of the process
contains a lytic enzyme, usually lysozyme or hyaluronidase.
Phage specificity . Phages have strict specificity. There are species specificity, i.e., the
ability to parasitize only in a certain type of microorganisms. Phages are usually
named after the host microbe (streptococcal, staphylococcal, cholera, dysentery,
etc.). Phages with more stringent specificity parasitize only on certain
representatives of a given species - these are typical phages. Phages that lyse
microorganisms of closely related species, for example, species belonging to the
genus of causative agents of dysentery (shigella), are called polyvalent.
The interaction of a phage with a sensitive cell passes through successive
stages. The whole cycle takes in different systems phage - bacterium from several
minutes to 1-2 hours. Let's analyze the sequence of this process using the example of
the T-even phage of Escherichia coli.
Stage I - adsorption of phage particles on the surface receptors of the cell is carried
out with the help of filaments of the tail process. Hundreds of phages can be
adsorbed on one cell for cell lysis, one is enough). Phage adsorption is specific.
Stage II - the penetration (injection) of the nucleic acid of the phage into cells in
different phages occurs in different ways. In E. coli T-phages, the spikes of the basal
lamina are in contact with the cell wall. The rod "pierces" the cell wall. An enzyme
found in the process, most often lysozyme, destroys the cytoplasmic membrane. At
the same time, the sheath of the process contracts, and the nucleic acid of the phage
is "injected" into the cell through the channel of the rod. The empty protein shell of
the phage ("shadow") remains outside.
Stage III - reproduction of the protein and nucleic acid of the phage inside the cell.
Stage IV - assembly and formation of mature phage particles.
Stage V - cell lysis and release of mature phage particles from it. Usually, the cell wall
ruptures and several hundred new phages are released into the environment,
capable of infecting fresh cells. Such lysis is called lysis from the inside (Fig. 23).
 Rice. 23. Scheme of the main stages of the interaction of a phage with a bacterial
cell. 1 - introduction of the nucleic acid of the phage into the cell; 2 - young,
multiplying phages; 3 - mature phages; 4 - isolation of phages

In contrast to lysis from the inside, lysis from the outside occurs when a very large
number of phages are adsorbed on the cell at once. They make numerous holes in
the cell wall through which the contents of the cell flow out. Thus, during lysis from
the outside, the phage does not multiply, and the number of its particles does not
increase.
According to the nature of the action on microorganisms, virulent and temperate
phages are distinguished.
Virulent phages cause the lysis of the infected cell with the release into the
environment of a large number of phage particles capable of infecting new cells. In
this case, the culture of microorganisms is lysed. The liquid medium becomes
transparent - the formation of phagolysate * occurs - a medium in which there is a
large number of phages. With the development of a virulent phage in bacteria
growing on a dense medium, either transparent areas of continuous lysis are formed,
or separate transparent formations grow - phage colonies. They are called negative
colonies (plaques). Colonies of different phages differ in size and structure (Fig. 24).
*
 ( With further incubation of the phagolysate in a thermostat, secondary turbidity of the medium may occur - the cells
of the Populations that remain alive, resistant to the phage, multiply.  )

Rice. 24. Colonies of Escherichia coli phages. 1 - large; 2 - small

Temperate phages do not lyse all cells in the population. With some of them, phages
enter into symbiosis: the nucleic acid of the phage (its genome) is integrated into the
cell chromosome and is called a prophage. A single chromosome is formed. The
bacterial cell does not die. A prophage that has become part of the cell genome can
be transmitted to an unlimited number of descendants, i.e., to new cells, during its
reproduction. The phenomenon of symbiosis of a microbial cell with a temperate
phage (prophage) is called lysogeny, and a culture in which there is a prophage is
called lysogenic. This name reflects the ability of the prophage to spontaneously
leave the cell chromosome and, passing into the cytoplasm, turn into a virulent
phage. Those culture cells in which the virulent phage was formed die (lyse), the rest
remain lysogenic.
Lysogenic cultures do not differ in their basic properties from the original ones, but
they are resistant to re-infection with the phage of the same name. When a
lysogenic culture is exposed to penetrating radiation (certain doses and exposure to
X-rays, cosmic rays), certain chemicals and a number of other factors, the production
of virulent phage and the lysis of culture cells by it increase significantly.
Temperate phages can be detrimental to microbiological production. For example, if
the strains that produce vaccines, antibiotics, and other biological substances are
lysogenic, there is a danger that the temperate phage will become virulent, which
will lead to the lysis of the production strain.
Temperate phages are a powerful factor in the variability of microorganisms. A
prophage can change some properties of a microbial culture, for example, make it
capable of producing toxin, which is observed among diphtheria bacilli, the causative
agent of scarlet fever, etc. In addition, by turning into a virulent form and lysing a
cell, a phage can capture a part of the host cell's chromosome and transfer this part
of the chromosome to another cell, where the phage will again turn into a prophage,
and the cell will acquire new properties (see Chapter 10).
The distribution of phages in nature is ubiquitous. Phages are found where
microorganisms sensitive to them are found: in water, soil, sewage, excretions of
humans and animals, etc. Almost all known bacteria are hosts of phages specific to
them.
The resistance of phages to physical and chemical factors is higher than that of the
vegetative forms of their hosts. Phages withstand heating up to 75°C, prolonged
drying, pH from 2.0 to 8.5. They are not sensitive to antibiotics, thymol, chloroform
and a number of other substances that destroy the accompanying
microflora. Therefore, these substances are used in the isolation and preservation of
phages. Acids and disinfectants are detrimental to phages.

Control questions

1. Define the concept of a phage.

2. Who first observed the action of a phage? Who discovered the phage and studied
its nature?
3. What is the nature, chemical composition and structure of phages?
4. What is the specificity of the action of the phage?
5. What is the difference between the action of virulent and temperate phages?

Methods for studying virulent phages

 Material preparation
The material from which the phage is isolated are usually filtrates obtained using
bacterial filters from environmental objects, human and animal organs and
secretions, cultures of microorganisms, etc.
Before filtration, the test material is prepared as follows:
Liquids (blood, urine, water, washings from objects, etc.) are freed from large
particles using a paper filter or centrifugation so that they do not clog the pores of
the bacterial filter.
Viscous material (pus, feces) is emulsified in isotonic sodium chloride solution or
broth, after which it is freed from large particles, as described above.
Dense material (pieces of organs, food, etc.) is pre-crushed - usually ground in a
mortar with sterile quartz sand. Instead of sand, sterile Pasteur pipette tips or broken
coverslips can be used. The ground material is carefully emulsified in an isotonic
sodium chloride solution or broth and freed from a large suspension.
The prepared material is passed through a bacterial filter, freeing from extraneous
microflora.
Cultures of microorganisms (known or under study) . When working with phage, 20-
24-hour cultures grown on agar or 2-6-hour cultures in liquid media are used. The
purity of the culture is established in smears stained with Pfeiffer magenta or
Gram. Suspension is prepared from cultures grown on agar slant. 3-5 ml of isotonic
sodium chloride solution is poured into a culture tube. Rotating the tube between
the palms, carefully wash off the culture from the surface of the medium so as not to
wet the stopper. The suspension is transferred into a sterile test tube and diluted
with isotonic sodium chloride solution 1:10 (0.5 ml of suspension in 4.5 ml of
solution).
When working with phage, sterile utensils (graduated and Pasteur pipettes, Petri
dishes, test tubes, flasks, mortars), a thermostat, a filter device, a centrifuge, tripods,
and a colony counting device are required.
Attention! All work with phage requires asepsis.
Qualitative Methods
The presence of a phage in a particular substrate is recognized by the lysis of a
microbial culture sensitive to it (test culture).
Phage detection on dense media . The test culture is seeded with "lawn" (see
Chapter 7) on the surface of the agar in a Petri dish. The inoculation is dried in a
thermostat for 30-40 minutes with the lid open, after which a drop of the studied
material is applied to it. After a few minutes, when the liquid is absorbed, the cups
are placed in a thermostat for 18-20 hours. If there is a phage in the material under
study, the culture will lyse and in the place where the drop was applied, the culture
will either not grow at all (solid lysis) or separate colonies will form. phage.
Phage detection in liquid media. In two test tubes with the same amount of broth,
one drop of the culture, the microbe in relation to which the phage is being studied,
is introduced. The studied phage or the filtrate of the material in which it is
determined is added to one of them. The second tube serves as a culture growth
control. The tubes are placed in a thermostat for 12-20 hours. The results are
recorded only if there is growth of the culture in the control (turbidity of the
medium). The absence of visible growth or the subsequent clarification of the
medium in the test tube with the test material indicates the presence of a phage. If
the contents of this tube are turbid, the study must be supplemented by inoculation
on a solid medium: turbidity could occur from the growth of a phage-resistant
culture. Only if the phage is not detected in the agar culture, it can be concluded that
it is not present in the studied material.
Quantitative Methods
Quantitative methods are of great importance in many studies, including those of
phage. The doctor needs to know the activity of the phage in order to correctly
determine its dose for therapeutic and prophylactic use, the researcher needs to
know how the activity of the phage changes in the experiment.
Phage activity is expressed by the concept of titer, distinguishing the phage titer in
liquid and solid media.
In the study in a liquid medium (according to Appelman), the phage titer is its highest
dilution (or the smallest amount), which inhibits the growth of the test culture under
the conditions of this experiment.
On a dense medium (titration according to Gracia), the phage titer is the number of
particles (corpuscles) of the phage in 1 ml of the starting material.
Phage titration according to Appelman (in liquid medium) . When titrating a phage,
the volume and composition of the medium, temperature, aeration, the number of
microbes, and the test tubes in which titration is carried out (diameter, bottom
configuration, glass thickness) must be the same. Only the amount of phage changes.
All components involved in biological experiments are subject to mandatory control
for the correctness of their work.
When titrating the phage, the control of the phage and the broth is set for sterility,
the control of the culture is for its viability.
Setting up the experience. 1. Pour 4.5 ml of sterile broth into 12 sterile tubes. The
liquid level in all test tubes must be the same. If this is not the case, then a mistake
has been made. In this case, the non-standard tube is replaced with a new one and
refilled with broth. When spilling the broth, use pipettes with a capacity of 5-10 ml.
2. The test tubes are placed in a rack and numbered from 1 to 10, on one of the
remaining test tubes they write "KK" (culture control), on the other - "KF" (phage
control). All test tubes in which the experiment is carried out must be labeled.
3. Sequential tenfold dilutions of the phage from 10 -1 to 10 -10 are prepared in ten
test tubes (Table 9). 0.5 ml of phage is added to the 1st tube. The same amount is
added to the phage control tube. After changing the pipette, thoroughly mix the
contents of the 1st test tube with a new pipette and transfer 0.5 ml of it into the 2nd
test tube; 0.5 ml of the contents of the 2nd tube is transferred to the 3rd and so on
up to the 10th tube, changing the pipettes each time. After mixing the contents of
the 10th tube, 0.5 ml of the liquid from it is poured into a disinfectant solution. Also
come with the contents of the phage control tube. To dilute the phage, pipettes with
a capacity of 1-2 ml are used. Check the liquid level in the test tubes. It must be the
same.

Table 9. Phage titration scheme according to Appelman

Symbols: + culture growth, - lack of culture growth.
Note. Arrows indicate the transfer of phage from tube to tube. From tube 10 and
from the phage control tube, 0.5 ml is poured into the disinfectant solution.
4. Add 1 drop (0.05 ml) of the test culture suspension to all tubes, except for the
phage control tube. The tubes are shaken. A slight haze should appear in all tubes
except the phage control tube. If this does not happen, all operations with a non-
standard tube are repeated again.
5. The test tubes are placed in a thermostat for 18-20 hours, after which the result of
titration is taken into account. Accounting for results must begin with controls.
With the correct setting of the experiment in the control of the culture, there should
be a multiplication of bacteria - the contents of the test tube become cloudy. This
control allows us to conclude that the culture taken in the experiment is viable and
that the nutrient medium ensures its development under the given conditions of the
experiment. This means that if the culture did not grow in test tubes with phage, it
was affected by the phage.
The liquid in the phage control tube should remain clear. This control allows us to
conclude that the dishes, broth and phage are sterile.
If the results in the controls are correct, the nature of the changes in the first ten test
tubes is recorded. The growth of the culture in test tubes of the "experimental"
series indicates the absence or insufficient amount of phage in them. The phage titer
is established, i.e. its highest dilution, in which the culture was lysed (the contents of
the tube are transparent). The titer is denoted by the degree of dilution of the phage
with the opposite sign, which corresponds to the ordinal number of the tube. For
example, if the culture does not grow in the first seven tubes, the phage titer is 10 -
7
 (see Table 9).
Phage titration according to Grace (on solid medium) using the agar layer
method allows determining the number of phage particles in the material being
titrated. The method is based on the fact that each phage particle gives a zone of
enlightenment (lysis) on a cup with a lawn of a microbe sensitive to it, i.e. forms a
separate colony.
Setting up the experience. Cups with 20-25 ml of MPA are covered with sterile filter
paper and dried in a thermostat or under a bactericidal lamp (the distance from the
lamp is not more than 2 m). The titratable phage is diluted from 10 -1 to 10 -10 (as in
the previous experiment) and 1 ml is transferred into other numbered test tubes
(respectively, from the 1st to the 1st and so on up to the 10th), into which 2.5 ml of
0.7% MPA, melted and cooled to 45°C. Add 0.1 ml of the test culture to each of these
tubes. The contents of the test tubes are quickly mixed (do not allow the agar to
solidify) and poured onto the surface of the medium into Petri dishes with numbers
corresponding to the numbers of the test tubes. After 30 minutes, the cups are
placed in a thermostat.
The results are recorded after 18-20 hours. At a high concentration of phage (in the
first plates), a continuous lysis of the culture will occur. In those dilutions of phage in
which there was a small amount of phage particles, isolated colonies will appear,
which are counted. In order not to make a mistake in the count, each counted colony
is marked from the side of the bottom of the dish. The colony counting apparatus
makes the work much easier (see Fig. 54). To establish the number of phage particles
in 1 ml of phagolysate, use the formula: n = y×x, where n is the desired number; y is
the number of phage colonies grown on the plate; x is the dilution of the phage in
the dish in which the colonies were counted.
For example, if 25 colonies grew in a dish with a phage dilution of 10 -8 (1:10 8 ), then
1 ml of the initial liquid contains 25×10 8 or 2.5×10 9 phage particles.
More accurate results are obtained if the number of phage particles in the initial
liquid is determined by several dilutions and the arithmetic mean is calculated. For
example, when diluting the phage 10 -6 , 320 colonies grew, therefore, in 1 ml of the
initial liquid there were 320×10 or 3.2×10 8 phage particles. When diluted phage 10 -
7
 grew 42 colonies, therefore, in the original liquid was 4.2×10 8 /ml of phage
particles. When the phage was diluted 10 -8 , 5 colonies grew, therefore, in the initial
liquid there were 5 × 10 8/ml phage particles. By adding the values obtained in these
calculations and dividing the sum by 3 (the number of calculations performed), the
number of phage particles in 1 ml of the titrated preparation is determined. In our
example, it is equal to 4.1×10 8 .
It is best to count colonies on plates where at least 5 and no more than 50 colonies
have grown. Otherwise, the accuracy of the calculation suffers. If there are many
colonies on the dish, the dish can be divided into several sectors, the colonies on one
of them can be counted and the resulting figure multiplied by the number of sectors.
As a rule, all biological studies are carried out in three parallel experiments. In this
example, each phage dilution is simultaneously titrated three times.

Phage isolation methods

direct method . The phage is obtained and studied directly in the filtrates of the test
material. The presence and activity of a phage is known by the lysis of a culture
sensitive to it.
As a rule, the direct method does not give convincing results due to the small
amount of phage contained in the filtrate. To increase its quantity, enrichment
methods are used.
enrichment method . The prepared filtrate is introduced into a 2-3-hour broth
culture of the corresponding microorganisms. Seeding is incubated in a
thermostat. The phage multiplies in the culture cells and its titer increases
significantly. After that, the broth is filtered and the properties and activity of the
phage are determined in the filtrate.

Practical applications of phages

The use of phages is based on their strict specificity and ability to destroy microbial
cells or enter into symbiosis with them.
Phage prophylaxis and phage therapy - the prevention and treatment of infections
with the help of phages - is based on the fact that, meeting a pathogen in the
patient's body, the phage destroys it. Currently, phages are widely used in the
treatment and prevention of staphylococcal and streptococcal infections, even those
that do not respond to antibiotics, as well as cholera, plague, and a number of other
infections, such as infections caused by Escherichia coli and Proteus.
Phage diagnosticsincludes: a) identification of isolated cultures using known
(diagnostic) phages. The culture corresponds to the phage that lysed it. For example,
if a cholera phage caused lysis, then this is a culture of vibrio cholerae. The strict
specificity of typical phages makes it possible to type variants within a species
(fagovars). Phage typing is of great importance in epidemiology, as it makes it
possible to establish the source of infection and resolve a number of other issues
(see pp. 243, 292); b) identification of an unknown phage by a microbial test
culture. If phage lyzes the culture of the causative agent of dysentery, then this is a
dysentery phage; c) an accelerated method of diagnostics using the reaction of
increasing the titer of the RNTF phage does not require the isolation of a pure culture
of the pathogen. The studied material (from the patient or from environmental
objects) and indicator phage, the titer of which is strictly established, put into the
broth. After incubation in a thermostat, the phage titer is determined according to
Gracia. An increase in titer (the number of phage corpuscles) by 5 times or more
indicates that the material under study contains the corresponding pathogens in
which the phage has multiplied.
Temperate phages are widely used in solving cardinal problems of biology. With their
help, the genetic code has been studied, great success has been achieved in genetic
engineering, they are used to study tumor growth, as a factor in the variability of
microorganisms, and in other studies. Since lysogenic cultures, in contrast to
"healthy" cultures, are sensitive to radiation, they serve to determine the reliability
of protection of spacecraft from cosmic rays: in case of unreliable protection, the
prophage passes into a virulent form and lyses the culture.

Phage preparations

In the production of phage preparations, well-studied strains of microorganisms and

phages are used, which are usually grown in reactors, which makes it possible to
obtain large amounts of phagolysate.
Phages are produced in liquid form (ampoules and vials), in tablets and
suppositories. Phage tablets intended for oral administration are coated with an
acid-resistant coating that protects the phages from the action of gastric
hydrochloric acid.
All phage preparations are subject to mandatory control for the absence of foreign
flora, harmlessness and activity (titer), which is carried out at the production facility
that produces them. Selective control is carried out at the State Research Institute
for Standardization and Control of Medical Biological Preparations named after V.I. L.
A. Tarasevich. The released phage is provided with a label, which indicates: the
institution that produces it, the name of the phage, series, control number and
expiration date. Each package is supplied with instructions for the use and storage of
the phage.

Control questions
1. What properties of the phage underlie its production and use?
2. Why is it necessary to titrate phage under sterile conditions?
3. What test tubes are used to start recording the experience of phage titration
according to Appelman?

Exercise

1. Study and describe the nature of the action of the phage on cultures of
microorganisms grown on a liquid and solid medium (obtain the cultures from the
teacher).
2. Perform an Appelman titration of the phage and record the results.
3. Count the phage colonies on the plate (Grazia method). Knowing the dilution of
the phage, determine the number of phage corpuscles in 1 ml of the initial
preparation.
4. Examine various phage preparations.

Chapter 9 Chemoprophylaxis and chemotherapy

General characteristics - N. A. Belskaya

Antibiotics (from the Greek anti - against, bios - life) are the waste products of living
organisms that can selectively kill microorganisms or suppress their growth.
The production of antibiotics by microorganisms is one of the most important
manifestations of microbial antagonism (from the Greek antagonizomai - I fight, I
compete). The largest number of microorganisms with antagonistic properties is
found in the soil, especially among fungi, actinomycetes, and spore-bearing
bacteria. Antagonists are also detected in water bodies (rivers, lakes), as well as
among representatives of the normal microflora of humans and animals. For
example, E. coli, bifidum bacteria, lactobacilli in the intestines of people (see chapter
6). The first attempts at the practical use of microbial antagonism belong to L.
Pasteur and I. I. Mechnikov.
L. Pasteur in 1877 found that putrefactive bacteria inhibit the growth of anthrax
bacilli when they are grown together on a nutrient medium. As a result of his
observations, Pasteur suggested the possibility of using the phenomenon of bacterial
antagonism to treat infectious diseases.
II Mechnikov (1894), studying the role of putrefactive intestinal bacteria, found that
they systematically poison the body with the products of their vital activity and this
contributes to premature aging of people. He also discovered that lactic acid bacteria
(Bulgarian bacillus) found in yogurt inhibit the development of putrefactive intestinal
bacteria and suggested using antagonistic relationships of microorganisms as one of
the methods of combating old age.
Russian scientists V. A. Manassein and A. G. Polotebnov (1871-1872), many years
before the discovery of antibiotics, used the green mold penicillium to treat purulent
wounds and other skin lesions.
The idea to use one type of microorganism in the fight against another (antagonism)
has brought significant results. From Pseudomonas aeruginosa, the first antibiotic,
pyocyonase (R. Emmerich, O. Lev), was obtained, but it did not find wide application.
The beginning of the doctrine of antibiotics was laid in 1929, when the English
scientist A. Fleming discovered lysis of colonies near the accidentally grown mold
Penicillium notatum on cups with inoculations of Staphylococcus aureus. Fleming
found that mold broth culture filtrate kills not only staphylococci, but also other
microorganisms. For 10 years, Fleming tried to get penicillin in a chemically pure
form. However, he did not succeed. A purified preparation of penicillin suitable for
clinical use was obtained by English researchers E. Chain and G. Flory in 1940.
The Soviet microbiologist Z. V. Ermolyeva used another type of mold, Penicillium
crustosum (1942), to obtain penicillin, and was one of the organizers of the
production of penicillin during the Great Patriotic War.
The discovery of penicillin and its successful use for the treatment of
pyoinflammatory processes and a number of other infectious diseases prompted
scientists to search for new antibiotics that have a detrimental effect on various
microorganisms. Currently, more than 2000 different antibiotics have been
obtained. However, not all of them are used in clinical practice, since some turned
out to be toxic, while others were inactive in the conditions of the human body.
The source of antibiotics are a variety of microorganisms with antimicrobial
activity. Antibiotics are isolated from mold fungi (penicillin, etc.), actinomycetes
(streptomycin, tetracycline, etc.), bacteria (gramicidin, polymyxins); Substances with
antibiotic action are also obtained from higher plants (phytoncides of onion, garlic)
and animal tissues (lysozyme, ecmolin, interferon).
Antibiotics can have a bacteriostatic and bactericidal effect on microorganisms. The
bactericidal action of antibiotics causes the death of microorganisms, and the
bacteriostatic action inhibits or delays their reproduction. The nature of the action
depends on both the antibiotic and its concentration.
The classification of antibiotics can be based on various principles: according to the
source of production, chemical structure, mechanism and spectrum of antimicrobial
activity, method of production. Most often, antibiotics are classified according to the
spectrum of antimicrobial activity and sources of production.
The mechanism of the antimicrobial action of antibiotics is diverse: some disrupt the
synthesis of the bacterial cell wall (penicillin, cephalosporins), others inhibit the
processes of protein synthesis in the cell (streptomycin, tetracycline,
chloramphenicol), others inhibit the synthesis of nucleic acids in bacterial cells
(rifampicin, etc.).
Each antibiotic is characterized by a spectrum of action, i.e. the drug can have a
detrimental effect on certain types of microorganisms. Broad-spectrum antibiotics
are active against various groups of microorganisms (tetracyclines) or inhibit the
reproduction of many gram-positive and gram-negative bacteria (streptomycin,
etc.). A number of antibiotics act against a narrower range of microorganisms, for
example, predominantly gram-negative bacteria are sensitive to polymyxin.
According to the spectrum of action, antibiotics are divided into antibacterial,
antifungal and antitumor.
Antibacterial antibiotics inhibit the development of bacteria and constitute the most
extensive group of drugs that differ in chemical composition. For the treatment of
infectious diseases caused by bacteria, broad-spectrum antibiotics are more often
used: tetracyclines, chloramphenicol, streptomycin, gentamicin, kanamycin, semi-
synthetic penicillins and cephalosporins and other drugs.
Antifungal antibiotics (nystatin, levorin, amphotericin B, griseofulvin) have an
inhibitory effect on the growth of microscopic fungi, as they violate the integrity of
the cytoplasmic membrane of microbial cells. Used to treat fungal diseases.
Antitumor antibiotics (rubomycin, bruneomycin, olivomycin) inhibit the synthesis of
nucleic acids in animal cells and are used to treat various forms of malignant
neoplasms.
The biological activity of antibiotics is measured in international units of action
(ED). The smallest amount of the drug that has an antimicrobial effect on test
bacteria sensitive to it is taken as a unit of antibiotic activity (for example, for
penicillin - Staphylococcus aureus, streptomycin - Escherichia coli, etc.). Currently,
units of antibiotic activity are expressed in micrograms * of pure drug. Thus, 0.6 μg of
penicillin is taken per unit of activity, and for most antibiotics, 1 unit corresponds to
1 μg (streptomycin, etc.).
*
 ( 1 mcg - 10  -6  g .)
A powerful industry for the production of antibiotics has been created in our
country. Natural antibiotics are obtained biosynthetically: strains-producers of fungi,
actinomycetes, bacteria are grown in a liquid nutrient medium of the appropriate
composition, at a certain pH value, optimal temperature and aeration. Antibiotic
substances are end products of microbial metabolism and are produced by cells into
the nutrient medium, from where they are extracted by chemical methods.
The study of the chemical structure of antibiotics made it possible to obtain synthetic
drugs by chemical synthesis (levomycetin).
A great achievement is the development of methods for obtaining semi-synthetic
antibiotics based on a change in the chemical structure of a natural drug. As a result,
it was possible to expand the spectrum of antimicrobial action, eliminate some of the
shortcomings of natural antibiotics. In recent years, semi-synthetic penicillins,
cephalosporins, tetracyclines, rifampicin and other drugs have been widely used in
clinical practice.
Antibiotic therapy can sometimes be accompanied by complications from the
macroorganism, and also cause changes in various properties of microorganisms.
Possible complications with antibiotic therapy . Some antibiotics (penicillin,
streptomycin, etc.), introduced into the patient's body, cause a state of
hypersensitivity (allergy), which increases with the use of the drug. Allergic reactions
develop in the form of a rash-urticaria, swelling of the eyelids, lips, nose,
dermatitis. The most formidable complication is anaphylactic shock (see Chapter 13),
from which the death of the patient can occur * .
*
 ( The better the antibiotic is purified from ballast substances, the less often and to a lesser extent it causes pronounced
allergic actions. )

Attention! Before using an antibiotic parenterally, it is necessary to identify the

absence of hypersensitivity to it of the patient's body. This is determined using an
intradermal test with this drug: 0.1 ml of an antibiotic is injected into the skin of the
inner side of the forearm and observed for 20-30 minutes. If the reaction is positive
(the diameter of the papule is more than 1 cm and a large area of redness), then the
antibiotic cannot be administered.
The introduction of large doses of broad-spectrum antibiotics into the body, as a
rule, is accompanied by the death of representatives of the normal microflora of the
respiratory tract, intestines and other organs. This leads to a change in the usual
antagonistic relationship between microorganisms in vivo. As a result, opportunistic
bacteria (staphylococci, proteus) and fungi of the genus Candida, resistant to these
antibiotics, can become active and cause secondary infections. This is how fungal
infections occur - candidiasis of the skin, mucous membranes, internal
organs; dysbacteriosis (violations of the normal composition of the microflora).
To prevent the development of candidiasis, antibiotics are administered with
antifungal drugs, such as nystatin, etc. The use of drugs prepared from
representatives of the normal microflora (colibacterin, bifidumbacterin, bifikol) after
taking antibiotics prevents the development of dysbacteriosis.
Long-term treatment and use of antibiotics can have a toxic effect on the patient's
body: tetracyclines can cause liver damage, levomycetin - hematopoietic organs,
streptomycin in some cases affects the vestibular and auditory analyzers,
cephalosporins can disrupt kidney function (nephrotoxicity). Many antibiotics often
cause hypovitaminosis and irritation of the mucous membrane of the
gastrointestinal tract.
Antibiotics can have a harmful effect on fetal development, especially in women who
used antibiotics during the first period of pregnancy. Tetracycline group antibiotics
have a direct effect on the fetus.
Resistance of microorganisms to antibiotics . Often, during antibiotic treatment,
antibiotic-sensitive microorganisms become resistant (resistant) forms. Acquired
bacterial resistance to an antibiotic is inherited by new populations of bacterial cells.
The mechanism of resistance formation is diverse (see Chapter 10). In most cases,
resistance is associated with the ability of bacteria to synthesize enzymes that
destroy certain antibiotic substances. For example, the resistance of staphylococci to
penicillin is explained by their ability to produce the enzyme penicillinase, which
destroys the antibiotic. At the same time, for Escherichia coli, Proteus and other
bacteria of the intestinal family, penicillinase is a constitutive (permanent) enzyme
and determines their natural resistance to penicillin.
Some bacteria are found to be multidrug resistant, i.e. a bacterial cell can be
resistant to several antibiotics. Resistance to penicillin and streptomycin, which were
the first to be used in clinical practice, is especially pronounced.
The effectiveness of antibiotic therapy is determined mainly by the degree of
sensitivity of bacteria to the drug used. Therefore, the sensitivity of cultures of
microorganisms isolated from patients is checked for various antibiotics that are
used for treatment.
During the action of antibiotics, changes in the morphological, cultural, and biological
properties of bacteria are possible; L-shapes may form (see chapter 3).
Antibiotics isolated from mushrooms . Penicillin was obtained from some strains of
fungi of the genus Penicillium (Penicillium notatum, Penicillium chrysogenum).
Penicillin - highly active against pathogenic cocci: gram-positive staphylococci,
streptococci, pneumococci; gram-negative - meningo- and gonococci. It is used to
treat anthrax, tetanus, gas gangrene, syphilis and other diseases. Penicillin is
administered parenterally. The drug can not be used orally, as it loses its activity in
acidic and alkaline environments and is destroyed in the gastrointestinal tract.
Already at the very beginning of the use of penicillin, it was noticed that it is quickly
excreted from the body, and to maintain the concentration of penicillin in the blood
necessary for the therapeutic effect, it is administered every 3-4 hours.
Subsequently, penicillin preparations with a prolonged (prolonged) action were
created. These include ecmonovocillin, bicillin-1, bicillin-3, bicillin-5. Bicillin-1, 3, 5
are antibiotics that are successfully used to treat rheumatism and syphilis.
At present, semi-synthetic penicillins have been obtained: methicillin, oxacillin,
cloxacillin, which are not destroyed by penicillinase and are used to treat infections
caused by penicillin-resistant staphylococci; ampicillin is active not only against
gram-positive, but also gram-negative bacteria (causative agents of typhoid fever,
dysentery, etc.). Oxacillin and ampicillin are resistant to the acidic environment of
the stomach, which allows them to be used orally.
Fungi of the genus Cephalosporium produce the antibiotic cephalosporin. Its semi-
synthetic derivatives, of which ceporin (cephaloridin) and cefomezin have found the
greatest use, are low toxic, have a wide spectrum of action, are not destroyed by
penicillinase, do not cause allergic reactions in persons sensitive to penicillin, and are
widely used to treat many infectious diseases.
Antibiotics produced by actinomycetes . For the first time, the antagonistic action of
radiant fungi (actinomycetes) was established by N. A. Krasilnikov
(1939). Streptomycin was isolated from Actinomyces globisporus by the American
scientist A. Waksman (1943). The discovery of streptomycin marked a new era in the
fight against tuberculosis, as Mycobacterium tuberculosis was found to be
susceptible to the drug. Streptomycin has a detrimental effect on many gram-
positive and gram-negative bacteria and is used to treat plague, tularemia,
brucellosis, etc. An antibiotic is administered parenterally.
Bacteria quickly become resistant to streptomycin. Some microorganisms form
streptomycin-dependent forms that can multiply on nutrient media only when an
antibiotic is added.
Actinomycetes are producers of natural antibiotics of the tetracycline group
(tetracycline, chlortetracycline, oxytetracycline). All drugs have a wide spectrum of
action, inhibit the reproduction of many types of gram-positive and gram-negative
bacteria, rickettsia, some protozoa (dysentery amoeba). Tetracycline is rapidly
absorbed from the gastrointestinal tract, it is prescribed with nystatin for the
prevention of candidiasis.
In recent years, semi-synthetic derivatives of oxytetracycline (metacycline,
doxycycline, etc.) have been widely used, which turned out to be more effective than
natural preparations.
Levomycetin is a synthetic drug identical to natural chloramphenicol isolated from
the culture fluid of Streptomyces venezuelae. The antimicrobial spectrum of
levomycetin includes many gram-positive and gram-negative bacteria, rickettsia,
spirochetes. Most often, chloramphenicol is used to treat intestinal infections -
typhoid fever, paratyphoid fever, dysentery, as well as various rickettsiosis - typhus
and other diseases.
Antibiotics were obtained from actinomycetes: erythromycin, oleandomycin,
kanamycin, rifampicin, lincomycin, etc. These drugs are classified as "reserve"
antibiotics and are used to treat diseases caused by bacteria resistant to other
antibiotics.
Antibiotics produced by bacteria . Polymyxins and gramicidin C are of the greatest
practical importance.
Polymyxins combine a group of related antibiotics produced by spore-forming soil
bacilli, B. polimixa. Polymyxins B, M and E are active mainly against gram-negative
bacteria (enterobacteria, Pseudomonas aeruginosa, etc.).
Gramicidin C was isolated by Soviet scientists G. M. Gause and M. G. Brazhnikova
(1942) from various strains of soil bacilli - B. brevis. It is susceptible to Gram-
fermenting bacteria. Gramicidin C can cause hemolysis of erythrocytes, therefore it is
used only topically for the treatment of suppurative processes.
Antibiotic substances derived from higher plants . Soviet researcher T. P. Tokin
(1928) discovered that many higher plants form volatile substances with
antimicrobial activity (phytoncides). They protect plants from
pathogens. Phytoncides are volatile essential oils that are extremely unstable, as a
result of which it is very difficult to obtain pure phytoncides preparations.
Phytoncides are isolated from onion juice, garlic, eucalyptus and lichen leaves, St.
John's wort. They are also found in the juice of horseradish, radish, aloe and other
plants. The use of phytoncides in medical practice is limited, since it is not possible to
obtain well-purified, stable and low-toxic preparations.
Antimicrobial substances isolated from animal tissues . Lysozyme was first
discovered by the Russian scientist N. P. Lashchenkov (1909) in the protein of a
chicken egg. Later, lysozyme was found in milk, lacrimal fluid, saliva and tissues of
various organs (kidneys, spleen, liver); found that it, as a natural protective factor of
the body, has a bacteriolytic (dissolving bacteria) effect on many pathogenic and
saprophytic microorganisms. It is used to treat eye and skin diseases.
Ekmolin was isolated by Z. V. Ermoleva from fish tissues. It is used in combination
with penicillin (ecmonovocillin), as it enhances and prolongs its action in the body.
Of particular interest is interferon, which is formed in the cells of the body under the
influence of viruses and is a factor in the natural protection of the cell from the
reproduction of viruses. Interferon, discovered by Isaacs and Lindemann (1957), has
a wide antiviral spectrum. The study of the mechanism of action of interferon
showed that it interferes with the synthesis of nucleic acids of many viruses and
causes their death. Interferon is species-specific: human interferon does not affect
viruses in animals.
Interferon is isolated from human leukocytes and designated as If-α. It is used to
prevent and treat influenza and other viral respiratory diseases. In recent years,
there have been reports of the effective action of interferon in some malignant
neoplasms.

Control questions

1. What are antibiotics?

2. What phenomenon underlies the action of antibiotics?
3. What are the sources of antibiotics?
4. How do antibiotics differ in terms of the mechanism of antimicrobial action?
5. What is the nature of the action of antibiotics?
6. What is called the antimicrobial spectrum of antibiotics?
7. What are the possible complications from the macroorganism during antibiotic
therapy?
8. What properties can change in microorganisms under the influence of antibiotics?

The sensitivity of microorganisms to antibiotics - N.

A. Belskaya

( According to the Order of the Ministry of Health of the USSR No. 250 dated March 13, 1975 "On the unification of
methods for determining the sensitivity of microorganisms to chemotherapeutic drugs".  )

In clinical practice, antibiotic-sensitive microorganisms are considered to be those

microorganisms on which antibiotics have a bacteriostatic or bactericidal effect.
In any laboratory study, the criterion for the sensitivity of microorganisms to
antibiotics is the minimum concentration of the antibiotic that inhibits (delays) the
growth of the pathogen under standard experimental conditions.
To determine drug sensitivity, it is optimal to use a pure culture of the pathogen. It is
necessary to isolate cultures of microbes from the body for sensitivity testing before
starting antibiotic treatment, since under their influence the growth of the causative
agent of the disease can be completely inhibited. The sensitivity of microorganisms
to antibiotics is determined by diffusion into agar using standard disks or by serial
dilution in liquid and solid nutrient media.
Methods of determination
disk method. A suspension of the studied culture is sown with a "lawn" (see Chapter
7). As inoculum, a daily broth culture or 1 billion microbial suspension prepared
according to the optical standard of turbidity No. 10 (see below) can be used. The
seeded cups are dried for 30-40 minutes at room temperature. Then paper discs
impregnated with solutions of various antibiotics are placed on the surface of the
seeded agar with tweezers. Each disc is pressed lightly with the jaws of the tweezers
so that it fits snugly against the surface of the agar. The discs are placed at equal
distances from each other and at a distance of 2 cm from the edge of the cup. One
plate can be used to study the sensitivity of one strain to 4-5 antibiotics.
The seeded cups with discs applied to them are placed in a thermostat at 37 ° C for
18-24 hours. The cups are placed upside down to avoid condensation water getting
on the surface of the crops.
Accounting for results. The action of antibiotics is assessed by the phenomenon of
growth retardation around the disk (Fig. 25). The diameter of the microbial growth
inhibition zones around the discs is determined using a ruler, including the diameter
of the disc itself. Between the degree of sensitivity of the microbe to antibiotics and
the size of the zone of no growth, there are the following relationships (Table 10).
 Rice. 25. Determination of the sensitivity of bacteria to antibiotics (disc method)

Table 10. Determination of the degree of sensitivity of microorganisms to antibiotics

by the size of the zone of no growth

The answer indicates what sensitivity the studied strain has, and not the size of the
zone of growth inhibition.
In some cases, determine the sensitivity of microorganisms to antibiotics in the
native material (pus, wound discharge, etc.). In this case, the material is applied to
the surface of nutrient agar and evenly rubbed over the surface with a sterile glass
spatula * , and then discs are applied. The disk method for determining the sensitivity
of microorganisms due to its simplicity and accessibility is widely used in practical
laboratories and is regarded as a qualitative method.
*
 ( For those types of microorganisms that do not grow on meat peptone agar, such as streptococci, pneumococci and
others, use agar with blood or serum. )
Method of serial dilutions in a liquid nutrient medium . This method is an accurate
quantitative method, it is used in scientific work and in especially important cases in
the laboratories of hospitals and preventive institutions.
To set up the experiment, it is necessary to have a pure culture of the tested
microorganism, the main solution of the antibiotic, meat-peptone broth on
Hottinger's digest, containing 1.2-1.4 g/l of amine nitrogen.
The activity of antibiotics is expressed in units/ml or mcg/ml. To prepare the stock
solution of the antibiotic, antibiotics are used that are commercially available with an
indication of their number in the vial.
If on the label, instead of the number of units in the vial, the dosage is indicated in
units of mass, then it should be borne in mind that 1 g of activity for most antibiotics
corresponds to 1 million units. From this solution, the required dilutions of
antibiotics should be prepared. Instructions for preparing the stock solution of
antibiotics using penicillin as an example are given in Table. eleven.

Table 11 Preparation of penicillin stock solution

A suspension of a culture of microorganisms grown on a dense nutrient medium is

prepared. The resulting suspension is compared with the optical turbidity standard
No. 10 (see below), and then diluted with sterile isotonic sodium chloride solution to
10 6 microbial bodies in 1 ml. To obtain the appropriate dilution of the microbial
suspension, a series of consecutive tenfold dilutions is prepared (see below).
Setting up the experience. In 12 sterile test tubes pour 1 ml of liquid nutrient
medium. In the 1st test tube, 1 ml of the stock solution of the antibiotic is added,
containing, for example, 32 IU per 1 ml. The contents of the 1st tube are mixed and 1
ml is transferred to the 2nd tube, from the 2nd to the 3rd, from the 3rd to the 4th,
and so on up to the 10th, from which 1 ml is removed. Thus, the 1st tube will contain
16 units, the 2nd - 8 units, the 3rd - 4 units, etc. A separate pipette is used to prepare
each dilution. The contents of the 11th tube serves as a control for bacterial growth,
and the 12th tube serves as a control for the sterility of the nutrient medium. Into all
test tubes, except for the 12th, add 0.1 ml of the test culture of a certain density. The
inoculation is incubated in a thermostat for 18-24 hours and the results of the
experiment are recorded.
The results are recorded in the presence of growth in the culture control and the
absence of growth in the medium control. Then note the last tube with a complete
visible growth inhibition of microbes. The amount of antibiotic in this tube is the
minimum inhibitory concentration for the tested strain and determines the degree of
its sensitivity to this antibiotic. The response issued by the laboratory indicates the
minimum inhibitory concentration.
Method of serial dilutions on solid nutrient medium . Prepare two-fold dilutions of
the antibiotic, as in the method of serial dilutions in a liquid nutrient medium. Then
take 1 part of each antibiotic dilution and 9 parts of nutrient agar, melted and cooled
to 42 ° C (at the rate of 1 ml of antibiotic + 9 ml of MPA), mix well and pour into Petri
dishes.
The density (concentration) of the culture is determined according to the optical
turbidity standard No. 10 and diluted with a sterile isotonic solution to 10 7 microbial
bodies in 1 ml. The test cultures are applied with a bacterial loop to the surface of
nutrient agar with an antibiotic. 20-25 strains are inoculated per cup. Seeded cups
are placed in a thermostat at 37 ° C for 16-20 hours for most types of
microorganisms. The nutrient agar plate without antibiotic, on which the test
cultures are applied, is the control.
The results are recorded in the presence of growth in the control dish, and the
minimum inhibitory concentration of the antibiotic is determined by the last Petri
dish, where a complete delay in bacterial growth is noted.
Fleming's track method . The method is used to determine the spectrum of action of
an antibiotic. In a Petri dish with MPA, a path 1 cm wide is cut out with a sterile
scalpel and removed. Then, a certain concentration of antibiotic solution is
introduced into a test tube with melted and cooled to 42-45 ° C meat-peptone
agar. The contents of the tube are mixed and poured into the lane so that the liquid
does not go beyond its limits. After solidification of the agar, cultures of several
studied microorganisms are inoculated with a loop perpendicular to the lane. Crops
are placed in a thermostat for 18-24 hours.
Accounting for results. Cultures sensitive to the drug begin to grow only at a certain
distance from the lane, insensitive cultures grow to the very edge.
Optical turbidity standard procedure
Optical turbidity standards are used to determine the number of microbial bodies in
1 ml. They are manufactured by the State Research Institute for Standardization and
Control of Medical Biological Preparations of the Ministry of Health of the USSR. L. A.
Tarasevich (GISK). The following turbidity standards exist:
0.5 billion microbes in 1 ml - No. 5 (5 units of turbidity)
0.9" "" 1" - No. 9 (9" ")
1 " " " 1 " - No. 10 (10 " " )
1.1 "" "1" - No. 11 (11 "")
Before determining the number of microbial bodies in 1 ml, a microbial suspension is
first obtained. To do this, pour 5-6 ml of isotonic sodium chloride solution into a test
tube with a culture grown on slant agar and, rotating the tube between the palms,
wash off the culture from the surface of the medium. Part of the resulting
suspension is transferred with a sterile pipette into a sterile test tube, the wall
thickness and diameter of which corresponds to the test tube of the optical
standard. Then, the density of the resulting microbial suspension is compared with
one of the optical turbidity standards. If necessary, the microbial suspension is
diluted by adding isotonic sodium chloride solution to the desired turbidity. If the
turbidity of the obtained microbial suspension coincides with the turbidity of the
optical standard, then the number of microbial bodies in it corresponds to the
number of the standard.

Control questions

1. What is the criterion for the sensitivity of microorganisms to antibiotics in a

laboratory study?
2. When should cultures of microorganisms be isolated from the body of patients to
determine sensitivity to antibiotics?
3. What are the methods for determining the sensitivity of microorganisms to
antibiotics?

Exercise

1. Take a bottle of penicillin containing 1 ml of 300,000 IU from the teacher and

prepare a stock solution of antibiotic in 32 U/ml.
2. Determine the sensitivity of microorganisms to antibiotics using the paper disc
method, consider the results and give an answer.
3. Determine the sensitivity of the isolated culture of staphylococci to penicillin by
the method of serial dilutions in a liquid nutrient medium, take into account the
results and give an answer.

Chemoprophylaxis and chemotherapy

In medical practice, chemicals have long been used to prevent and treat infectious
diseases. The Indians used cinchona bark to fight malaria, and in Europe as early as
the 16th century, mercury was used to treat syphilis. Chemotherapy is the use for
the treatment of a disease of chemicals that have a specific effect on the cells of the
causative agent of the disease and do not damage human cells and tissues. The
foundations of scientific chemotherapy were formulated by P. Ehrlich. He received
the first chemotherapy drugs - salvarsan and neosalvarsan containing arsenic. For
several decades, they have been used in the treatment of syphilis.
Chemoprophylaxis is the use of chemicals to prevent infectious diseases.
The action of chemotherapeutic drugs on the cells of pathogens is based on the
similarity of their molecules with a number of substances necessary for the
metabolism of microorganisms: amino acids, vitamins, enzymes, etc. The drug is
absorbed by the bacterial cell instead of the component it needs and begins its
destructive effect. As a result of violation of the most important systems of the cell, it
dies (bactericidal action), and if the violations are weak, then a bacteriostatic effect is
noted.
An important step in the development of chemotherapy was the creation of
sulfanilamide preparations (streptocide, norsulfazol, sulfadimezin, etc.). They give a
good therapeutic effect in sore throat, purulent-inflammatory infections, intestinal
diseases. Synthetic chemotherapeutic drugs PASK (para-aminosalicylic acid), tibon,
ftivazide, etc. helped in the fight against tuberculosis. Currently, chemical antiviral
and antitumor drugs are being developed and used. Of great importance are
antibiotics - chemotherapeutic drugs of biological origin.
However, chemotherapy drugs have a number of negative properties. Influencing a
certain chain of metabolism, they can, along with the pathogen cell, also affect
human cells. As a result of treatment with chemotherapy drugs, a large number of
intermediate products with side effects accumulate in the human body. Cases of
changes in blood composition, cell mutations and other functional disorders of the
human body as a result of the use of chemotherapeutic drugs are described.

Chapter 10. Genetics of microorganisms - F. K. Cherkes

The ability of living organisms to maintain certain characteristics over many
generations is called heredity.
In the process of studying heredity, it turned out that each subsequent generation,
under the influence of various factors, can acquire features that distinguish them
from previous generations. This property is called variability. Thus, heredity and
variability are closely related.
The science that studies the heredity and variability of living organisms is called
genetics (from the Greek genos - birth).
Back in the 19th century, Charles Darwin proved that all existing species of living
organisms originated from a few forms through variability, and the resulting changes
that are inherited are the basis of the evolutionary process. Darwin's theory received
the highest rating from the classics of Marxism-Leninism. F. Engels regarded it as one
of the greatest discoveries of the 19th century.
The study of heredity and variability in higher organisms is associated with great
difficulties because of their long lifespan and the small number of offspring.
A convenient object for this study are microorganisms, which are characterized by a
short life cycle, rapid reproduction and the ability to produce numerous offspring. In
addition, they have a pronounced morphology that can be studied visually using a
light microscope. Microorganisms are biochemically active, which is easy to take into
account when using special nutrient media.
The ability of microorganisms to change their properties under the influence of
various factors (temperature, ultraviolet and X-ray radiation, etc.) allows them to be
widely used as a model in the study of heredity and variability.
The first object of genetic research was Escherichia coli, which is well cultivated in
the laboratory. It was also important that the morphological, cultural, and
biochemical properties of this bacterium are well studied. In the future, other
bacteria, as well as viruses, became the object of genetic research.
Studies of the genetics of microorganisms have shown that their role as a carrier of
genetic information is played by DNA (for some viruses, RNA).
The DNA molecule in bacteria consists of two strands, each of which is spirally
twisted relative to the other. When a cell divides, the filamentous spiral doubles;
each of the threads serves as a template or matrix on which a new thread is
built. Moreover, each strand that has arisen in the process of cell division contains a
newly formed double-stranded DNA molecule.
The composition of DNA includes four nitrogenous bases - adenine, guanine, cytosine
and thymine, the order of arrangement in the chain in different organisms
determines their hereditary information encoded in DNA.
The functional unit of heredity is the gene, which is a segment of the DNA
strand. The genes contain all the information regarding the properties of the cell.
The complete set of genes that a cell possesses is called the genotype. Genes are
divided into structural genes, which carry information about specific proteins
produced by the cell, and regulator genes, which regulate the work of structural
genes. For example, a cell produces those proteins that it needs under given
conditions, but when conditions change, regulator genes change the properties of
the cell, adapting them to new conditions.
Changes in the morphological, cultural, biochemical and other properties of
microorganisms that occur under the influence of external factors are
interrelated. For example, changes in morphological properties are usually
accompanied by changes in the physiological characteristics of the cell.
In the process of studying the variability of microorganisms, a special form of
variability, dissociation, was discovered. This type of variability was described by P.
de Kruy and J. Arkwright and is expressed in the fact that when some crops are sown
on solid nutrient media, the colonies are divided into two types: smooth, round,
shiny colonies with even edges - S-shape (from the English . smooth - smooth), and
flat, opaque colonies of irregular shape, with jagged edges - R-shape (from English
rough - rough). There are also transitional forms: M-forms (slimy) and g-forms
(dwarf).
Colonies belonging to the smooth S-form can, under certain conditions, change to
the R-form and vice versa, but the transition from the R-form to the S-form is more
difficult.
Dissociation is observed in a number of bacteria, in particular, in the pathogens of
anthrax, plague, etc.
Characterization of S- and R-forms of colonies
S-shape R-shape
Colonies are smooth, shiny, Irregular shaped colonies,
regular convex shape cloudy, rough
When growing in broth - Grow in broth as a precipitate
uniform turbidity Motile bacteria have flagella
Motile bacteria present may be absent
flagella No capsules
Capsular bacteria have biochemical properties expressed
capsule weakly
Biochemically active Most bacteria are less
disease-causing disease-causing
They are isolated more often in acute cases They are usually isolated in chronic
period of the disease form of the disease
Pathogenic bacteria are more often in the S-shape. An exception are the causative
agents of tuberculosis, plague, anthrax, in which the R-form is pathogenic (Fig. 26).
 Rice. 26. Growth of causative agents of tuberculosis on solid medium (R-form)

Changes that occur in bacterial cells can be non-inherited - phenotypic variability and
inherited - genotypic variability.

Phenotypic variability (modification)

Modification of microorganisms occurs as a response of the cell to the unfavorable

conditions of its existence. It is an adaptive response to external stimuli. The
modification is not accompanied by a change in the genotype, and therefore the
changes that have arisen in the cell are not inherited. When optimal conditions are
restored, the resulting changes are lost. Modification may relate to different
properties of microorganisms - morphological, cultural, biochemical, etc.
Morphological modification is expressed in changes in the shape and size of
bacteria. For example, when penicillin is added to a nutrient medium, the cells of
some bacteria elongate. The lack of calcium salts in the environment causes
increased sporulation in the anthrax bacillus. With an increased concentration of
calcium salts, the ability to form spores is lost, etc. With prolonged growth of
bacteria in the same medium, polymorphism arises due to the influence of the
products of their vital activity accumulated in it.
Cultural modification consists in changing the cultural properties of bacteria with a
change in the composition of the nutrient medium. For example, with a lack of
oxygen, Staphylococcus loses its ability to form a pigment. The miraculous rod at
room temperature forms a bright red pigment, but at 37 ° C the ability to form this
pigment is lost, etc.
Biochemical (enzymatic) modification. Each type of bacteria has a specific set of
enzymes, thanks to which they absorb nutrients. These enzymes are produced on
certain nutrient substrates and are predetermined by the genotype.
During the life of bacteria, not all genes responsible for the synthesis of the
corresponding enzymes usually function. In the genome of bacteria there are always
spare possibilities, i.e., genes that determine the production of adaptive
enzymes. For example, E. coli growing on a medium that does not contain the
carbohydrate lactose does not produce the enzyme lactase, but if it is subcultured on
a medium with lactose, it begins to produce this enzyme. Adaptive enzymes allow
you to adapt to certain conditions of existence.
Thus, modification is a way of adapting a microorganism to environmental
conditions, providing them with the opportunity to grow and multiply in changed
conditions. Acquired properties are not inherited, so they do not play a role in
evolution, but mainly contribute to the survival of microbial populations.

Genotypic (inherited) variability

Genotypic variability can arise as a result of mutations and genetic recombinations.

Mutations (from Latin mutatio - change) are inherited structural changes in genes.
Large mutations (genomic rearrangements) are accompanied by the loss or change
of relatively large sections of the genome - such mutations are usually irreversible.
Small (point) mutations are associated with the loss or addition of individual DNA
bases. In this case, only a small number of features change. Such altered bacteria can
completely return to their original state (revert).
Bacteria with altered traits are called mutants. Factors that cause the formation of
mutants are called mutagens.
Bacterial mutations are divided into spontaneous and induced. Spontaneous
(spontaneous) mutations occur under the influence of uncontrolled factors, that is,
without the intervention of the experimenter. Induced (directed) mutations appear
as a result of the treatment of microorganisms with special mutagens (chemicals,
radiation, temperature, etc.).
As a result of bacterial mutations, the following can be noted: a) changes in
morphological properties; b) change in cultural properties; c) the emergence of
resistance to drugs in microorganisms; d) loss of ability to synthesize amino acids,
utilize carbohydrates and other nutrients; e) weakening of pathogenic properties,
etc.
If a mutation leads to the fact that mutagenic cells acquire advantages over other
cells of the population, then a population of mutant cells is formed and all acquired
properties are inherited. If the mutation does not give the cell advantages, then the
mutant cells, as a rule, die.
Genetic recombinations . Transformation. Cells that are able to accept the DNA of
another cell during transformation are called competent. The state of competence
often coincides with the logarithmic growth phase.
Transduction is the transfer of genetic information (DNA) from a donor bacterium to
a recipient bacterium with the participation of a bacteriophage. Transducing
properties are mainly temperate phages. Reproducing in a bacterial cell, phages
incorporate a part of bacterial DNA into their DNA and transfer it to the
recipient. There are three types of transduction: general, specific and abortive.
1. General transduction is the transfer of various genes located in different parts of
the bacterial chromosome. At the same time, donor bacteria can transfer various
characteristics and properties to the recipient - the ability to form new enzymes,
resistance to drugs, etc.
2. Specific transduction is the transfer by the phage of only some specific genes
localized in special regions of the bacterial chromosome. In this case, only certain
characteristics and properties are transferred.
3. Abortive transduction - the transfer by a phage of a single fragment of the donor's
chromosome. Usually, this fragment is not included in the chromosome of the
recipient cell, but circulates in the cytoplasm. When the recipient cell divides, this
fragment is transferred only to one of the two daughter cells, and the second cell
receives the unchanged chromosome of the recipient.
With the help of transducing phages, a number of properties can be transferred from
one cell to another, such as the ability to form toxin, spores, flagella, produce
additional enzymes, drug resistance, etc.
Conjugation is the transfer of genetic material from one bacterium to another by
direct cell contact. Cells that donate genetic material are called donors, and those
that receive it are called recipients. This process is one-sided - from the donor cell to
the recipient cell.
Donor bacteria are designated F+ (male type) and recipient bacteria are designated
F- (female type). When F+ and F- cells come close together, a cytoplasmic bridge
appears between them. The formation of the bridge is controlled by factor F (from
the English fertility - fertility). This factor contains genes responsible for the
formation of genital villi (sex-pili). The function of a donor can be performed only by
those cells that contain factor F. The recipient cells are deprived of this factor. When
crossing, the F factor is transferred from the donor cell to the recipient. Having
received the F factor, the female cell itself becomes a donor (F +).
The conjugation process can be interrupted mechanically, for example by shaking. In
this case, the recipient receives incomplete information contained in the DNA.
The transfer of genetic information by conjugation is best studied in
Enterobacteriaceae.
Conjugation, like other types of recombination, can occur not only between bacteria
of the same species, but also between bacteria of different species. In these cases,
recombination is called interspecific.
 Plasmids

Plasmids are relatively small extrachromosomal DNA molecules of a bacterial

cell. They are located in the cytoplasm and have a circular structure. The plasmids
contain several genes that function independently of the genes contained in the
chromosomal DNA.
A typical feature of plasmids is their ability to self-reproduce (replicate).
They can also move from one cell to another and include new genes from the
environment. Plasmids include:
Prophages , causing a number of inherited changes in a lysogenic cell, such as the
ability to form a toxin (see transduction).
F-factor , which is in an autonomous state and takes part in the conjugation process
(see conjugation).
R-factor , which gives the cell resistance to drugs (for the first time, the R-factor was
isolated from Escherichia coli, then from Shigella). Studies have shown that the R-
factor can be removed from the cell, which is generally characteristic of plasmids.
The R-factor has intraspecific, interspecific and even intergeneric transmissibility,
which can cause the formation of difficult-to-diagnose atypical strains.
Bacteriocinogenic factors (col-factors) , which were first discovered in the culture of
Escherichia coli (E. coli), in connection with which they are called colicins. Later they
were also found in other bacteria: vibrio cholerae - vibriocins, staphylococci -
staphylocins, etc.
Col-factor is a small autonomous plasmid that determines the synthesis of protein
substances capable of causing the death of bacteria of its own species or a closely
related one. Bacteriocins are adsorbed on the surface of sensitive cells and cause
metabolic disorders, which leads to cell death.
Under natural conditions, only a few cells in a population (1 per 1000) spontaneously
produce colicin. However, under some influences on the culture (treatment of
bacteria with UV rays), the number of colicin-producing cells increases.

The practical meaning of variability

Even Pasteur artificially obtained irreversible changes in the pathogens of rabies and
anthrax and prepared vaccines that protect against these diseases. Further research
in the field of genetics and variability of microorganisms made it possible to obtain a
large number of bacterial and viral strains used to obtain vaccines.
The results of the study of the genetics of microorganisms were successfully used to
elucidate the patterns of heredity in higher organisms.
A new branch of genetics, genetic engineering, is also of great scientific and practical
importance.
Genetic engineering methods make it possible to change the structure of genes and
include genes of other organisms responsible for the synthesis of important and
necessary substances into the bacterial chromosome. As a result, microorganisms
become producers of such substances, the production of which by chemical means is
a very difficult and sometimes even impossible task. In this way, such medicines as
insulin, interferon, etc. are currently obtained. Using mutagenic factors and
selection, mutants-producers of antibiotics were obtained, which are 100-1000 times
more active than the original ones.

Control questions

1. What is the functional unit of heredity?

2. What is the role of regulatory genes?
3. What is dissociation and what forms of dissociation do you know?
4. What does phenotypic variability mean and what properties can it be expressed
in?
5. What does genotypic variability mean and in what forms can it be expressed?
6. What are plasmids?
7. What is the practical significance of variability?

Chapter 11. The doctrine of infection - F. K. Cherkes

An infection or an infectious process (from Latin infectio - to infect, pollute) is a set of
phenomena that arise and develop in a macroorganism during the introduction and
reproduction of pathogenic microorganisms in it.
Manifestations of infection are diverse, depending on the properties of the
microorganism, the state of the macroorganism and environmental conditions.
The extreme degree of severity of the infectious process is an infectious disease.
Infectious diseases have been known for a long time. The peoples of ancient times
could not have a correct idea of the causes of these diseases and considered them
"God's punishment." However, even Hippocrates, and in the 16th century D.
Frakaetoro and others already suggested that contagious diseases are associated
with some creatures that are transmitted from the sick to the healthy.
In the middle of the 19th century, L. Pasteur, R. Koch, I. I. Mechnikov, D. I. Ivanovsky
and other scientists established that microorganisms are the causative agents of
infectious diseases.
The relationship between microorganisms and the macroorganism is a symbiosis,
which is characterized by the following forms: mutualism, commensalism and
parasitism.
Mutualism (from Latin mutuus - mutual) is a cohabitation that is beneficial for both
cohabitants. For example, lactic acid bacteria live at the expense of the
macroorganism and are antagonists of the putrefactive microflora of the human
intestine.
Commensalism (from the French commensal - cohabitant, companion) is a form of
cohabitation in which one cohabitant (microorganism) lives at the expense of the
host (macroorganism), without harming him. Commensal microbes include
representatives of the normal microflora of the body, such as non-disease-causing
staphylococci, Escherichia coli, etc. However, when the macroorganism falls into
favorable conditions or when these microorganisms from their natural habitat enter
other organs, they can cause disease.
Parasitism (from the Greek parasitos - parasite) characterizes the relationship when
one organism (parasite) lives at the expense of another (host) and harms it.
The transition of microorganisms from saprophytism to parasitism was accompanied
by a change in a number of their properties. The basis of such changes was the
constant variability of microorganisms, followed by the natural selection of those
forms that are more adapted to the new conditions of life. First, parasites developed
that did not completely lose the ability to exist independently in the environment
(facultative). Then obligate (obligate) parasites appeared, reproducing only in the
body of their host.
The evolutionary nature of the formation of parasitism in microorganisms is also
manifested in the fact that some of their species have acquired the ability to live and
reproduce only in the body of a certain species. For example, the causative agents of
typhoid fever, gonorrhea parasitize only in the human body.
Later, by differentiating, microorganisms adapted to certain organs and tissues. For
example, pneumococci mainly affect the mucous membranes of the respiratory
tract, gonococci - the mucous membrane of the genital organs, pathogens of typhoid
and dysentery - the intestinal mucosa, etc.

Pathogenicity and virulence of microorganisms

The ability of microorganisms to cause pathological processes in the macroorganism,

i.e., diseases, is called pathogenicity (from Latin pathos - suffering, genos -
birth). Microorganisms with this ability are called pathogens. Pathogenicity is a
genetically determined species trait. Most pathogenic microorganisms are
characterized by specificity - the ability of a given type of microbe to cause a specific
disease. For example, cholera is caused by vibrio cholerae, gonorrhea by gonococcus,
etc.
Different strains of the same species may have different pathogenic effects. The
degree or measure of pathogenicity is called virulence.
Virulence, like any property of a microorganism, can change. These changes are
either phenotypic in nature, or are the result of disturbances in the cell genome -
then they are inherited. Phenotypic changes leading to a weakening of virulence
occur when microorganisms are exposed to unfavorable conditions, for example,
when they are exposed to various physical and chemical factors. These changes are
restored, virulence increases again when microbes enter favorable conditions of
existence. A stable decrease in virulence can be obtained with prolonged action of
various substances. So, Calmette and Guerin received BCG - a live vaccine from
tuberculosis bacteria. For 13 years, scientists have subcultured the culture on media
containing bovine bile. At the same time, selection (selection) of avirulent bacterial
cells with high resistance to bile took place. Their number in the original culture was
small (their properties were not manifested in the population).
Virulence can be enhanced by passage of microorganisms through susceptible
animals. In this case, the selection of virulent individuals of the population takes
place.
The virulence of microorganisms is due to their ability to adhere (adhere), colonize
(reproduce), invade (penetrate tissues, macroorganism cells) and suppress
phagocytosis.
Adhesion - the ability to be adsorbed on certain cells of the host organism that are
sensitive to a given microbe. It is due, on the one hand, to the surface structures of
the microbial cell (drank, etc.), and on the other hand, to the presence of
macroorganism cell receptors that are capable of joining with the microbial cell.
Colonization can be on the surface of cells to which microbes have adhered (for
example, V. cholerae multiply on enterocytes) or within cells into which adherent
microbes penetrate (for example, dysenteric bacilli multiply in cells of the large
intestine).
Invasiveness is associated with the ability of microbes to produce enzymes that
disrupt (increase) the permeability of connective and other tissues. These enzymes
include: a) hyaluronidase (spreading factor), which destroys the hyaluronic acid of
the connective tissue and thereby promotes the penetration of microbes into
tissues; b) neuraminidase, which cleaves neuraminic acid from glycoproteins,
glycolipids, polysaccharides, which are part of different tissues, and thus increases
their permeability.
Suppression of phagocytosis is carried out by bacterial capsules. The substances that
make up the capsules of various microorganisms are not the same, and their
functions are also different. So, the polypeptide of the capsules of the anthrax
pathogen protects it from capture by phagocytes; Pseudomonas aeruginosa
polysaccharide inhibits both capture and intracellular digestion of bacteria.
In addition to these factors, microbes are protected from phagocytosis by certain
enzymes. For example, staphylococcal coagulase promotes plasma clotting, which
leads to the formation of a protective "sheath" around the microbial cell; fibrinolysin
dissolves fibrin, thereby promoting the spread of microbes.
Of particular importance in virulence is the ability of microorganisms to synthesize
toxins (poisons). Toxins formed by microorganisms are divided into two groups -
exotoxins and endotoxins.
Exotoxins are products of microbial metabolism that are secreted into the
environment. They are of protein origin, which causes their low resistance to
external influences. The exception is the neurotoxin of botulism bacillus,
enterotoxins of staphylococcus aureus, vibrio cholerae, which can withstand short-
term boiling.
Microorganisms that form exotoxin are usually localized at the site of penetration (in
the gate), and the exotoxin produced by them circulates in the macroorganism, for
example, tetanus, diphtheria, etc.
Exotoxins are characterized by high toxicity and pronounced specificity -
organotropism. Each type of toxin affects specific organs or tissues. For example,
tetanus toxin affects the nervous system, and diphtheria toxin affects the muscles of
the heart, etc.
According to their biological activity, toxins are not the same: some of them
completely determine the clinical picture of the disease, for example, tetanus,
diphtheria, botulinum toxins. Others take a more limited part in the infectious
process, cause atypical reactions in terms of clinical manifestations, for example,
hemolytic toxins of staphylococci, Escherichia coli, etc.
Exotoxins diffuse into the environment. They are obtained by sowing a toxigenic
culture in a liquid nutrient medium and growing it under conditions of maximum
accumulation of the toxin. After filtration through bacterial filters, a filtrate
containing exotoxin is obtained.
At present, a number of exotoxins have been obtained in pure form and are well
studied. Purified toxins are more toxic.
The toxic effect of exotoxins is removed if the active center of the poison is blocked
by acting on it with chemical and physical factors. Under the action of 0.4% formalin,
keeping at 39-40 ° C for 3-4 weeks, exotoxins lose their toxic properties, but retain
their antigenic properties. Such drugs are prepared as vaccines and are called
toxoids.
Endotoxins are a lipopolysaccharidoprotein complex closely associated with the cell
of a microorganism. They are not specific. The clinical picture caused by endotoxins
of different microorganisms is of the same type: the reaction of the body is usually
accompanied by general symptoms of intoxication - fever, headache, etc.
The close relationship of endotoxin with the cells of the microorganism determines
its resistance to temperature and other external factors. To obtain endotoxin, it is
necessary to destroy the cell of the microorganism.
Properties of exo- and endotoxins
Exotoxins Endotoxins
Protein nature Lipopolysaccharidoprotein complex
Diffuse out of the cell into Associated with the microbial body
cell environment
Highly toxic Low toxic
Selectively act on Cause general phenomena
organs and tissues (specific) intoxication
Thermolabile Thermostable
under the influence of formalin under the influence of formalin
turn into anatoxin partially neutralized
Formed mainly Formed mainly
Gram-positive bacteria Gram-negative bacteria
The action of the toxin is determined on animals sensitive to this toxin. For example,
diphtheria toxin is tested on guinea pigs, botulinum toxin is tested on white mice,
etc.
To determine the virulence and strength of the toxin (toxicity) of microbes, the
symbols are used: DLM, DCL, LD 50 .
DLM (Dosis letalis minima) - the smallest dose of microbes or toxin that kills most
experimental animals. DCL (Dosis certe letalis) - the smallest dose of microbes or
toxin that kills all animals taken in the experiment. LD 50 (Dosis letalis) - the dose of
microbes or toxin, which leads to the death of 50% of experimental animals.
The doses that determine the virulence or strength of the toxin depend on the type
and strain of the microbe, the type of toxin, and also on the route of
administration. To determine the strength of the toxin, a series of serial dilutions are
made from the test material, each of them is tested on a group of animals sensitive
to this type of toxin. To obtain comparative results in determining doses (DLM, DO,
LD 50 ), the study is carried out on animals of the same species and sex, having the
same body weight.

The role of the macroorganism in the infectious

process

The emergence of an infectious disease largely depends on the reactivity of the

macroorganism, its readiness to neutralize pathogenic microbes and poisons that
have entered its internal environment. In this case, the following factors play an
important role.
Age. The value of age is determined by the physiological characteristics of the
organism, in particular the nature of metabolism. It is known that children are more
sensitive to the causative agents of certain infections. There are so-called "children's
infections" - scarlet fever, whooping cough, measles, chicken pox, mumps, etc.
Elderly people have a hard time with pneumonia. Along with this, there are
infectious agents that equally affect people of any age, such as the influenza virus.
The state of the nervous system. It has been established that depression of the
nervous system contributes to the occurrence and more severe course of infectious
diseases, since the activity of protective mechanisms in the macroorganism is
reduced.
State of the endocrine system. In people suffering from endocrine diseases
(diabetes, dysfunction of the thyroid gland, etc.), purulent-inflammatory processes
often occur, which is also the result of a decrease in the body's defenses.
Nutrition. With malnutrition, a person often develops infectious diseases. The result
of malnutrition is increased morbidity and mortality from tuberculosis, cholera,
dysentery and other infections in a number of capitalist countries, where part of the
poor population is systematically starving.
Proteins and vitamins are essential in food. So, starvation, and sometimes just an
insufficient amount of proteins, leads to a violation of protein metabolism. This
entails a decrease in the synthesis of immunoglobulins, a decrease in the activity of
phagocytes. The loss of phagocytic activity of cells also occurs with a lack of vitamin
A, which often leads to the occurrence of inflammatory processes on the skin and
mucous membranes. Lack of B and C vitamins increases susceptibility to tuberculosis,
diphtheria, streptococcal, staphylococcal and other diseases.
In the body's resistance to pathogens of many infections, a large role belongs to
trace elements, the lack of which in food leads to metabolic disorders, increased
susceptibility to infectious diseases.
"Normal microflora" plays a significant role in the implementation of the protective
functions of the body. Representatives of this microflora are often pronounced
antagonists of pathogenic microbes. For example, Escherichia coli - a permanent
inhabitant of the large intestine - inhibits the development of typhoid and other
intestinal pathogens.

The influence of the environment on the emergence

and development of the infectious process

Refrigeration reduces resistance to many pathogenic and opportunistic

microorganisms. For example, the action of cold and at the same time moist air
reduces the resistance of the mucous membrane of the respiratory tract, which leads
to disease.
The development of infectious diseases can be promoted by overheating, prolonged
and intense exposure to sunlight, ionizing radiation in high doses, occupational
hazards (high temperature in hot shops, exposure, chemical poisoning, lack of
oxygen, physical and mental overwork, etc.). Poor sanitary and hygienic conditions
reduce the overall resistance of the body
Thus, the ratio of the virulence of microorganisms, the state of the macroorganism
and environmental conditions determine the possibility of occurrence and the nature
of the course of the infectious process.

Mechanism of transmission

The source of infectious agents are humans and animals. Sick people and animals are
of the greatest importance, however, convalescents (convalescents), patients with a
latent form of the disease, microcarriers can be a source.
All human infectious diseases are divided into two groups according to the nature of
the sources: anthroponoses, in which the source of pathogens is a person, and
zoonoses - diseases inherent in animals, but to which humans are also susceptible.
The mechanisms of transmission of infectious agents are different, but certain for
each type of microorganisms and are due to localization in the body of the patient
(or carrier), as well as by isolation. In accordance with the primary localization of
pathogens in the body, 4 types of transmission mechanisms are distinguished:
1) fecal-oral - pathogens are localized in the intestines (typhoid fever, dysentery,
cholera), transmitted by the alimentary route - with food, water;
2) airborne - pathogens are localized in the respiratory tract (flu, whooping cough,
etc.), transmitted by airborne, airborne dust;
3) transmissible - pathogens are localized in the circulatory system (malaria, typhus,
relapsing fever, etc.), transmitted by blood-sucking insects;
4) contact: a) direct - the transmission of pathogens occurs through direct contact
(venereal disease); b) indirect - through contaminated environmental objects (toys
that may contain pathogens, for example, dysentery). Pathogens are localized on the
skin, mucous membranes, wound surfaces.
Of great importance for the occurrence of an infectious disease is the place of
penetration of the pathogen - the entrance gate, as well as the infectious dose.
Entrance gates are those organs and tissues of the host organism through which
pathogenic microorganisms penetrate. For example, the causative agent of typhoid
fever causes the disease only when it enters through the mouth, and gonococcus -
when it enters the mucous membrane of the genital tract, or the conjunctiva of the
eye. If these pathogens enter the body in a different way, not through their entrance
gates, then the disease does not develop, and the microorganisms die.
However, some microorganisms (for example, plague, tularemia, anthrax) can cause
disease by entering the host organism in various ways. In these cases, the entrance
gate determines only the form of the clinical course (cutaneous form, pulmonary,
intestinal, etc.).
For the occurrence of an infectious disease, pathogens must enter the body in a
certain "critical dose". Its value is not the same for various pathogens. For example,
for the onset of a disease with dysentery, the infectious dose is on average
10 2 virulent pathogens, typhoid fever - 10 5 , cholera - 10 7 , etc.

Forms of the infectious process

Depending on the route of penetration of the pathogen into the host organism,
exogenous and endogenous infections are distinguished.
Exogenous infections occur as a result of the entry of pathogens from the
environment.
With endogenous (autoinfection) infection, the pathogens are in the body as part of
the obligate or transient flora. When the protective properties of the body are
weakened, they can cause the onset of the disease.
According to the duration of the course, acute and chronic infections are
distinguished. Acute ones are characterized by a relatively short-term (from 1 week
to 1 month) course (for example, influenza, measles, cholera, typhoid fever, etc.),
chronic - a protracted course (for months - years) (for example, malaria, syphilis,
tuberculosis, brucellosis , leprosy).
If the infection is caused by one type of pathogen, such an infection is called a
monoinfection. When the body is infected simultaneously with 2-3 different
pathogens (for example, diphtheria bacillus and streptococcus), they speak of a
mixed infection.
A secondary infection is distinguished from mixed infections, when an infection
caused by another pathogen (for example, staphylococcus or streptococcus) joins
the main disease (for example, influenza).
Reinfection is a condition when a second disease has arisen as a result of a new
infection with the same type of pathogen. If the disease resumed before recovery as
a result of infection with the same pathogen, they speak of superinfection. Such
infections are observed with syphilis, gonorrhea.
Relapse - the return of symptoms of the disease (relapsing fever, malaria), which
occur without re-infection due to pathogens remaining in the body.
Some infectious diseases can be hidden, without clinical manifestations. Such forms
of infection are called latent (for example, tuberculosis can be asymptomatic).
One of the forms of infection that occurs without signs of illness is microcarriage. It is
formed more often after the illness, when clinical recovery occurs, but the pathogens
continue to remain in the body of the sick person and are released into the
environment (for example, carriage of typhoid, dysentery bacilli). In some cases,
microcarriage develops in healthy individuals who have been in contact with sick or
even carriers of pathogenic microorganisms.
Depending on the localization of pathogens in the patient's body, a focal infection is
distinguished, in which microbes are located in the local focus, do not spread beyond
it (for example, tonsillitis, furunculosis), and generalized, when the forces of
aggression of microorganisms exceed the strength of the host's defense mechanisms
and pathogens from the local outbreaks spread throughout the body. The condition
when infectious agents circulate for a certain time in the blood, but do not multiply
in it (for example, typhoid fever), is called bacteremia.
In the event that the pathogen is in the blood for a long time, accumulates there and
even multiplies, sepsis or septicemia occurs (from the Latin sepsis - pus). Such a flood
of microorganisms occurs with plague, anthrax. Sepsis is also caused by pyogenic
cocci. A special feature of sepsis is that the clinical picture does not depend on the
type of pathogen.
The formation of purulent foci in various organs as a result of sepsis is called
septicopyemia. The circulation of a toxin in the blood is called toxinemia, the
circulation of viruses is called viremia.
 The dynamics of the development of an infectious
disease

The dynamics of the development of an infectious disease consists of several

periods.
The incubation period is the time from the introduction of a pathogenic microbe to
the appearance of the first signs of the disease. During this period, reproduction and
accumulation of pathogens and their toxins occurs. Its duration is not the same for
different diseases, but it is certain for individual infectious diseases. For example, the
average duration of the incubation period for typhoid fever is 14 days, scarlet fever
and diphtheria - 5-7 days, whooping cough - 9 days, measles - 10-11 days, toxic
infections occur a few hours after infection, influenza - after 2-3 days.
Clinical manifestations of the disease of a number of infections occur after a very
long incubation period (for example, leprosy develops several years - up to 30 years!
- after infection).
Prodromal period - the period of precursors occurs after the incubation period and is
manifested by symptoms common to various diseases (headache, weakness,
malaise, fever). The duration of this period is small - from several hours to 3 days.
The period of development of the main clinical phenomena is characterized by the
manifestation of a variety of symptoms, but specific to each disease, which depends
on the type of pathogen. This period is often accompanied by: fever, dysfunction of
the respiratory system, digestion, the appearance of vascular phenomena
(sometimes the appearance of a rash), a change in the blood picture, etc. The
duration of this period depends on the type of infection.
The change of periods of the disease is of great importance for laboratory
diagnostics, since each of them is characterized by a certain localization of the
pathogen and ways of its isolation from the body, which determines the type of
material taken and the tactics of laboratory research.
The period of recovery (reconvalescence) is characterized by the extinction of painful
phenomena, the gradual restoration of the physiological functions of the body.
Spread of infectious diseases . Infectious diseases are characterized by
contagiousness and can occur in the form of epidemics - mass diseases associated
with each other. Mass diseases spreading to several countries and continents are
called a pandemic. Infectious diseases that occur in isolated cases are called
sporadic. Diseases that occur only in a particular area are called endemic. They are
usually caused by the presence of a carrier and other reservoirs of the infectious
agent.
In the USSR over the past decades, infectious diseases rarely occur in the form of
epidemic outbreaks.
A number of diseases, such as smallpox and plague, have been eradicated. The
incidence of diphtheria, whooping cough, etc. has been sharply reduced. All this is
the result of preventive measures constantly taken by the Soviet public health
service.

Control questions

1. What is an infectious process?

2. What is pathogenicity and virulence?
3. How are exo- and endotoxins differentiated?
4. What are the mechanisms of transmission of infectious agents?
5. What is the role of the macroorganism and environmental factors in the
occurrence and course of the infectious process?
6. What is the dynamics of the development of an infectious disease?

Biological research methods

Biological methods are called research methods carried out on laboratory

animals. The purpose of these studies: the isolation of microorganisms from the test
material, especially in cases where the pathogen cannot be detected by seeding, for
example, in viral diseases, rickettsiosis, etc.; isolation of a pure culture from material
contaminated with other microorganisms that do not allow the desired pathogen to
multiply on an artificial nutrient medium; determination of some properties of
isolated microorganisms (virulence, etc.).
Experimental infection makes it possible to reproduce some infectious diseases and
solve a number of issues related to infection and immunity, the effectiveness of
immunobiological preparations, the determination of their reactivity and preventive
(preventive) properties.
When choosing a laboratory animal, it is necessary to take into account the degree of
its susceptibility to the infection under study and to determine whether this
pathogen causes the disease in natural conditions.
Types of laboratory animals
For experimental infection, white mice, rats, guinea pigs and rabbits are most often
used. Monkeys, cats, dogs, horses, small and large cattle, wild animals (hamsters,
ground squirrels, wild rats, voles), as well as birds (chickens, pigeons, etc.) serve as
laboratory animals for some special studies.
Currently, purebred (inbred) animals are often used, obtained by inbreeding
(brothers, sisters) for 20-40 generations with the selection of animals according to a
certain trait. The genetic homogeneity of mice ensures the uniformity of responses,
so a smaller number of animals can be taken into the experiment.
There are special laboratories in which research is conducted on microbial animals
(they are kept under aseptic conditions). The science that studies the microbial life of
macroorganisms is called gnotobiology. One of the objectives of the study is to
reveal the role of normal and altered microflora in the physiological and pathological
processes of the macroorganism.
Maintenance of laboratory animals
Animals used for biological experiment are kept in special conditions. For this, there
are special rooms - vivariums. In the vivarium, laboratory animals are kept in cages or
jars mounted on racks. Each type of laboratory animal is kept separately. The
vivarium room should be warm, bright and dry. Animals are bred in special nurseries
in conditions close to natural. Animals arriving from the nursery are previously
placed in a quarantine department.
The vivarium should have a number of special and ancillary sections: quarantine, for
keeping healthy animals, a room for autopsies, and a kitchen (a room for cooking).
In a well-arranged vivarium, there should be two exits - to isolate part of the room in
the event of a spontaneous infection, that is, an infection that occurs without
artificial infection.
The general condition of the animals is monitored daily. All changes in the state of
animals (lethargy, lack of appetite, the presence of infiltrates) or their death are
noted in a special registration journal.
Care of laboratory animals and cleaning of the vivarium . Cleaning the vivarium
requires special clothing: a bathrobe, an apron, rubber gloves, a scarf and
slippers. When working with animals intended for experimentation with pathogens
of dangerous infections, they additionally put on an oilcloth apron, rubber boots,
oversleeves, a mask, and goggles.
Cleaning begins with an inspection of cages and jars to identify all diseased and dead
animals. Then they take out the drinkers and feeders, clean them of food debris,
wash them thoroughly, and boil the glass ones. After that, special metal scrapers
clean the cage of debris and install drinkers and feeders in place. Once a week, cages
and jars should be washed with hot water, disinfected or autoclaved. After cleaning
the cells, they begin to clean the room.
At the end of the cleaning, all the collected garbage is burned in the oven. Dead
animals are opened and also burned under the control of the person responsible for
this (doctor or laboratory assistant). Vivarium workers thoroughly disinfect and wash
their hands.
Animal feeding . Proper and complete feeding of animals is very important. Rational
nutrition standards have been developed for each type of animal, which are
approved by the USSR Ministry of Health. These norms include cereal mixtures, root
crops, hay, bran, vegetables, animal products, etc.
The quantity and nature of the products depends on the type of animal and the
purpose of the experiment.
Poor maintenance and malnutrition of animals lead to a decrease in their resistance
to infection, which affects the results of experiments.
Animal selection and preparation for experiment
Each type of animal is best obtained from one nursery. For the experiment, animals
of a certain breed, the same weight, which approximately corresponds to the same
age, and the same sex are selected.
Selected animals are placed in clean cages or jars. Hay or sawdust is placed at the
bottom of the cage. Small animals, such as white mice, can be planted in 5-8
pieces. Animals are transferred to the laboratory in cages or jars.
Animals are weighed before the experiment. For weighing it is more convenient to
use decimal (without weights) or tared scales.
For some experiments, animals are thermometered daily. The temperature is
measured with a special mercury thermometer, which is disinfected before use,
wiped dry and lubricated with petroleum jelly. After use, the thermometer is
disinfected again. The thermometer is inserted into the lumen of the rectum, and the
depth of insertion for each type of animal should always be the same, for example,
for a guinea pig - 3.5 cm. For this purpose, a limiter is put on a glass column above
the tank - a ring. The temperature measurement is carried out within 5 minutes. The
results are recorded in a special journal.
Animal labeling . Each animal taken in the experiment is marked. The type of
marking depends on the type of animal. For example, white rats and mice are
marked with paints: a saturated alcohol solution of fuchsin, picric acid, gentian violet,
potassium permanganate, chrysoidine, etc. They paint different parts of the body:
head, back, front right paw, back left paw, etc. Places of coloring mean the
conditional numbers of animals, for example, the color of the front right paw is No.
3. Using a combination of color and coloring place, several hundred animals can be
marked.
For marking rabbits and guinea pigs, metal plates are used, on which the number is
engraved. These plates have an expanded central part and two outgoing spikes to
the sides. Before use, the plates are immersed in alcohol for 2-3 hours. The ear of the
experimental animal is wiped with alcohol, ether, and only after that the spikes are
threaded into the ears of the animal, brought to the inner surface of the ear and
bent (Fig. 27). Birds (hens and pigeons) are usually put on an aluminum ring on the
right foot, on which a number is affixed.

Rice. 27. Plate with a number for labeling laboratory animals

 Experimental infection of animals
Animal immobilization . Before the experiment, the animal must be removed from
the cage. To prevent the animal from biting or scratching the experimenter, there
are various methods: mice and rats are taken by the end of the tail, rabbits and
guinea pigs are taken by the back skin.
Then the animal is fixed, i.e., its mobility is limited, and the most convenient position
for manipulation is created.
There are various devices for fixing animals: boards of various types, boxes, boxes,
plates, machines, etc.
The type of fixation depends on the nature of the manipulation. For example, when
introducing material into a rabbit ear vein, a wooden box can be used, the front wall
of which has a hole for the head and consists of two halves, one of which is
retractable. The animal is placed in a box, the head is pushed into the hole and fixed
with a retractable half of the wall (Fig. 28). To fix the animal in a supine position, a
board is used, which must correspond to the size of the animal's body. On the side of
the board at the level of the paws, four rings or hooks are strengthened. The animal
is placed belly up or down, depending on the nature of the manipulation, loops are
put on the paws, made of a gauze bandage or rope, the other end of which is fixed
on a hook or in a ring.

Rice. 28. Box for fixing the head of a rabbit

Simpler forms of immobilization are also used: the animal can be swaddled tightly in
a towel or gown, or kept in a position that restricts movement. For example, the
assistant takes the guinea pig with his right hand by the hind legs, with his left hand
by the chest. A white mouse can be taken by the tip of the tail, placed on the table,
and when the tail is stretched when the animal moves, grab the skin of the head or
the back of the head between the ears with the left hand. Rats are fixed in the same
way, but usually using forceps.
You can work with small animals, such as mice, alone, without an assistant: to do
this, grab the skin of the back of the mouse's head between the ears with the index
and thumb of the left hand and, turning the hand with the palm up, hold the left paw
and tail between the little finger and the soft part of the palm. The necessary
manipulations are performed with the free hand (Fig. 29).

Rice. 29. Fixing the mouse without an assistant

Preparation of instruments for the experiment . Syringes, needles, tweezers,

scalpels intended for infecting animals are sterilized by boiling. Syringes are
preliminarily disassembled, placed in the sterilizer mesh, boiled for 30 minutes, then
sterilely collected with tweezers.
An animal can be infected with native material: pus, sputum, blood, emulsion from
organs, etc., and a suspension of microbes, the density of which is determined using
an optical standard.
The material intended for injection is placed in a sterile container. To avoid splashing
of the test material, fill the syringe with extreme care. To do this, the needle hole is
lowered below the surface of the material and carefully, slowly draw a slightly larger
amount of material into the syringe barrel than required. Then the syringe is turned
vertically (needle up) and, covering the tip of the needle with sterile cotton, air
bubbles and excess material are pushed out of the syringe. Dirty cotton wool is
dropped with tweezers into a disinfectant solution.
 Control questions

1. What does the term "biological method" mean?

2. What should be considered when choosing a laboratory animal?
3. What animals are usually used for the experiment?
4. What requirements should the vivarium room meet? What departments does the
vivarium consist of?
5. How are laboratory animals marked?
6. Why are animals fixed? What devices are available for this?
7. How should the syringe be filled to avoid splashing of material?

Exercise

Sterilize the syringe. Collect it. Fill with isotonic sodium chloride solution, cover the
tip of the needle with cotton and push out air bubbles and excess material (isotonic
sodium chloride solution) from the syringe.
Methods of infection
There are the following ways of introducing the material: subcutaneous, intradermal,
dermal, intramuscular, intraperitoneal, intravenous, oral (through the mouth),
intranasal (through the nose into the respiratory tract); eye injection, central nervous
system injection, etc.
The area where an injection, incision or puncture is to be made is called the
operating field. The most commonly used material is subcutaneous injection. For the
introduction of material into animals intradermally, subcutaneously or cutaneously,
it is necessary to free the surgical field from wool. Wool can be cut (for this, scissors
with loaded edges are used to avoid cuts), plucked or removed with a razor,
stretching the skin for this with the thumb and forefinger of the left hand (so as not
to injure the skin).
In some cases, a depilatory *
 is used to completely remove hair , but it can only be used 2-3 days
before the experiment, as it sometimes causes skin irritation. After applying the
depilatory, the skin is cleaned of wool, washed with warm water and lubricated with
petroleum jelly.
*
 ( Composition of the depilatorium: 7 parts of talc, 7 parts of white flour, 1 part of soap powder and 3 parts of sodium
sulfide alloy. )

With any method of hair removal, the surgical field is disinfected with alcohol or an
alcoholic solution of iodine before injection.
Subcutaneous administration . The injection site is chosen depending on the type of
animal. In rabbits, the material is usually injected under the skin of the back, in
guinea pigs - under the skin of the abdomen or flank, in mice and rats - under the
skin of the back or neck.
For subcutaneous injection, the skin of the animal is captured in a fold, the needle is
injected at the base of the formed fold, it is slowly inserted to half, slightly deviating
to the side so that the injected material does not spill out, then the fold is lowered,
cotton wool moistened with alcohol or an alcoholic solution of iodine is applied to
the needle, and withdraw the needle quickly.
Intradermal administration . This method is most often used for allergic tests and
the introduction of a toxin (for example, diphtheria). It is necessary to have thin,
sharp needles (No. 18-20) with a short beard. It is convenient to use a tuberculin
syringe. The skin, freed from wool, is stretched with the thumb and forefinger; the
needle is inserted at an acute angle with the cut up. A correctly inserted needle
shines through the epidermis. The test material is injected slowly; a bubble forms at
the injection site, which quickly resolves.
skin method . The surface of the skin after removing the hair is scarified * . The test
material is applied to the scarified area and rubbed with a spatula or a sterile
stick. The animal must be immobilized until the applied material dries.
*
 ( Scarification is damage to the surface of the skin, which is produced by any pointed instrument: a scalpel, a needle or
a special scarifier. )

Intramuscular administration . The material is injected into the thickness of the

muscle, more often into the muscle of the thighs. To do this, it is convenient to grab
the muscle with the index and thumb of the left hand; the right hand at a right angle
insert the needle.
Intraperitoneal administration . With this method of administration, the animal
should be held vertically with its head down (to move the internal organs to the
diaphragm). The needle is inserted into the lower abdomen, slightly receding from
the midline - this position reduces the possibility of injuring the intestines. Rabbits
and guinea pigs are preliminarily made with a small incision in the upper layer of the
skin. Use needles with a short beard, which are injected at an acute angle. After
puncturing the skin, the syringe is transferred to a horizontal position and the needle
is pushed at a right angle to the abdominal wall until a "failure" is felt, then it is again
transferred to a vertical position and the test material is injected.
Intravenous administration . With this method, the choice of vein depends on the
type of animal: in rabbits, the material is injected into the marginal vein of the ear, in
mice and rats - into the vein of the tail, in guinea pigs - directly into the heart, since
their veins are inaccessible for injections.
Long-barbed needles are used for intravenous injections. Animals must be
immobilized. After fixing the animal, to better identify the vein, the skin is wiped with
cotton wool moistened with xylene or warm water. You can also click on the area of
the skin where the vein is located with your fingertips and press the vein at the root
of the tail or ear - these manipulations contribute to the swelling of the vein. Then
the experimenter fixes the injection site with his left hand, pierces the skin with a
syringe in his right hand, and at a very sharp angle, almost parallel to the vein, inserts
the needle with the cut up. If the needle does not enter the vein, then when the
piston is pressed, difficulty is felt and a white bubble appears at the injection site,
formed by fluid that has entered the tissue. In this case, the needle is removed and a
new injection is made closer to the root of the tail (if injected into the vein of the tail)
or to the base of the ear (if injected into the vein of the ear). When a needle enters a
vein, the fluid passes freely into it. After the material is introduced, the needle is
pressed slightly below the injection site, a piece of sterile cotton wool is applied to
this place, after which the needle is removed. The injection site is wiped with alcohol
or an alcohol solution of iodine.
The test material is injected into the heart of guinea pigs. To do this, with two fingers
of the left hand, a cardiac impulse is probed and a needle is injected through the
intercostal space into the place of the impulse. When the needle enters the heart,
blood is shown in the syringe, and then all the material is injected.
When injecting, especially into the blood stream (into a vein, into the heart), the
injected fluid must be free from air bubbles that can cause a gas embolism.
Introduction through the digestive tract. There are several ways of oral (through the
mouth) charging. The simplest of them is the admixture of the test material with the
animal's food. This method is good because it is close to natural, but in this case it is
difficult to take into account the amount of material introduced. The second method
is the introduction of material into the stomach using a probe, the diameter and
length of which depend on the type of animal. The probe should be inserted after
the mouth expander is inserted into the animal's mouth. Previously, the probe is
lubricated with petroleum jelly. When the inserted probe reaches the nasopharynx, a
few drops of water are poured into the mouth and the probe is advanced at the time
of swallowing. A properly inserted probe should move easily and freely. After the
tube reaches the stomach, the required amount of the test material is injected
through its outer end with a syringe without a needle. The third way is instillation
with a syringe with a needle. To do this, the animal is fixed in a vertical position, the
mouth is opened with tweezers or by pressing two fingers on the cheeks, and the
test material is injected drop by drop, with each drop being administered only after
the animal has swallowed the previous one. This slow administration is necessary to
eliminate the possibility of fluid entering the respiratory tract. Finally, the test
material can be injected into the esophagus using a syringe with a needle ending in
an olive. The diameter of the needle should be 1 mm, the length depends on the
type of animal: 30-40 mm for mice, 70-80 mm for rats, etc. This slow administration
is necessary to eliminate the possibility of fluid entering the respiratory tract. Finally,
the test material can be injected into the esophagus using a syringe with a needle
ending in an olive. The diameter of the needle should be 1 mm, the length depends
on the type of animal: 30-40 mm for mice, 70-80 mm for rats, etc. This slow
administration is necessary to eliminate the possibility of fluid entering the
respiratory tract. Finally, the test material can be injected into the esophagus using a
syringe with a needle ending in an olive. The diameter of the needle should be 1 mm,
the length depends on the type of animal: 30-40 mm for mice, 70-80 mm for rats,
etc.
With this method of introduction, the animal is also fixed in a vertical position, its
mouth is opened with tweezers and a needle is inserted, leading it along the back
wall of the pharynx, then the needle is moved to a vertical position and the liquid is
poured very slowly.
Introduction through the respiratory tract . Intranasal (through the nose) infection is
carried out in two different ways. The simplest way is to instill the test material
through the nose with a syringe (in order not to injure the mucous membrane, use a
blunt needle). Infection is carried out under light ether anesthesia; cotton wool
moistened with ether is applied to the nose, under the influence of which the animal
begins to breathe deeply, while drawing in the injected material.
The second method is instillation of the test material into the nostrils with a pipette
(drop by drop); with this method, the animal is fixed with the abdomen up.
Introduction to the Eye . There are several ways to enter material. Produce infection
through the conjunctiva. To do this, while holding the eyelid, the test material is
instilled into the eye with a pipette (1-2 drops). The second way is
subconjunctival. The material is injected with a thin needle under the
conjunctiva. This way you can enter 0.1-0.2 ml. The third method is the introduction
of material into the scarified cornea; with this method, the animal must be fixed. The
eye is pressed with tweezers, the cornea is scarified with the end of a scalpel or a
regular needle; the test material is rubbed into the scarified cornea with a thin glass
spatula or cotton swab. This method is used mostly in experiments on rabbits and
dogs, less often on guinea pigs and other animals.

Control questions

1. What is a surgical field and how is it processed?

2. What methods of animal infection do you know?
3. What determines the amount of material administered to an animal?

Exercise

Draw a diagram of the markings of a white mouse. Apply paint to the diagram so that
the number of the animal corresponds to 3.
In table. 12 shows the values of the maximum amount of liquid to be administered
depending on the type of animal and the method of administration.
 Table 12. Scheme of the introduction of infected material to animals

Autopsy and microbiological examination of dead animals

In case of experimental infection, the autopsy of the animal after its death, the
nature of the changes and the result of bacteriological examination of the internal
organs make it possible to determine the cause of death of the animal, the ways in
which pathogens spread in the body and the place of their localization. In some
cases, animals have to be sacrificed at certain stages of the experiment. There are
several ways to kill. The most commonly used is the following: the animal is placed in
a jar or tightly closed vessel, into which a piece of cotton wool moistened with ether
or chloroform is dipped. The death of the animal occurs in a few minutes. One way
to kill small animals, such as mice, is decapitation (the heads of mice are cut off). An
air embolism is used to kill rabbits: a needle is inserted into a vein, connected to a
syringe without liquid and, pressing the piston, introduce air into a vein; the animal
dies instantly.
An autopsy should be performed immediately after the death of the animal in order
to avoid the penetration of microorganisms from the intestine into the blood and
other organs. In the absence of such an opportunity, the corpses are kept in the cold.
An autopsy is performed on a special table, in a separate room, if any.
The table should contain everything necessary for autopsy, bacteriological
examination of the pathological and anatomical material: a board or cuvette, filled
with paraffin, for fixing the animal; gas burner or spirit lamp; a vessel with alcohol
and cotton at the bottom, which contains sterilized instruments (scissors, scalpel,
surgical and anatomical tweezers, etc.); sterile cotton swabs, bacterial loop, sterile
Pasteur pipettes, glass slides, Petri dishes, test tubes with nutrient media. The choice
of media depends on the type of microorganisms that are considered the cause of
death of the animal.
All observations made during the autopsy are recorded. The type of animal, number,
time and place of infection, material used for infection, time of death, detected
changes, etc. are noted.
During the autopsy, care must be taken to ensure that liquid and pieces of tissues
and organs do not fall on the table. After each manipulation, the instruments are
washed with water or alcohol and burned. The crops made are inscribed. Smears are
fixed in the flame of a burner or in a special fixing liquid (for example, in Nikiforov's
mixture).
The autopsy consists of the following stages: 1) fixation of the animal; 2) examination
of the outer covers; 3) opening and examination of the chest cavity; 4) opening and
examination of the abdominal cavity.
Fixation. Animals are placed belly up. Rabbits and guinea pigs are tied by their paws
to hooks or rings located in the corners of the board. Mouse and rat paws can be
pinned with buttons or needles. At the same time, the paws are widely pushed apart.
Inspection of the outer covers. Cotton wool moistened with a disinfectant solution is
wiped over the entire surface of the body. Examine the outer covers and make a skin
incision along the midline, starting from the pubis to the lower jaw, trying not to
damage the side incisions. The instruments used to open the skin are wiped, burned
or changed, and only then the opening is continued. The edge of the skin flap is
grasped with tweezers and it is separated from the subcutaneous tissue with a
scalpel. They note the condition of the inguinal and axillary lymph nodes,
subcutaneous tissue, vasodilation, hemorrhage, suppuration, etc.
If changes in the lymph nodes (swelling, edema, suppuration) are detected, smears
are made and their contents are sown on special media.
To open the chest cavity, the xiphoid process is grasped with tweezers, an incision is
made under it, into which scissors are inserted, and the ribs are cut on both sides at
the junction with the cartilages of the sternum. The sternum is thrown up and the
chest cavity is examined. When examining the lungs and heart, their color, size, and
consistency are noted. If there are changes, culture is performed and smears are
made from the altered part of the lung. Next, cut off a piece of the lung and lower it
into a jar or test tube with water. A healthy lung floats to the surface, a diseased one
sinks. Be sure to do a blood culture from the heart. To do this, hold the heart with
tweezers at its base, cauterize the top of the heart with a hot scalpel and pierce the
burned area with a sterile Pasteur pipette, while blood rises into the capillary.
When opening the abdominal cavity, the skin of the abdominal wall is lifted with
tweezers, an incision is made with scissors from the pubis to the diaphragm, then
two lateral incisions are made - at the pubis and diaphragm, thereby widely opening
the abdominal cavity. All organs of the abdominal cavity are examined, from the
altered organs they are sown on nutrient media, smears and smears-imprints. In the
absence of external changes, it is necessary to sow and make smears from the
spleen, liver and mesenteric nodes. For sowing, the cauterized surface of the organ is
incised with a sterile scalpel, a piece is cut out from the depth of the organ, part of it
is lowered into a nutrient medium, smears are made from the other part, applying
the cut part to the glass, and smears. Imprints of all organs can be made on one
glass, following a certain sequence in their arrangement. This method has been
adopted in animal experiments, infected with pathogens of especially dangerous
infections. Knowing the order in which the imprints of various organs are located, it
is possible not to inscribe each imprint (Fig. 30).

Rice. 30. Preparation of smears-imprints of organs and a blood smear on one glass. 1

- lymph nodes; 2 - lung; 3 - spleen; 4 - liver; 5 - blood smear

After completion of the opening, a thorough cleaning of the workplace is carried

out. The corpse of an animal is placed in a jar or a special vessel (for example, a
bucket) and, under the control of a responsible person, is burned or autoclaved. If it
is impossible to destroy the corpse immediately after opening, it is poured with a
disinfectant solution (5% carbolic acid or 5% lysol). The board or cuvette is wiped
with alcohol and burned or poured for a day with a disinfectant solution. The
instruments are carefully placed in the sterilizer using clean tweezers and boiled for
30-40 minutes. After working with pathogenic spore-forming bacteria, the
instruments are autoclaved. Crops are placed in a thermostat.
Fixed smears are stained and examined.

Control questions

1. What is the purpose of autopsy of laboratory animals?

2. What are the stages of the autopsy of laboratory animals?
3. How are smears and smears-prints from organs and tissues prepared? What rules
should be observed when preparing prints on a single slide?
4. How are equipment and instruments disinfected during and after the autopsy?
In microbiological practice, animals are also used as donors. Animal blood is used
both in whole form and to obtain its individual components (serum, plasma,
erythrocytes, etc.). Blood and blood serum can be used as media ingredients, as well
as to study the immune properties after vaccination.
More often they use the blood of a rabbit, guinea pigs, less often - rats and mice,
from large animals - the blood of horses, sheep, bulls.
The method of obtaining blood depends on the type of animal and the purpose of
the study.
Technique for taking blood from animals
In a rabbit, a small amount of blood is usually taken from the marginal vein of the
ear, in rats and mice - from the vein of the tail, in guinea pigs, due to poorly
developed superficial veins, directly from the heart. Blood from the heart can also be
taken from other laboratory animals.
In the case when donors are large animals (horses, sheep), blood is taken from the
jugular vein.
To take a small amount of blood from a rabbit, the hair is plucked from the outer
surface of the ear, the skin is disinfected and hyperemia is caused by clicking on the
vein with fingers or rubbing the ear with cotton moistened with warm water (xylene
should not be used, as it can cause lysis - dissolution of erythrocytes).
In addition, you can press the vein at the root of the ear, which also contributes to
hyperemia. Then the experimenter grabs the tip of the ear with the thumb and
forefinger (fixing it) and inserts the needle without a syringe along the vein. The
blood dripping from the needle is collected in a sterile test tube, after which the
needle is removed, the injection site is disinfected with alcohol or an alcohol solution
of iodine.
When taking blood from a tail vein in mice and rats, the tip of the tail is
amputated. The blood flowing from the stump is collected in a sterile tube or sucked
with a pipette. To stop bleeding, the stump is cauterized with hydrogen peroxide or
in a burner flame.
When taking blood from the heart, the animal is fixed in a horizontal position with its
belly up, the hair is removed, the injection site is disinfected, then the heart beat is
felt with the fingertips of the left hand and a needle is inserted in this place. If the
needle enters the heart, the blood itself enters the syringe, raising the piston. The
amount of blood taken depends on the type of animal and its weight (Table 13).

Table 13. Maximum amount of blood obtained from laboratory animals

After simultaneous bloodletting, the animal should be injected under the skin with
isotonic sodium chloride solution, heated to body temperature, in a volume equal to
the volume of blood taken.
If necessary, to obtain the maximum amount of blood, total bloodletting (complete
bleeding) is performed. With total bloodletting, blood is most often taken from the
carotid artery. The animal is fixed in a horizontal position with its belly up,
anesthetized by bringing cotton soaked with ether to the nose, and after processing
the surgical field, a skin incision is made on the neck. The carotid artery is exposed,
cut off, two silk ligatures are applied and the artery is cut between them. The
peripheral end of the artery is quickly clamped, and the central end is grasped with
tweezers and lowered into a sterile vessel. When the blood stream dries up, you can
additionally collect blood by massaging the area of \u200b\u200bthe heart, liver and
other organs. The disadvantage of total bloodletting is the death of the animal.
Processing and isolation of blood constituents
Obtaining defibrinated blood . The blood is placed in a sterile flask or jar with glass
beads and shaken vigorously for 15-20 minutes; while fibrin settles on the beads in
the form of a clot. Fibrin-free blood is poured into a sterile container.
Obtaining citrated blood . A substance that prevents its coagulation is added to the
blood - 5% sodium citrate solution in a ratio of 1:10 (1 ml of 5% sodium citrate
solution per 10 ml of blood).
Getting plasma . The liquid part of the blood is obtained from citrated blood, which
is centrifuged or placed in a refrigerator for 18-20 hours. As a result, a layer of
yellowish liquid - plasma - forms above the sediment.
Obtaining blood serum (see chapter 12).
Obtaining a suspension of erythrocytes . A suspension of erythrocytes is obtained
from whole and defibrinated blood.
The blood is centrifuged for 15-20 minutes at 2000-3000 rpm. Erythrocytes settle to
the bottom, the yellowish-red liquid formed above them is drained, and sterile
isotonic sodium chloride solution is added to the test tubes to the original volume
and centrifuged again. This washing of erythrocytes is performed 2-3 times until the
supernatant becomes completely colorless. The last portion of the supernatant is
removed, and a suspension of erythrocytes remains in the test tube, which can be
used within 2-3 days. To preserve red blood cells for a longer period, they are
treated with formalin. To obtain a 50% suspension, two volumes of a buffer solution
having a pH of 7.2 are added to one volume of erythrocytes, and with constant
stirring, the same amount of a 3% formalin solution. The resulting mixture is kept in a
water bath at 37°C for 2-3 hours, stirring it every 15-20 minutes, and then in a
thermostat (20 hours at 37°C in total). The next day, the mixture is centrifuged in the
same solution, the supernatant is drained, and the precipitate is brought to its
original volume with a buffer solution, poured into vials, tightly closed and stored at
4°C.

Control questions
1. What is the technique for taking blood from rabbits, guinea pigs, rats, mice, and
which of them serve as donors more often?
2. What is total bloodletting?
3. What is the maximum amount of blood that can be obtained from laboratory
animals with simultaneous and total bloodletting?
4. How to get nitrate and defibrinated blood and blood components: plasma, serum,
erythrocyte suspension?

Exercise

Take a test tube with blood and extract serum from it.

Chapter 12 immune reactions. Immunoprophylaxis and

immunotherapy of infectious diseases - L. B.
Bogoyavlenskaya, G. I. Katz
The concept of immunity means the body's immunity to any genetically alien agents,
including pathogens and their poisons (from Latin immunitas - liberation from
something).
When genetically alien structures (antigens) enter the body, a number of
mechanisms and factors come into action that recognize and neutralize these
substances alien to the body.
The system of organs and tissues that carries out protective reactions of the body
against violations of the constancy of its internal environment (homeostasis) is called
the immune system.
The science of immunity - immunology studies the body's reactions to foreign
substances, including microorganisms; body reactions to foreign tissues
(compatibility) and to malignant tumors; determines the immunological blood
groups, etc. The foundations of immunology were laid by the spontaneous
observations of the ancients about the possibility of artificially protecting a person
from an infectious disease. Observations of people who were in the focus of the
epidemic led to the conclusion that not everyone gets sick. So, those who have
recovered from this disease do not get sick with the plague; measles usually gets sick
once in childhood; those who have had cowpox do not get sick with smallpox, etc.
There are known methods of ancient peoples to protect against snake bites by
rubbing plants rubbed with snake venom into notches on the skin; to protect herds
from peripneumonia of cattle, also making notches on the skin with a dagger,
previously immersed in the lungs of a bull that died from this disease.
E. Jenner (1876) made the first artificial vaccination to prevent infection. However,
only L. Pasteur was able to scientifically substantiate the principles of artificial
protection against infectious diseases. He proved that infection with weakened
pathogens leads to immunity of the body upon repeated encounters with these
microorganisms.
Pasteur developed drugs that prevented anthrax and rabies.
Immunology received further development in the works of I. I. Mechnikov on the
importance of cellular immunity (phagocytosis) and P. Ehrlich on the role of humoral
factors (body fluids) for the development of immunity.
Currently, immunology is a science in which protection against infectious diseases is
only one of the links. It explains the causes of tissue compatibility and rejection
during organ transplantation, fetal death in a Rh-conflict situation, complications
during blood transfusion, solves the problems of forensic medicine, etc.
The main types of immunity are shown in the diagram.

Types of immunity

Hereditary (species) immunity

Hereditary (species) immunity is the most durable and perfect form of immunity,
which is due to inherited factors of resistance (resistance).
It is known that man is immune to the plague of dogs and cattle, and animals do not
get sick with cholera and diphtheria. However, hereditary immunity is not absolute:
by creating special, unfavorable conditions for a macroorganism, one can change its
immunity. For example, overheating, cooling, beriberi, the action of hormones lead
to the development of a disease that is usually unusual for a person or animal. So,
Pasteur, cooling chickens, caused them with artificial infection anthrax, which they
do not get sick under normal conditions.

acquired immunity

Acquired immunity in a person is formed during life, it is not inherited.

natural immunity . Active immunity is formed after a disease (it is called post-
infectious). In most cases, it persists for a long time: after measles, chicken pox,
plague, etc. However, after some diseases, the duration of immunity is short and
does not exceed one year (flu, dysentery, etc.). Sometimes natural active immunity
develops without visible disease. It is formed as a result of latent (latent) infection or
repeated infection with small doses of the pathogen that do not cause a pronounced
disease (fractional, household immunization).
Passive immunity is the immunity of newborns (placental), acquired by them through
the placenta during fetal development. Newborns can also get immunity from their
mother's milk. This type of immunity is short-lived and by 6-8 months, as a rule,
disappears. However, the importance of natural passive immunity is great - it
ensures the immunity of infants to infectious diseases.
artificial immunity . A person acquires active immunity as a result of immunization
(vaccinations). This type of immunity develops after the introduction into the body of
bacteria, their poisons, viruses, weakened or killed in various ways (vaccinations
against whooping cough, diphtheria, smallpox).
At the same time, an active restructuring takes place in the body, aimed at the
formation of substances that have a detrimental effect on the pathogen and its
toxins (antibodies). There is also a change in the properties of cells that destroy
microorganisms and their metabolic products. The development of active immunity
occurs gradually over 3-4 weeks and it persists for a relatively long time - from 1 to 3-
5 years.
Passive immunity is created by introducing ready-made antibodies into the
body. This type of immunity occurs immediately after the introduction of antibodies
(sera and immunoglobulins), but lasts only 15-20 days, after which the antibodies are
destroyed and excreted from the body.
The concept of "local immunity" was introduced by A. M. Bezredka. He believed that
individual cells and tissues of the body have a certain susceptibility. By immunizing
them, they create, as it were, a barrier to the penetration of infectious agents. At
present, the unity of local and general immunity has been proven. But the
significance of the immunity of individual tissues and organs to microorganisms is
beyond doubt.
In addition to the above division of immunity by origin, there are forms of immunity
directed to different antigens.
Antimicrobial immunity develops in diseases caused by various microorganisms or
with the introduction of corpuscular vaccines (from live, weakened or killed
microorganisms).
Antitoxic immunity is developed in relation to bacterial poisons - toxins.
Antiviral immunity is formed after viral diseases. This type of immunity is mostly
long and persistent (measles, chicken pox, etc.). Antiviral immunity also develops
when immunized with viral vaccines.
In addition, immunity can be divided depending on the period of release of the body
from the pathogen.
Sterile Immunity . Most pathogens disappear from the body when a person
recovers. This type of immunity is called sterile (measles, smallpox, etc.).
Non-sterile immunity . Susceptibility to the causative agent of infection persists only
during its stay in the host organism. Such immunity is called non-sterile or
infectious. This type of immunity is observed in tuberculosis, syphilis and some other
infections.
Control questions
1. What is immunity?
2. What forms of immunity do you know?
Human immunity to infectious diseases is due to the combined action of nonspecific
and specific protective factors.
Nonspecific are the innate properties of the body that contribute to the destruction
of a wide variety of microorganisms on the surface of the human body and in the
cavities of its body.
The development of specific defense factors occurs after the body comes into
contact with pathogens or toxins; the action of these factors is directed only against
these pathogens or their toxins.

Nonspecific body defense factors

There are mechanical, chemical and biological factors that protect the body from the
harmful effects of various microorganisms.
Skin . Intact skin is a barrier to the penetration of microorganisms. In this case,
mechanical factors are important: the rejection of the epithelium and the secretion
of sebaceous and sweat glands, which contribute to the removal of microorganisms
from the skin.
The role of chemical protection factors is also performed by the secretions of the
glands of the skin (sebaceous and sweat). They contain fatty and lactic acids, which
have a bactericidal (killing bacteria) effect.
Biological protection factors are due to the detrimental effect of the normal
microflora of the skin on pathogenic microorganisms.
The mucous membranes of various organs are one of the barriers to the penetration
of microorganisms. In the respiratory tract, mechanical protection is carried out with
the help of ciliated epithelium. The movement of the cilia of the epithelium of the
upper respiratory tract constantly moves the mucus film along with various
microorganisms towards the natural openings: the oral cavity and nasal
passages. The hairs of the nasal passages have the same effect on bacteria. Coughing
and sneezing help remove microorganisms and prevent their aspiration (inhalation).
Tears, saliva, breast milk and other body fluids contain lysozyme. It has a destructive
(chemical) effect on microorganisms. The acidic environment of gastric contents also
affects microorganisms.
The normal microflora of the mucous membranes, as a factor of biological
protection, is an antagonist of pathogenic microorganisms.
Control questions
1. What are non-specific protective factors?
2. What factors prevent the penetration of pathogenic microorganisms through the
skin and mucous membranes?
Inflammation is the reaction of a macroorganism to foreign particles penetrating
into its internal environment. One of the causes of inflammation is the introduction
of infectious agents into the body. The development of inflammation leads to the
destruction of microorganisms or release from them.
Inflammation is characterized by a violation of the circulation of blood and lymph in
the lesion. It is accompanied by fever, swelling, redness and pain.
Cellular non-specific defense factors
Phagocytosis
One of the main mechanisms of inflammation is phagocytosis - the process of
absorption of bacteria.
The phenomenon of phagocytosis was first described by I. I. Mechnikov. He began
studying phagocytosis from a single-celled amoeba, for which phagocytosis is a way
of digesting food. Having traced this process at different stages of the development
of the animal world, I. I. Mechnikov completed it with the discovery of specialized
human cells, with the help of which the destruction of bacteria, the resorption of
dead cells, foci of hemorrhages, etc. is of great importance.
Various cells of the body (blood leukocytes, endothelial cells of blood vessels) have
phagocytic activity. This activity is most pronounced in mobile polymorphonuclear
leukocytes, blood monocytes and tissue macrophages, and to a lesser extent in bone
marrow cells. All mononuclear phagocytic cells (and their bone marrow precursors)
are combined into a system of mononuclear phagocytes (MPS).
Phagocytic cells have lysosomes that contain more than 25 different hydrolytic
enzymes and proteins with antibacterial properties.
Stages of phagocytosis . Stage 1 - the approach of the phagocyte to the object due to
the chemical influence of the latter. This movement is called positive chemotaxis
(towards the object).
Stage 2 - adhesion of microorganisms to phagocytes.
Stage 3 - the absorption of microorganisms by the cell, the formation of
phagosomes.
Stage 4 - the formation of a phagolysosome, where enzymes and bactericidal
proteins enter, the death and digestion of the pathogen.
The process that ends with the death of phagocytosed microbes is called complete
phagocytosis.
However, some microorganisms, being inside phagocytes, do not die, and sometimes
even multiply in them. These are gonococci, Mycobacterium tuberculosis,
Brucella. This phenomenon is called incomplete phagocytosis; while phagocytes die.
Like other physiological functions, phagocytosis depends on the state of the body -
the regulatory role of the central nervous system, nutrition, age.
The phagocytic activity of leukocytes changes in many and often non-infectious
diseases. By determining a number of indicators of phagocytosis, it is possible to
establish the course of the disease - recovery or deterioration of the patient's
condition, the effectiveness of the treatment, etc.
To assess the functional state of phagocytes, the absorption activity is most often
determined by two tests: 1) phagocytic index - the percentage of phagocytic cells
(the number of leukocytes with absorbed microbes out of 100 observed); 2)
phagocytic number - the average number of microbes or other objects of
phagocytosis absorbed by one leukocyte.
The bactericidal capabilities of phagocytes are determined by the number of
lysosomes, the activity of intracellular enzymes, and other methods.
The activity of phagocytosis is associated with the presence of antibodies in the
blood serum - opsonins. These antibodies enhance phagocytosis, preparing the cell
surface for absorption by the phagocyte.
The activity of phagocytosis largely determines the body's immunity to a particular
pathogen. In some diseases, phagocytosis is the main protective factor, in others it is
an auxiliary one. However, in all cases, the lack of phagocytic ability of cells
dramatically worsens the course and prognosis of the disease.
Cellular reactivity
The development of the infectious process and the formation of immunity are
completely dependent on the primary sensitivity of cells to the pathogen. Hereditary
species immunity is an example of the lack of sensitivity of cells of one animal
species to microorganisms that are pathogenic for others. The mechanism of this
phenomenon is not well understood. It is known that cell reactivity changes with age
and under the influence of various factors (physical, chemical, biological).
Control questions
1. What is phagocytosis?
2. What stages of phagocytosis do you know?
3. What is complete and incomplete phagocytosis?
Humoral factors of nonspecific protection
In addition to phagocytes, there are soluble non-specific substances in the blood that
have a detrimental effect on microorganisms. These include complement, properdin,
β-lysines, x-lysines, erythrin, leukins, plakins, lysozyme, etc.
Complement (from Latin complementum - addition) is a complex system of protein
blood fractions that has the ability to lyse microorganisms and other foreign cells,
such as red blood cells. There are several complement components: C 1 , C 2 , C 3 , etc.
Complement is destroyed at a temperature of 55 ° C for 30 minutes. This property is
called thermolability. It is also destroyed by shaking, under the influence of UV rays,
etc. In addition to blood serum, complement is found in various body fluids and in
inflammatory exudate, but is absent in the anterior chamber of the eye and
cerebrospinal fluid.
Properdin (from Latin properde - to prepare) is a group of components of normal
blood serum that activates complement in the presence of magnesium ions. It is
similar to enzymes and plays an important role in the body's resistance to
infection. A decrease in the level of properdin in the blood serum indicates an
insufficient activity of immune processes.
β-lysines are thermostable (temperature-resistant) substances of human blood
serum that have an antimicrobial effect, mainly against gram-positive
bacteria. Destroyed at 63 ° C and under the action of UV rays.
X-lysine is a thermostable substance isolated from the blood of patients with high
fever. It has the ability to complement lyse bacteria, mainly gram-negative ones,
without participation. Withstands heating up to 70-100°C.
Erythrin isolated from animal erythrocytes. It has a bacteriostatic effect on
diphtheria pathogens and some other microorganisms.
Leukins are bactericidal substances isolated from leukocytes. Thermostable,
destroyed at 75-80 ° C. Found in the blood in very small quantities.
Plakins are substances similar to leukins isolated from platelets.
Lysozyme is an enzyme that destroys the membrane of microbial cells. It is found in
tears, saliva, blood fluids. The rapid healing of wounds of the conjunctiva of the eye,
mucous membranes of the oral cavity, nose is largely due to the presence of
lysozyme.
The constituent components of urine, prostatic fluid, extracts of various tissues also
have bactericidal properties. Normal serum contains a small amount of interferon.
Control questions
1. What are humoral nonspecific defense factors?
2. What humoral factors of nonspecific defense do you know?

Specific body defense factors (immunity)

The components listed above do not exhaust the entire arsenal of humoral
protection factors. Chief among them are specific antibodies - immunoglobulins,
formed when foreign agents - antigens - are introduced into the body.
Antigens
Antigens are substances that are genetically alien to the body (proteins,
nucleoproteins, polysaccharides, etc.), to the introduction of which the body
responds with the development of specific immunological reactions. One of these
reactions is the formation of antibodies.
Antigens have two main properties: 1) immunogenicity, i.e., the ability to cause the
formation of antibodies and immune lymphocytes; 2) the ability to enter into a
specific interaction with antibodies and immune (sensitized) lymphocytes, which
manifests itself in the form of immunological reactions (neutralization, agglutination,
lysis, etc.). Antigens that have both traits are called complete antigens. These include
foreign proteins, sera, cellular elements, toxins, bacteria, viruses.
Substances that do not cause immunological reactions, in particular the production
of antibodies, but enter into a specific interaction with ready-made antibodies, are
called haptens - defective antigens. Haptens acquire the properties of full-fledged
antigens after combining with large molecular substances - proteins,
polysaccharides.
The conditions that determine the antigenic properties of various substances are:
foreignness, macromolecularity, colloidal state, solubility. Antigenicity is manifested
when a substance enters the internal environment of the body, where it meets with
the cells of the immune system.
The specificity of antigens, their ability to combine only with the corresponding
antibody, is a unique biological phenomenon. It underlies the mechanism of
maintaining the constancy of the internal environment of the body. This constancy is
ensured by the immune system, which recognizes and destroys genetically alien
substances (including microorganisms, their poisons) that are in its internal
environment. The human immune system has a constant immunological
surveillance. It is able to recognize foreignness when cells differ in just one gene
(cancerous).
Specificity is a feature of the structure of substances in which antigens differ from
each other. It is determined by the antigenic determinant, i.e., a small section of the
antigen molecule, which is connected to the antibody. The number of such sites
(groupings) is different for different antigens and determines the number of antibody
molecules with which the antigen can combine (valency).
The ability of antigens to combine only with those antibodies that have arisen in
response to the activation of the immune system by this antigen (specificity) is used
in practice: 1) diagnosis of infectious diseases (determination of specific pathogen
antigens or specific antibodies in the patient's blood serum); 2) prevention and
treatment of patients with infectious diseases (creation of immunity to certain
microbes or toxins, specific neutralization of poisons of pathogens of a number of
diseases during immunotherapy).
The immune system clearly differentiates "self" and "foreign" antigens, reacting only
to the latter. However, reactions to the body's own antigens - autoantigens and the
emergence of antibodies against them - autoantibodies are possible. "Barrier"
antigens become autoantigens - cells, substances that during the life of an individual
do not come into contact with the immune system (eye lens, spermatozoa, thyroid
gland, etc.), but come into contact with it in case of various injuries, usually being
absorbed into the blood. And since during the development of the organism these
antigens were not recognized as "our own", natural tolerance (specific
immunological non-response) did not form, i.e. cells of the immune system remained
in the body capable of an immune response to these own antigens.
As a result of the appearance of autoantibodies, autoimmune diseases can develop
as a result of: 1) the direct cytotoxic effect of autoantibodies on the cells of the
corresponding organs (for example, Hashimoto's goiter - damage to the thyroid
gland); 2) mediated action of autoantigen-autoantibody complexes, which are
deposited in the affected organ and cause damage (for example, systemic lupus
erythematosus, rheumatoid arthritis).
Antigens of microorganisms . A microbial cell contains a large number of antigens
that have different locations in the cell and different significance for the
development of the infectious process. Different groups of microorganisms have
different composition of antigens. In intestinal bacteria, O-, K-, H-antigens are well
studied.
The O antigen is associated with the cell wall of the microbial cell. It was usually
called "somatic", since it was believed that this antigen is enclosed in the body
(soma) of the cell. The O-antigen of gram-negative bacteria is a complex
lipopolysaccharide-protein complex (endotoxin). It is heat-stable, does not collapse
when treated with alcohol and formalin. Consists of the main nucleus (core) and side
polysaccharide chains. The specificity of O-antigens depends on the structure and
composition of these chains.
K antigens (capsular) are associated with the capsule and cell wall of the microbial
cell. They are also called shells. K antigens are located more superficially than O
antigens. They are mainly acidic polysaccharides. There are several types of K-
antigens: A, B, L, etc. These antigens differ from each other in resistance to
temperature effects. A-antigen is the most stable, L - the least. Surface antigens also
include the Vi antigen, which is present in pathogens of typhoid fever and some
other intestinal bacteria. It is destroyed at 60°C. The presence of the Vi-antigen was
associated with the virulence of microorganisms.
H-antigens (flagellate) are localized in the flagella of bacteria. They are a special
protein - flagellin. They break down when heated. When processed with formalin,
they retain their properties (see Fig. 70).
Protective antigen (protective) (from Latin protectio - patronage, protection) is
formed by pathogens in the patient's body. The causative agents of anthrax, plague,
brucellosis are able to form a protective antigen. It is found in exudates of affected
tissues.
Detection of antigens in pathological material is one of the methods of laboratory
diagnosis of infectious diseases. Various immune responses are used to detect the
antigen (see below).
With the development, growth and reproduction of microorganisms, their antigens
can change. There is a loss of some antigenic components, more superficially
located. This phenomenon is called dissociation. An example of it is "S" - "R"-
dissociation.
Control questions
1. What are antigens?
2. What are the main properties of antigens?
3. What microbial cell antigens do you know?
Antibodies
Antibodies are specific blood proteins - immunoglobulins that are formed in
response to the introduction of an antigen and are able to specifically react with it.
There are two types of proteins in human serum: albumins and globulins. Antibodies
are associated mainly with globulins modified by antigen and called
immunoglobulins (Ig). Globulins are heterogeneous. According to the speed of
movement in the gel when an electric current is passed through it, they are divided
into three fractions: α, β, γ. Antibodies belong mainly to γ-globulins. This fraction of
globulins has the highest speed of movement in an electric field.
Immunoglobulins are characterized by molecular weight, sedimentation rate during
ultracentrifugation (centrifugation at a very high speed), etc. The differences in these
properties made it possible to divide immunoglobulins into 5 classes: IgG, IgM, IgA,
IgE, IgD. All of them play a role in the development of immunity against infectious
diseases.
Immunoglobulins G (IgG) make up about 75% of all human immunoglobulins. They
are most active in the development of immunity. The only immunoglobulins cross
the placenta, providing passive immunity to the fetus. They have a small molecular
weight and a sedimentation rate during ultracentrifugation.
Immunoglobulins M (IgM) are produced in the fetus and are the first to appear after
infection or immunization. This class includes "normal" human antibodies, which are
formed during his life, without visible manifestations of infection or during domestic
repeated infection. They have a high molecular weight and sedimentation rate
during ultracentrifugation.
Immunoglobulins A (IgA) have the ability to penetrate the secrets of mucous
membranes (colostrum, saliva, bronchial contents, etc.). They play a role in
protecting the mucous membranes of the respiratory and digestive tracts from
microorganisms. In terms of molecular weight and sedimentation rate during
ultracentrifugation, they are close to IgG.
Immunoglobulins E (IgE) or reagins are responsible for allergic reactions (see Chapter
13). They play a role in the development of local immunity.
Immunoglobulins D (IgD). Found in small amounts in serum. Not studied enough.
Structure of immunoglobulins . Molecules of immunoglobulins of all classes are
constructed in the same way. IgG molecules have the simplest structure: two pairs of
polypeptide chains connected by a disulfide bond (Fig. 31). Each pair consists of a
light and heavy chain, differing in molecular weight. Each chain has constant sites
that are genetically predetermined, and variables that are formed under the
influence of the antigen. These specific regions of an antibody are called active
sites. They interact with the antigen that caused the formation of antibodies. The
number of active sites in an antibody molecule determines the valency - the number
of antigen molecules that the antibody can bind to. IgG and IgA are divalent, IgM are
pentavalent.

Rice. 31. Schematic representation of immunoglobulins

Immunogenesis - antibody formation depends on the dose, frequency and method
of antigen administration. There are two phases of the primary immune response to
the antigen: inductive - from the moment the antigen is introduced until the
appearance of antibody-forming cells (up to 20 hours) and productive, which begins
by the end of the first day after the introduction of the antigen and is characterized
by the appearance of antibodies in the blood serum. The amount of antibodies
gradually increases (by the 4th day), reaching a maximum on the 7-10th day and
decreasing by the end of the first month.
A secondary immune response develops when the antigen is re-introduced. At the
same time, the inductive phase is much shorter - antibodies are produced faster and
more intensively.
Control questions
1. What are antibodies?
2. What classes of immunoglobulins do you know?
Cellular mechanisms of the immune response
Lymphoid cells of the body perform the main function in the development of
immunity - immunity, not only in relation to microorganisms, but also to all
genetically alien cells, for example, during tissue transplantation. Lymphoid cells
have the ability to distinguish "own" from "foreign" and eliminate "foreign"
(eliminate).
The ancestor of all cells of the immune system is the hematopoietic stem cell. In the
future, two types of lymphocytes develop: T and B (thymus-dependent and bursa-
dependent). These cell names are derived from their origin. T cells develop in the
thymus (goiter, or thymus) and under the influence of substances secreted by the
thymus in peripheral lymphoid tissue.
The name B-lymphocytes (bursa-dependent) comes from the word "bursa" - a bag. In
the bursa of Fabricius, birds develop cells similar to human B-lymphocytes. Although
no organ analogous to the Bag of Fabricius has been found in humans, the name is
associated with this bag.
During the development of B-lymphocytes from a stem cell, they go through several
stages and are converted into lymphocytes capable of forming plasma cells. Plasma
cells, in turn, form antibodies and on their surface there are three classes of
immunoglobulins: IgG, IgM and IgA (Fig. 32).
 Rice. 32. Abbreviated scheme of development of immunocytes

The immune response in the form of the production of specific antibodies occurs as
follows: a foreign antigen, having penetrated into the body, is primarily
phagocytosed by macrophages. Macrophages, processing and concentrating the
antigen on their surface, transmit information about it to T-cells, which begin to
divide, "mature" and secrete a humoral factor that includes B-lymphocytes in
antibody production. The latter also "mature", develop into plasma cells, which
synthesize antibodies of a given specificity.
So, by joint efforts, macrophages, T- and B-lymphocytes carry out the immune
functions of the body - protection from everything genetically alien, including
pathogens of infectious diseases. Protection with antibodies is carried out in such a
way that immunoglobulins synthesized to a given antigen, connecting with it
(antigen), prepare it, make it sensitive to destruction, neutralization by various
natural mechanisms: phagocytes, complement, etc.
 Control questions
1. What is the role of macrophages in the immune response?
2. What is the role of T-lymphocytes in the immune response?
3. What is the role of B-lymphocytes in the immune response?
Theories of Immunity . The importance of antibodies in the development of
immunity is undeniable. What is the mechanism of their formation? This issue has
been the subject of controversy and discussion for a long time.
Several theories of antibody formation have been created, which can be divided into
two groups: selective (selection - selection) and instructive (instruct - instruct,
direct).
Selective theories suggest the existence in the body of ready-made antibodies to
each antigen or cells capable of synthesizing these antibodies.
Thus, Ehrlich (1898) assumed that the cell has ready-made "receptors" (antibodies)
that are connected to the antigen. After combining with the antigen, antibodies are
formed in even greater quantities.
The same opinion was shared by the creators of other selective theories: N. Jerne
(1955) and F. Burnet (1957). They argued that already in the body of the fetus, and
then in the adult body, there are cells capable of interacting with any antigen, but
under the influence of certain antigens, certain cells produce the "necessary"
antibodies.
Instructive theories [Gaurowitz F., Pauling L., Landsteiner K., 1937-1940] consider the
antigen as a "matrix", a stamp on which specific groups of antibody molecules are
formed.
However, these theories did not explain all the phenomena of immunity, and at
present the most accepted is the clonal selection theory of F. Burnet
(1964). According to this theory, in the embryonic period in the body of the fetus
there are many lymphocytes - precursor cells, which are destroyed when they
encounter their own antigens. Therefore, in an adult organism there are no longer
cells for the production of antibodies to its own antigens. However, when an adult
organism encounters a foreign antigen, selection (selection) of a clone of
immunologically active cells occurs and they produce specific antibodies directed
against this "foreign" antigen. When meeting this antigen again, the cells of the
"selected" clone are already larger and they quickly form a larger amount of
antibodies. This theory most fully explains the basic phenomena of immunity.
The mechanism of interaction between antigen and antibodies has various
explanations. So, Ehrlich likened their connection to the reaction between a strong
acid and a strong base with the formation of a new substance such as a salt.
Borde believed that antigen and antibodies mutually adsorb each other like paint
and filter paper or iodine and starch. However, these theories did not explain the
main thing - the specificity of immune reactions.
The most complete mechanism for connecting an antigen and an antibody is
explained by the hypothesis of Marrek (the "lattice" theory) and Pauling (the "farm"
theory) (Fig. 33). Marrek considers the combination of antigen and antibodies in the
form of a lattice, in which the antigen alternates with the antibody, forming lattice
conglomerates. According to Pauling's hypothesis (see Fig. 33), antibodies have two
valences (two specific determinants), and an antigen has several valences - it is
polyvalent. When antigen and antibodies are combined, agglomerates are formed
that resemble "farm" buildings.

Rice. 33. Schematic representation of the interaction of antibodies and antigen. A -

according to the Marrsk scheme: B - according to the Pauling scheme. The structure
of the complex: a - at optimal ratios; b - with an excess of antigen; c - with an excess
of antibodies

With an optimal ratio of antigen and antibodies, large strong complexes are formed
that are visible to the naked eye. With an excess of antigen, each active center of
antibodies is filled with an antigen molecule, there are not enough antibodies to
combine with other antigen molecules, and small, invisible complexes are
formed. With an excess of antibodies, there is not enough antigen to form a lattice,
there are no antibody determinants and there is no visible manifestation of the
reaction.
Based on the above theories, the specificity of the antigen-antibody reaction is today
presented as the interaction of the determinant group of the antigen and the active
centers of the antibody. Since antibodies are formed under the influence of an
antigen, their structure corresponds to the determinant groups of the antigen. The
determinant group of the antigen and fragments of the active sites of the antibody
have opposite electrical charges and, when combined, form a complex, the strength
of which depends on the ratio of the components and the environment in which they
interact.
The doctrine of immunity - immunology - has achieved great success over the past
decades. The disclosure of the patterns of the immune process has made it possible
to solve various problems in many areas of medicine. Methods for the prevention of
many infectious diseases have been developed and are being improved; treatment of
infectious and a number of other (autoimmune, immunodeficiency)
diseases; prevention of fetal death in Rh-conflict situations; transplantation of tissues
and organs; fight against malignant neoplasms; immunodiagnostics - the use of
immunity reactions for diagnostic purposes.
Immunity reactions are reactions between an antigen and an antibody or between
an antigen and sensitized * lymphocytes that occur in vivo and can be reproduced in
the laboratory.
*
 ( Sensitized - hypersensitive. )
Immunity reactions entered the practice of diagnosing infectious diseases in the late
19th and early 20th centuries. Due to their high sensitivity (they capture antigens in
very large dilutions) and, most importantly, their strict specificity (they make it
possible to distinguish antigens that are similar in composition), they have found
wide application in solving theoretical and practical problems of medicine and
biology. These reactions are used by immunologists, microbiologists, infectious
disease specialists, biochemists, geneticists, molecular biologists, experimental
oncologists, and doctors of other specialties.
Antigen-antibody reactions are called serological (from lat. serum - serum) or
humoral (from lat. humor - liquid), because the antibodies (immunoglobulins)
involved in them are always found in the blood serum.
Antigen reactions with sensitized lymphocytes are called cellular.
Control questions
1. How are antibodies formed?
2. What theories of antibody formation do you know?
3. What is the mechanism of interaction between an antigen and an antibody?

Serological reactions

Serological reactions - reactions of interaction between an antigen and an antibody

proceed in two phases: 1st phase - specific - the formation of a complex of an
antigen and its corresponding antibody (see Fig. 33). There is no visible change in this
phase, but the resulting complex becomes sensitive to nonspecific factors in the
environment (electrolytes, complement, phagocyte); 2nd phase - non-specific. In this
phase, the specific antigen-antibody complex interacts with non-specific factors of
the environment in which the reaction takes place. The result of their interaction can
be seen with the naked eye (gluing, dissolving, etc.). Sometimes these visible
changes are absent.
The nature of the visible phase of serological reactions depends on the state of the
antigen and the environmental conditions in which it interacts with the
antibody. There are reactions of agglutination, precipitation, immune lysis,
complement fixation, etc. (Table 14).
 Table 14. Serological reactions depending on the components involved and
environmental conditions

Application of serological reactions . One of the main applications of serological

reactions is the laboratory diagnosis of infections. They are used: 1) to detect
antibodies in the patient's serum, i.e. for serodiagnosis; 2) to determine the type or
type of antigen, for example, a microorganism isolated from a diseased
microorganism, i.e., to identify it.
In this case, the unknown component is determined by the known one. For example,
to detect antibodies in the patient's serum, a known laboratory culture of a
microorganism (antigen) is taken. If the serum reacts with it, then it contains the
corresponding antibodies and one can think that this microbe is the causative agent
of the disease in the patient being examined.
If it is necessary to determine which microorganism is isolated, it is tested in reaction
with a known diagnostic (immune) serum. A positive reaction result indicates that
this microorganism is identical to the one with which the animal was immunized to
obtain serum (Table 15).
 Table 15. Application of serological tests

Serological reactions are also used to determine the activity (titer) of sera and in
scientific research.
Carrying out serological reactions requires special preparation.
Vessels for serological reactions must be clean and dry. Test tubes (bacteriological,
agglutination, precipitating and centrifuge), graduated pipettes of various sizes and
Pasteur * , flasks, cylinders, slides and coverslips, Petri dishes, plastic plates with
holes are used.
*
 ( Each ingredient of the reaction is poured with a separate pipette. Pipettes should be kept until the end of the
experiment. To do this, it is convenient to place them in sterile test tubes marked where which pipette is.  )

Tools and equipment: loop, tripods, magnifying glass, agglutinoscope, thermostat,

refrigerator, centrifuge, chemical balance with weight.
Materials: antibodies (immune and test sera), antigens (cultures of microorganisms,
diagnosticums, extracts, lysates, haptens, erythrocytes, toxins), complement,
isotonic sodium chloride solution.
Attention! In serological reactions, only chemically pure sodium chloride is used.
Serums . Serum of the patient. Serum is usually obtained in the second week of
illness, when antibodies can be expected in it, sometimes sera of convalescents
(recovering) and those who have been ill are used.
Most often, to obtain serum, blood is taken from a vein in an amount of 3-5 ml into a
sterile tube and sent to the laboratory, accompanied by a label indicating the
patient's surname and initials, the alleged diagnosis and date.
Blood should be taken on an empty stomach or not earlier than 6 hours after a
meal. Blood serum after eating may contain droplets of fat, which make it cloudy and
unsuitable for research (such serum is called chylous).
Attention! When taking blood, it is necessary to follow the rules of asepsis.
To obtain serum, the blood is left for 1 hour at room temperature or placed in a
thermostat at 37 ° C for 30 minutes to form a clot.
Attention! Serum should not be kept in a thermostat for more than 30 minutes -
hemolysis may occur, which will interfere with research.
The resulting clot is separated from the walls of the test tube with a Pasteur pipette
or a loop ("circle"). The test tube is placed in a refrigerator for some time (usually 1
hour, but not more than 48 hours) for better separation of serum from a clot that
has contracted in the cold. The serum is then aspirated with a sterile Pasteur pipette
fitted with a rubber balloon or hose.
Serum should be sucked out very carefully so as not to capture the formed
elements. Serum should be completely transparent without any admixture of
cells. Turbid sera are sucked off again after the cells have settled. Serum can be freed
from formed elements by centrifugation.
Attention! Serum can remain on the clot for no more than 48 hours at + 4 ° C.
To obtain serum, blood can be taken from a puncture of the pulp of a finger or
earlobe with a Pasteur pipette. In infants, blood is taken from a U-shaped incision in
the heel.
When using a Pasteur pipette, blood is sucked into the pipette from the
puncture. The sharp end of the pipette is sealed. The pipette is placed in the test
tube with the sharp end down. So that it does not break, a piece of cotton wool is
placed at the bottom of the test tube. The appropriately labeled tube is sent to the
laboratory. The serum accumulated at the wide end of the pipette is sucked off.
Immune sera are obtained from the blood of people or animals (usually rabbits and
horses) immunized according to a certain scheme with the appropriate antigen
(vaccine). In the resulting serum, its activity (titer) is determined, i.e., the highest
dilution in which it reacts with the corresponding antigen under certain experimental
conditions.
Whey is usually prepared in production. They are poured into ampoules, which
indicate the name and title. In most cases, sera are dried. Before use, dry whey is
dissolved in distilled water to the original volume (also indicated on the label). Store
all dry (lyophilized) diagnostic preparations at 4-10°C.
For serological studies, native (not adsorbed) and adsorbed immune sera are
used. The disadvantage of native sera is the presence of group antibodies in them,
i.e. antibodies to microorganisms that have common antigens. Typically, such
antigens are found in microbes belonging to the same group, genus,
family. Adsorbed sera are highly specific: they react only with a homologous
antigen. Antibodies to other (heterogeneous) antigens are removed by
adsorption. The antibody titer of adsorbed sera is low (1:40, 1:320), so they are not
diluted * .
*
 ( Currently, special cells (hybridomas) have been obtained by biotechnology that produce monoclonal antibodies in
vitro, i.e., antibodies that react strictly specifically (with one antigen).  )

Agglutination reaction

The agglutination reaction (RA) is the agglutination and precipitation of microbes or

other cells under the action of antibodies in the presence of an electrolyte (isotonic
sodium chloride solution). The resulting precipitate is called agglutinate. For the
reaction you need:
1. Antibodies (agglutinins) - are in the patient's serum or in the immune serum.
2. Antigen - a suspension of live or killed microorganisms, erythrocytes or other cells.
3. Isotonic solution.
The agglutination reaction for serodiagnosis is widely used for typhoid fever,
paratyphoid fever (Vidal reaction), brucellosis (Wright reaction), etc. In this case, the
patient's serum is the antibody, and the known microbe is the antigen.
When microbes or other cells are identified, their suspension serves as an antigen,
and a known immune serum serves as an antibody. This reaction is widely used in
the diagnosis of intestinal infections, whooping cough, etc.
Preparation of ingredients: 1) obtaining serum, see p. 200; 2) preparation of the
antigen. Suspension of live microbes should be homogeneous and correspond (in 1
ml) to about 30 units. turbidity according to the GISK optical standard. For its
preparation, a 24-hour culture grown on agar slant is usually used. The culture is
washed off with 3-4 ml of isotonic solution, transferred to a sterile test tube, its
density is determined and, if necessary, diluted.
The use of a suspension of killed microbes - diagnosticums - facilitates the work and
makes it safe. Usually they use diagnosticums prepared at the factory.
Reaction setting. There are two methods for carrying out this reaction: the
agglutination reaction on glass (sometimes called the approximate one) and the
extended agglutination reaction (in test tubes).
Agglutination reaction on glass . 2 drops of specific (adsorbed) serum and a drop of
isotonic solution are applied to a fat-free glass slide. Non-adsorbed sera are pre-
diluted in a ratio of 1:5 - 1:25. Drops are applied to the glass so that there is a
distance between them. With a wax pencil on the glass, they mark where which drop
is. The culture is thoroughly rubbed with a loop or pipette on a glass, and then added
to a drop of isotonic solution and one of the drops of serum, stirring in each until a
homogeneous suspension is formed. The serum drop without culture is the serum
control.
Attention! Serum culture should not be transferred to a drop of isotonic saline, which
is an antigen control.
The reaction proceeds at room temperature for 1–3 min. The serum control should
remain clear and uniform haze should be observed in the antigen control. If flakes of
agglutinate appear against the background of a clear liquid in a drop where the
culture is mixed with serum, the reaction result is considered positive. If the reaction
result is negative, there will be a uniform turbidity in the drop, as in the antigen
control.
The reaction is more clearly visible when viewed against a dark background in
transmitted light. When studying it, you can use a magnifying glass.
Extended agglutination reaction . Sequential, most often two-fold dilutions of serum
are prepared. The patient's serum is usually diluted from 1:50 to 1:1600, the immune
one - up to a titer or up to half a titer. The titer of agglutinating serum is its maximum
dilution in which it agglutinates homologous cells.
Serum dilution: 1) put in a rack the required number of test tubes of the same
diameter, height and bottom configuration;
2) on each test tube indicate the degree of dilution of the serum, in addition, on the
1st test tube write the number of experience or the name of the antigen. On test
tubes of controls write "KS" - serum control and "KA" - antigen control;
3) pour 1 ml of isotonic solution into all test tubes;
4) prepare the initial (working) serum dilution in a separate tube. For example, to
prepare a working dilution of 1:50, 4.9 ml of isotonic solution and 0.1 ml of serum
are poured into a test tube. The degree of its dilution must be indicated on the test
tube. The initial serum dilution is added to the first two tubes and to the serum
control tube;
5) prepare serial two-fold dilutions of serum.
An approximate scheme of its breeding is given in table. 16.

Table 16. Serum dilution scheme for deployed RA

Note. The arrows indicate the transfer of liquid from tube to tube; from the 5th tube
and the serum control tube, 1.0 ml is poured into the disinfectant solution.
Attention! All tubes must contain the same volume of liquid.
After serum dilutions are made, 1-2 drops of antigen (diagnosticum or freshly
prepared suspension of bacteria) are added to all test tubes, except for serum
control. In the test tubes, a small uniform turbidity should appear. The serum control
remains transparent.
The tubes are thoroughly shaken and placed in a thermostat (37°C). Preliminary
accounting of the results of the reaction is carried out after 2 hours, and the final one
- after 18-20 hours (keeping at room temperature).
Accounting for results, as always, begins with controls. The serum control should
remain clear, the antigen control uniformly cloudy. The test tubes are viewed in
transmitted light (very convenient on a dark background) with the naked eye, using a
magnifying glass or an agglutinoscope.
Agglutinoscope - a device consisting of a hollow metal tube mounted on a stand. On
top of it is an eyepiece with an adjusting screw. A rotating mirror is attached under
the tube. A test tube with the liquid under study is inserted from the side into the
opening of the tube at such a distance that the liquid in it is under the eyepiece. By
setting the illumination with a mirror and focusing the eyepiece, the presence and
nature of the agglutinate are determined.
With a positive result of the reaction, grains or flakes of agglutinate are visible in the
test tubes. The agglutinate gradually settles to the bottom in the form of an
"umbrella", and the liquid above the sediment becomes clear (compare with a
uniformly cloudy antigen control).
To study the size and nature of the precipitate, the contents of the test tubes are
shaken slightly. There are fine-grained and flaky agglutination. Fine-grained (O-
agglutination) is obtained when working with O-sera * . Flaky (H) - in the interaction
of motile microorganisms with flagellated H-sera.
*
 ( O-sera contain antibodies to O (somatic) antigen, H-sera - to flagellate. )
Flocculent agglutination occurs more quickly, and the resulting precipitate is very
loose and easily broken.
The intensity of the reaction is expressed as follows:
++++ all cells settled, the liquid in the test tube is completely transparent. The result
of the reaction is strongly positive.
+++ sediment is less, there is no complete enlightenment of the liquid. The result of
the reaction is positive.
++ the sediment is even less, the liquid is turbid. The result of the reaction is slightly
positive.
+ slight sediment, cloudy liquid. Doubtful response.
- there is no sediment, the liquid is uniformly turbid, as in the antigen
control. Negative reaction result.
Possible errors in the formulation of the agglutination reaction . 1. Spontaneous
(spontaneous) agglutination. Some cells, especially microbes in the R-form, do not
give a homogeneous (homogeneous) suspension, quickly precipitate. To avoid this,
use an S-form culture that does not spontaneously agglutinate.
2. In the serum of healthy people there are antibodies to certain microorganisms
(the so-called "normal antibodies"). Their titer is low. Therefore, a positive result of
the reaction in a dilution of 1:100 and above indicates its specificity.
3. Group reaction with microbes similar in antigenic structure. For example, the
serum of a patient with typhoid fever can also agglutinate paratyphoid A and B
bacteria. In contrast to the specific group reaction, it occurs in lower titers. Adsorbed
sera do not give a group reaction.
4. It should be taken into account that specific antibodies after an illness and even
after vaccinations can persist for a long time. They are called "anamnestic". To
distinguish them from "infectious" antibodies formed during the current illness, the
reaction is put in dynamics, that is, the patient's serum is examined, taken again after
5-7 days. An increase in antibody titer indicates the presence of a disease - the titer
of "anamnestic" antibodies does not increase, and may even decrease.
Control questions
1. What are immune reactions, what are their main properties?
2. What components are involved in serological reactions? Why are reactions called
serological, how many phases do they consist of?
3. What is an agglutination reaction? Its use and methods. What is a diagnosticum?
4. What antigen is used in the study of the patient's serum? What serum determines
the type of an unknown microbe?
5. What is O- and H-agglutination? In what cases is a flocculent precipitate formed
and when is it fine-grained?
 Exercise
1. Set up a detailed agglutination test to determine the antibody titer in the patient's
serum and take into account its result.
2. Put the agglutination reaction on the glass to determine the type of isolated
microorganism.

Hemagglutination reaction

In laboratory practice, two hemagglutination reactions (RHA) are used, which are
different in their mechanism of action.
The first RGA refers to serological. In this reaction, erythrocytes are agglutinated
when interacting with the corresponding antibodies (hemagglutinins). The reaction is
widely used to determine blood groups.
The second RHA is not serological. In it, gluing of red blood cells is caused not by
antibodies, but by special substances formed by viruses. For example, the influenza
virus agglutinates the erythrocytes of chickens and guinea pigs, the polio virus
agglutinates the erythrocytes of sheep. This reaction makes it possible to judge the
presence of a particular virus in the test material.
Reaction setting. The reaction is put in test tubes or on special plates with wells. The
material to be tested for the presence of the virus is diluted with isotonic solution
from 1:10 to 1:1280; 0.5 ml of each dilution is mixed with an equal volume of 1-2%
erythrocyte suspension. In the control, 0.5 ml of erythrocytes are mixed with 0.5 ml
of isotonic solution. The test tubes are placed in a thermostat for 30 minutes, and
the plates are left at room temperature for 45 minutes.
Accounting for results. With a positive result of the reaction at the bottom of the test
tube or well, a precipitate of erythrocytes with scalloped edges ("umbrella") falls,
covering the entire bottom of the well. With a negative result, erythrocytes form a
dense precipitate with smooth edges ("button"). The same precipitate should be in
control. The intensity of the reaction is expressed by plus signs. The titer of the virus
is the maximum dilution of the material in which agglutination occurs.
Hemagglutination inhibition reaction
This is a serological reaction in which specific antiviral antibodies, interacting with
the virus (antigen), neutralize it and deprive it of the ability to agglutinate red blood
cells, i.e., inhibit the hemagglutination reaction. The high specificity of the
hemagglutination inhibition reaction (HITA) allows using it to determine the type and
even the type of viruses detected during the HA.
Reaction setting. 0.25 ml of antiviral serum in consecutive two-fold dilutions from
1:10 to 1:2560 is mixed with an equal volume of material containing the virus,
diluted 4 times less than the titer established in the RGA. The mixture is shaken and
placed in a thermostat for 30 minutes, after which 0.5 ml of a 1-2% suspension of
erythrocytes is added.
The reaction is followed by three controls (Table 17).

Table 17 Hemagglutination Inhibition Controls

The results are recorded after repeated incubation in a thermostat for 30 or 45

minutes at room temperature. With the correct setting of the experiment in the
control of serum and erythrocytes, a “button” should form - there is no factor
agglutinating erythrocytes; in the control of the antigen, an "umbrella" is formed -
the virus caused erythrocyte agglutination.
In the experiment, if the serum is homologous to the virus being studied, a "button"
is formed - the serum neutralized the virus. Serum titer is its maximum dilution in
which hemagglutination is delayed.
Indirect hemagglutination reaction
The reaction of indirect (passive) hemagglutination (RIHA) is based on the fact that
erythrocytes, if a soluble antigen is adsorbed on their surface, acquire the ability to
agglutinate when interacting with antibodies to the adsorbed antigen. The RNGA
scheme is shown in fig. 34. RNHA is widely used in the diagnosis of a number of
infections.

Rice. 34. Scheme of the reaction of passive hemagglutination (RPHA). A - obtaining

an erythrocyte diagnosticum: B - RPHA: 1 - erythrocyte: 2 - antigen under study; 3 -
erythrocyte diagnosticum; 4 - antibody to the studied antigen: 5 - agglutinate

Reaction setting. The test serum is heated for 30 minutes at 56 ° C, diluted

sequentially in a ratio of 1:10 - 1:1280 and poured into test tubes or wells in 0.25 ml,
where then 2 drops of erythrocyte diagnosticum are added (erythrocytes with
antigen adsorbed on them).
Controls: a suspension of erythrocyte diagnosticum with obviously immune
serum; suspension of diagnosticum with normal serum; a suspension of normal
erythrocytes with the tested serum. In the first control, agglutination should occur, in
the second and third it should not be.
With the help of RIGA, it is possible to determine an unknown antigen if known
antibodies are adsorbed on erythrocytes.
The hemagglutination reaction can be set in a volume of 0.025 ml (micromethod)
using a Takachi microtiter.
Control questions
1. What does a positive RGA result between erythrocytes and the material tested for
the presence of the virus indicate?
2. Will agglutination of erythrocytes occur if a virus and the corresponding serum are
added to them? What is the name of the reaction that reveals this phenomenon?
Exercise
Consider and register the result of RIGA.

precipitation reaction

In the precipitation reaction, a specific immune complex is precipitated, consisting of

a soluble antigen (lysate, extract, hapten) and a specific antibody in the presence of
electrolytes.
The cloudy ring or precipitate formed as a result of this reaction is called a
precipitate. This reaction differs from the agglutination reaction mainly in the size of
the antigen particles.
The precipitation reaction is usually used to determine the antigen in the diagnosis of
a number of infections (anthrax, meningitis, etc.); in forensic medicine - to determine
the species of blood, sperm, etc.; in sanitary and hygienic studies - when establishing
falsification of products; with its help determine the phylogenetic relationship of
animals and plants. For the reaction you need:
1. Antibodies (precipitins) - immune serum with a high titer of antibodies (not lower
than 1:100,000). The titer of precipitating serum is determined by the highest
dilution of the antigen with which it reacts. Serum is usually used undiluted or
diluted 1:5 - 1:10.
2. Antigen - dissolved substances of a protein or lipoid polysaccharide nature
(complete antigens and haptens).
3. Isotonic solution.
The main methods for carrying out the precipitation reaction are: ring precipitation
reaction and precipitation reaction in agar (gel).
Attention! All components involved in the precipitation reaction must be completely
transparent.
Ring precipitation reaction . 0.2-0.3 ml (5-6 drops) of serum are added to the
precipitation tube using a Pasteur pipette (serum should not fall on the walls of the
tube). The antigen is carefully layered onto the serum in the same volume, pouring it
with a thin Pasteur pipette along the wall of the test tube. The test tube is kept in an
inclined position. With proper layering, a clear boundary should be obtained
between the serum and the antigen. Carefully, so as not to mix the liquid, place the
test tube in a tripod. With a positive result of the reaction, a cloudy "ring" is formed
at the border of the antigen and antibody - a precipitate (see Fig. 48).
The reaction is followed by a number of controls (Table 18). The sequence of
introducing the reaction ingredients into the test tube is very important. You can not
layer the serum on the antigen (in the control - on the isotonic solution), since the
relative density of the serum is greater, it will sink to the bottom of the tube, and the
boundary between the liquids will not be detected.

Table 18

Note. + the presence of a "ring"; - lack of "ring".

The results are recorded after 5-30 minutes, in some cases after an hour, as always,
starting with controls. The "ring" in the 2nd test tube indicates the ability of the
immune serum to enter into a specific reaction with the corresponding
antigen. There should be no "rings" in the 3rd-5th test tubes - there are no
antibodies and antigens corresponding to each other. "Ring" in the 1st test tube - a
positive reaction result - indicates that the test antigen corresponds to the taken
immune serum, the absence of a "ring" ("ring" only in the 2nd test tube) indicates
their inconsistency - a negative reaction result.
Precipitation reaction in agar (gel) . The peculiarity of the reaction is that the
interaction of the antigen and antibody occurs in a dense medium, i.e., in a gel. The
resulting precipitate gives a cloudy band in the thickness of the medium. The
absence of a band indicates a mismatch between the reaction components. This
reaction is widely used in medical and biological research, in particular in the study
of toxin formation in the causative agent of diphtheria.
Control questions
1. What is the main difference between the reaction of agglutination and
precipitation?
2. Why can't cloudy ingredients be used in the precipitation reaction?
Exercise
1. Set up the ring precipitation reaction and draw the result.
2. Study the nature of the interaction of the antigen with the antibody in the agar
precipitation reaction, draw the result (get the cup from the teacher).

Lysis reaction (immune cytolysis)

Immune lysis is the dissolution of cells under the influence of antibodies with the
obligatory participation of complement. For the reaction you need:
1. Antigen - microbes, erythrocytes or other cells.
2. Antibody (lysine) - immune serum, rarely the patient's serum. Bacteriolytic serum
contains antibodies involved in the lysis of bacteria; hemolytic - hemolysins that
contribute to the lysis of red blood cells; for the lysis of spirochetes, spirochetolizins
are needed, cells - itolizins, etc.
3. Complement. Most complement in the serum of guinea pigs. This serum (mixture
from several animals) is usually used as a complement. Fresh (native) complement is
unstable and easily destroyed by heating, shaking, storage, so it can be used no
longer than two days after receipt. To preserve the complement, 2% boric acid and
3% sodium sulfate are added to it. This complement can be stored at 4°C for up to
two weeks. Dry complement is more commonly used. Before use, it is dissolved in an
isotonic solution to the original volume (indicated on the label).
4. Isotonic solution.
Hemolysis reaction (Table 19). For the reaction you need:
1. Antigen - 3% suspension of washed sheep erythrocytes at the rate of 0.3 ml of
erythrocyte sediment and 9.7 ml of isotonic solution.
2. Antibody - hemolytic serum (hemolysin) against sheep erythrocytes; usually
prepared in production, lyophilized and the titer is indicated on the label.
The hemolysin titer is the highest serum dilution at which complete hemolysis of a
3% suspension of erythrocytes occurs in the presence of complement. For the
hemolysis reaction, hemolysin is taken in a triple titer, i.e., it is diluted 3 times less
than before the titer. For example, if the serum titer is 1:1200, the serum is diluted
1:400 (0.1 ml of serum * and 39.9 ml of isotonic saline). An excess of hemolysin is
necessary, since some of it can be adsorbed by other components of the reaction.
*
 ( Less than 0.1 ml of serum should not be taken - measurement accuracy suffers.  )
3. Complement is diluted 1:10 (0.2 ml of complement and 1.8 ml of isotonic saline).
4. Isotonic solution.

Table 19. Scheme of the hemolysis reaction

Accounting for results. With a correctly set reaction in the 1st test tube, hemolysis
will occur - its contents will become transparent. In the controls, the liquid remains
cloudy: in the 2nd tube, complement is missing for the onset of hemolysis, in the 3rd
tube, there is no hemolysin, in the 4th tube, neither hemolysin nor complement is
present, in the 5th tube, the antigen does not match the antibody,
If necessary, hemolytic serum is titrated according to the following scheme (Table
20).
Before titration, an initial serum dilution of 1:100 (0.1 ml of serum and 9.9 ml of
isotonic saline) is prepared, from which the necessary dilutions are made, for
example:

Of these dilutions, 0.5 ml of serum is added to the test tubes of the titration
experience, as shown in Table. 20.

Table 20. Titration scheme for hemolytic serum (hemolysin)

In the example given in Table. 20, the titer of hemolytic serum is 1:1200.

When using fresh hemolytic serum, it must be inactivated to destroy its
complement. To do this, it is heated for 30 minutes at 56 ° C in a water bath or in an
inactivator with a thermostat. The latter method is better: it eliminates the
possibility of serum overheating, i.e., its denaturation. Denatured sera are not
suitable for testing.
bacteriolysis reaction . In this reaction, bacteria are complemented in the presence
of the appropriate (homologous) serum. The reaction scheme is fundamentally
similar to the hemolysis reaction scheme. The difference is that after a two-hour
incubation, all test tubes are seeded on Petri dishes with a medium favorable for the
microorganism taken in the experiment to find out if it is lysed. With a correctly set
experience in crops from the 2nd-5th test tubes (controls), there should be abundant
growth. Lack of growth or weak growth in culture from the 1st test tube
(experiment) indicates the death of microbes, i.e., that they are homologous to the
antibody.
Attention! The bacteriolysis reaction must be carried out under aseptic conditions.
Control questions
1. What will happen to erythrocytes if distilled water is used instead of isotonic
sodium chloride solution? What underlies this phenomenon?
2. What reaction will occur when erythrocytes interact with homologous immune
serum in the absence of complement?
Exercise
Set up the hemolysis reaction. Record and draw the result.

Complement fixation reaction

The complement fixation reaction (RCC) is based on the fact that a specific antigen-
antibody complex always adsorbs (binds) complement on itself.
This reaction is widely used in the identification of antigens and in the serodiagnosis
of infections, especially diseases caused by spirochetes (Wassermann reaction),
rickettsia and viruses.
RSK is a complex serological reaction. It involves complement and two antigen-
antibody systems. Essentially, these are two serological reactions.
The first system - the main one - consists of an antigen and an antibody (one is
known, the other is not). A certain amount of complement is added to it. When the
antigen and antibody of this system match, they will combine and bind the
complement. The resulting complex is finely dispersed and is not visible.
The formation of this complex is known with the help of a second hemolytic or
indicator system. It includes sheep erythrocytes (antigen) and the corresponding
hemolytic serum (antibody), i.e., a ready-made immune complex. In this system,
erythrocyte lysis can only occur in the presence of complement. If the complement is
bound by the first system (if the antigen and antibody correspond in it), then there
will be no hemolysis in the second system - since there is no free complement. The
absence of hemolysis (the contents of the tube are cloudy or there is an erythrocyte
sediment at the bottom of the tube) is recorded as a positive result of RSK (Fig. 35).
 Rice. 35. Scheme of the complement fixation reaction (RCC). I - positive result (no
hemolysis); II - negative result (hemolysis)

If the antigen does not match the antibody in the first system, then the immune
complex is not formed and the complement remains free. Remaining free, the
complement participates in the second system, causing hemolysis - the result of RSC
is negative (the contents of the tubes are transparent - "lacquer blood").
Components of the complement fixation reaction:
1. Antigen - usually a lysate, extract, hapten;
 suspension of microorganisms
2. Antibody - serum of the patient system
3. Complement - guinea pig serum
4. Antigen - sheep erythrocytes
5. Antibody - hemolysin to sheep erythrocytes
6. Isotonic saline system
In view of the fact that a large number of complex components are involved in RSC,
they must be pre-titrated and taken into the reaction in exact quantities and in equal
volumes: 0.5 or 0.25, less often 0.2 ml. Accordingly, the entire experiment is carried
out in volumes of 2.5, 1.25 or 1.0 ml (larger volumes give a more accurate
result). The titration of the reaction components is carried out in the same volume as
the experiment, replacing the missing ingredients with an isotonic solution.
Preparation of ingredients
1. Hemolytic serum (hemolysin). Serum is diluted 3 times less than its titer. Prepare a
total serum dilution for the entire experiment; the volume of which is determined by
multiplying the volume of serum in one tube (for example, 0.5 ml) by the number of
tubes, slightly exceeding the number of them in the experiment * .
*
 ( An excess of liquid is necessary in the preparation of all components of the reaction: part of it remains on the walls of
test tubes, flasks, pipettes. )

2. Sheep erythrocytes . A 3% suspension of washed sheep erythrocytes is prepared

for the entire number of test tubes in the experiment.
To prepare the hemolytic system, 30 minutes before introducing it into the
experiment, equal volumes of diluted hemolysin and erythrocyte suspensions are
mixed, adding serum to the erythrocytes, thoroughly mixed and incubated for 30
minutes at 37 ° C (sensitized).
3. Complement is usually diluted 1:10. It must be titrated before each
experiment. The complement titer is its smallest amount, when added to the
hemolytic system, complete hemolysis occurs within 1 hour at 37 ° C. The
complement titration scheme is presented in Table. 21.
 Table 21. Complement titration scheme

Note. The total volume of liquid in the test tubes is 2.5 ml.

Attention! The complement is titrated in the same volume as the main experiment,
replacing the missing ingredients with an isotonic solution.
Accounting for results. There should not even be traces of hemolysis in the controls,
since one of them does not have a complement, while the other does not contain
hemolysin. Controls indicate the absence of hemotoxicity reactions (the ability to
spontaneously lyse erythrocytes) in the components.
In the table. 21 example, the complement titer in a 1:10 dilution is 0.15 ml. In the
experiment, complement activity may decrease due to its nonspecific adsorption by
other reaction components, therefore, for the experiment, the amount of
complement is increased: the dose following the titer is taken. This is the working
dose. In the given example, it is equal to 0.2 ml of complement in a 1:10
dilution. Since all components involved in the CSC must be taken in equal volumes (in
our example, it is 0:5 ml), it is necessary to add 0.3 ml of isotonic solution to the
working dose of complement (0.2 ml 1:10). For the whole experiment, the volume of
each of them (complement and isotonic saline) is multiplied by the number of tubes
involved in the CSC. For example, to conduct an experiment in 50 test tubes, you
need to take 10 ml of 1:10 complement (0.2 ml × 50) and 15 ml of isotonic solution
(0.3 ml × 50).
4. The antigen is usually obtained ready-made with an indication of its titer, i.e. the
amount that, after dilution of the antigen, should be contained in 1 ml. For example,
at a titer of 0.4, it is diluted in 0.96 ml of isotonic solution. In the experience take the
amount of antigen, equal to half the titer (0.5 ml). This is his working dose. Prepare a
total antigen dilution for the entire experiment by multiplying 0.5 ml by the number
of tubes in the experiment.
5. Antibody - patient's serum. Fresh serum is inactivated before the experiment in
order to destroy the complement present in it. To do this, it is heated for 30 minutes
at 56 ° C in a water bath or in an inactivator with a thermostat. The latter method is
preferable: it eliminates the possibility of serum overheating, i.e., its
denaturation. Denatured sera are not suitable for testing. The patient's serum is
usually used in a dilution of 1:10 to 1:160.
Immune sera are most often prepared under industrial conditions and released
inactivated. They are bred 1:50 and above.
Attention! All components are prepared with a slight excess.
Conducting the main experience
When setting up an experiment, the sequence of adding components is extremely
important. The experiment is carried out in two phases (Table 22).
 Table 22. Scheme of the main experience of the RSC
1
 ( In the experiment, serum can be studied in consecutive two-fold dilutions. )
Phase I The required amount of isotonic sodium chloride solution is poured into the
test tubes, then the required volume of diluted serum and the working doses of
antigen and complement in the same volume. The experience is necessarily
accompanied by the control of all the ingredients involved in it: serum, antigen,
hemolytic system and complement.
The tubes are thoroughly shaken and incubated at 37°C for 45 minutes - 1 hour or at
4°C ("CSC in the cold") for 18 hours. During this time, in the presence of a specific
complex, complement fixation occurs. Conducting the reaction "in the cold"
significantly increases its sensitivity and specificity.
Phase II . At the end of the incubation, 1 ml of the hemolytic system is added to all
test tubes, which is previously kept in a thermostat for 30 minutes (sensitized). The
tubes are shaken and put back into the thermostat.
Accounting for results. The tubes are left in a thermostat until complete hemolysis in
the 2nd, 3rd, 6th and 7th tubes (control of serum, antigen and complement for one
and two doses). First of all, hemolysis will occur in the 7th test tube, which contains a
double amount of complement. After hemolysis occurs in this tube and its contents
become completely transparent, you need to carefully monitor the rest of the
controls. As soon as the liquid in the 2nd, 3rd and 6th test tubes becomes
transparent, you should immediately remove the rack with test tubes from the
thermostat. The fact that the experiment was not kept in the thermostat longer than
necessary is indicated by the presence of slight turbidity (incomplete hemolysis) in
the 5th test tube - it contains only half of the working dose of complement and
complete hemolysis with the correct setting of the experiment cannot be.
Hemolysis in the serum and antigen controls (tubes 2 and 3) indicates that their
doses were chosen correctly and that neither serum nor complement antigen bind
by themselves.
In the control of the hemolytic system (tube 4), if it works properly, there should not
even be traces of hemolysis - it lacks complement.
After making sure that the controls were passed correctly, experience can be taken
into account. The absence of hemolysis in test tubes of experience is regarded as a
positive result of the reaction. It indicates that there are antibodies in the serum that
are specific for the antigen taken. The complex formed by them bound the
complement and prevented its participation in the hemolysis reaction. If hemolysis
occurs in the test tubes, the result of the reaction is assessed as negative. In this
case, there is no correspondence between the antigen and the antibody, the
complement is not bound and participates in the hemolysis reaction.
In parallel with the patient's serum, the same experiment is performed with a known
positive serum (that is, with serum in which there are antibodies to a given antigen)
and a known negative one, in which there are no specific antibodies. With the
correct setting of the experiment, in the first case there should be a delay in
hemolysis, and in the second case there will be hemolysis.
The intensity of the reaction is expressed as follows:
++++ Complete hemolysis delay. Erythrocytes form a uniform turbidity or settle to
the bottom. In this case, the liquid in the test tube becomes colorless;
+++ approximately 25% of erythrocytes were lysed. The sediment is smaller, the
liquid above it is slightly pink. The result of the RSC is also assessed as sharply
positive;
++ about 50% of erythrocytes were lysed. The sediment is small, the liquid is
pink. Positive RSK result;
+ about 75% of erythrocytes are lysed. Insignificant sediment, intensely colored
liquid above it. Doubtful result of RSK;
All erythrocytes were lysed. The liquid is intensely colored and completely
transparent. Negative RSK result.
Control questions
1. What is the RSC principle?
2. What systems are involved in RSC? What does the hemolytic system consist of and
what role does it play in the reaction?
3. What is the preparation for the basic experience of the RSC? In what order is it
carried out? How many phases are in the RSC?
4. What does the absence of hemolysis in CSC mean?
Exercise
1. Titrate complement and set its working dose.
2. Calculate all the ingredients for setting up the main experiment, conduct the
experiment, take into account and draw the result.

Immunofluorescence reaction

The immunofluorescence test (RIF) uses fluorescent microscopy (see Chapter 2) for
serological studies. The reaction is based on the fact that immune sera, to which
fluorochromes are chemically attached, when interacting with the corresponding
antigens, form a specific luminous complex visible in a fluorescent microscope. Such
serums are called luminescent * . The method is highly sensitive, simple, does not
require the isolation of a pure culture (you can detect microorganisms directly in the
material from the patient: feces in cholera, sputum in whooping cough, brain tissue
in rabies). The result can be obtained half an hour after applying the luminescent
serum to the preparation. Therefore, RIF is widely used in express (accelerated)
diagnostics of a number of infections.
*
 ( Fluorochromes: fluorescein gives a green glow, rhodamine - red. )
To prepare preparations, a slide with a fixed smear (imprint, cut) is placed in a humid
chamber. The chamber is prepared as follows. Wet filter paper is placed on the
bottom of the Petri dish. Two glass rods are placed on it in parallel (you can use the
wide part of Pasteur pipettes). A glass slide is placed on them with a smear up.
Attention! Do not forget to circle the smear on the reverse side with a wax pencil.
A drop of luminescent serum is applied to the smear. The cup is closed and placed in
a thermostat or left at room temperature for 20-30 minutes. After incubation, it is
washed with a buffered isotonic solution (pH 7.4), rinsed with distilled water, dried, a
drop of buffered glycerol is applied, covered with a coverslip (not thicker than 0.17
mm!) and examined in a fluorescent microscope. If the preparation contains
microbes that are homologous to luminescent serum antibodies, they glow brightly
against a dark background. This method is called direct (Fig. 36). The inconvenience
of the direct RIF method is that it requires luminescent sera for each determined
antigen, which is difficult to prepare, and there is no complete set of ready-made
luminescent sera for any antigen. Therefore, the indirect method is often used. It
consists in that at the first stage the drug is treated with non-luminescent immune
specific serum to the desired antigen. If the preparation contains the desired
antigens (microbes), then an antigen-antibody complex is formed that cannot be
seen. After drying, at the second stage, the preparation is treated with luminescent
serum containing antibodies not to the desired antigen, but to globulins of the
animal species from which the specific serum was obtained. For example, if the first
serum was obtained during the immunization of a rabbit, then the second should
contain antibodies to rabbit globulins (see Fig. 36). These antibodies combine with
specific serum globulins that have been adsorbed on the desired antigen, and the
complex glows when the preparation is viewed through a fluorescent microscope. if
the preparation contains the desired antigens (microbes), then an antigen-antibody
complex is formed that cannot be seen. After drying, at the second stage, the
preparation is treated with luminescent serum containing antibodies not to the
desired antigen, but to globulins of the animal species from which the specific serum
was obtained. For example, if the first serum was obtained during the immunization
of a rabbit, then the second should contain antibodies to rabbit globulins (see Fig.
36). These antibodies combine with specific serum globulins that have been
adsorbed on the desired antigen, and the complex glows when the preparation is
viewed through a fluorescent microscope. if the preparation contains the desired
antigens (microbes), then an antigen-antibody complex is formed that cannot be
seen. After drying, at the second stage, the preparation is treated with luminescent
serum containing antibodies not to the desired antigen, but to globulins of the
animal species from which the specific serum was obtained. For example, if the first
serum was obtained during the immunization of a rabbit, then the second should
contain antibodies to rabbit globulins (see Fig. 36). These antibodies combine with
specific serum globulins that have been adsorbed on the desired antigen, and the
complex glows when the preparation is viewed through a fluorescent
microscope. containing antibodies not to the desired antigen, but to the globulins of
the animal species from which the specific serum was obtained. For example, if the
first serum was obtained during the immunization of a rabbit, then the second
should contain antibodies to rabbit globulins (see Fig. 36). These antibodies combine
with specific serum globulins that have been adsorbed on the desired antigen, and
the complex glows when the preparation is viewed through a fluorescent
microscope. containing antibodies not to the desired antigen, but to the globulins of
the animal species from which the specific serum was obtained. For example, if the
first serum was obtained during the immunization of a rabbit, then the second
should contain antibodies to rabbit globulins (see Fig. 36). These antibodies combine
with specific serum globulins that have been adsorbed on the desired antigen, and
the complex glows when the preparation is viewed through a fluorescent
microscope.
Rice. 36. Scheme of immunofluorescence reaction (RIF). I - direct method: II - indirect
method: a - 1st stage of setting up the reaction; b - 2nd stage of setting the reaction:
1 - the studied antigen: 2 - luminescent antibody to the studied antigen; 3 - non-
luminescent antibody to the studied antigen; 4 - luminescent antibody to the
globulins of the animal from which the antibodies to the studied antigen were
obtained; 5 - luminous immune complex: 6 - non-luminous immune complex

Opsonophagocytic reaction

Opsonophagocytic reaction (OPR) is one of the methods for assessing the activity of
immune phagocytosis. The higher this activity, the higher the body's resistance to
infection. In the immune organism, under the influence of antibodies (opsonins),
phagocytosis proceeds more actively (more microbes are absorbed in a shorter
period). Therefore, indicators of phagocytic activity are not only of diagnostic value
(for example, in brucellosis), but also allow predicting the outcome of the infectious
process, evaluating the results of treatment and vaccination. For the reaction you
need:
1. Antigen - a suspension of live or killed microorganisms.
2. Antibody (opsonins) - test serum.
3. Phagocytes - usually neutrophils of the studied blood.
Reaction setting. Using a micropipette, 0.05 ml of 2% sodium citrate solution is
poured into small test tubes; 0.1 ml of the test blood and 0.05 ml of a suspension of
microorganisms, the density of which corresponds to 10 units in 1 ml. turbidity
according to the GISK optical standard.
Attention! A separate pipette must be used for each ingredient.
Mix the contents of the tubes. The test tubes are placed in a thermostat for 30
minutes, after which their contents are mixed again and thin smears are prepared
(like blood smears). Stained according to Romanovsky - Giemsa.
Accounting for results. In different places of the smear, 25 neutrophils are counted,
taking into account the number of captured microorganisms in each of them. The
index of opsonophagocytic reaction (POFR) is calculated by the formula:
POFR = 3a + 2b + 1c + 0,
where a is the number of neutrophils containing more than 41 bacteria; b - the
number of neutrophils containing from 21 to 40 bacteria; c is the number of
neutrophils containing from 1 to 20 bacteria; 0 - the number of neutrophils that do
not contain bacteria.
The maximum indicator of the opsonophagocytic reaction with this accounting
system is 75.
The result of the reaction is evaluated according to the following scheme:
with POFR from 1 to 24 - weakly positive;
with POFR from 25 to 49 - pronounced;
with POFR from 50 to 75 - sharply positive.
In healthy people, the POFR is 0-1, rarely 4-5. The clear and sharply positive results of
the reaction indicate a high opsonizing effect of the serum of the examined person
with a pronounced activity of blood phagocytes.
Determination of only the activity of antibodies - opsonins is carried out by the
experience of establishing the opsoic index - the ratio of the phagocytic index in the
presence of immune (tested) serum to the phagocytic index in serum, which
obviously does not contain antibodies to a given microbe. The experiment is set up
as follows: 2 test tubes are taken, into one of which (experimental) they are added in
equal amounts (usually 0.2 ml): 1) the serum of the person being examined; 2) a
suspension of microbes, in which the presence of opsonins is determined; 3)
leukocytes (possible from the abdominal cavity of the mouse). The following is added
to the control tube: 1) serum without opsonins (control); 2) the same microbes as in
the experimental one; 3) leukocytes (the same as in the test tube).
Both tubes are kept in a thermostat for 30 minutes, and then smears are prepared
from one and the other, fixed and stained according to Romanovsky-Giemsa. Smears
are microscoped and the phagocytic index is determined in experimental and control
tubes.
In the presence of opsonins in the test serum, the opsonic index will be greater than
one. The greater the number obtained from dividing the phagocytosis index of the
test serum by the phagocytic index of the control serum, the more pronounced the
effect of antibodies - opsonins.
Control questions
1. On what property of antibodies is OPA based? Is this reaction specific?
2. What does an OFR score of 75 indicate?
Exercise
Examine the OFR of blood taken from a finger. Draw phagocytes. Calculate PORF.

Immunity reactions in vivo (skin tests)

When applying the antigen to scarified skin or intradermally, both the immune state
and the state of hypersensitivity to this drug can be detected.
Skin test with toxin . A titrated amount of toxin is injected intradermally. If the body
is immune, that is, it has a certain level of antitoxin, the action of the toxin will not
manifest itself - the toxin will be neutralized by the antitoxin. In a non-immune
organism, an inflammatory infiltrate (redness, induration, etc.) will develop at the
injection site of the toxin.
Allergen skin tests (allergy skin tests) to study hypertype reactions (see Chapter
13). With increased sensitivity of the immediate type, the introduced allergen
(antigen) reacts with antibodies adsorbed on the cells of various
organs. Hypersensitivity of the delayed type is due to the reaction to the allergen of
sensitized T-lymphocytes. Such sensitization occurs in a number of infections in
patients who have been ill and vaccinated (tuberculosis, brucellosis, etc.). Therefore,
skin-allergic tests for these infections are of diagnostic value.
Preparations for skin tests are prepared by special manufacturers, providing
instructions for their use.
Control questions
1. What is an antibody in a toxin skin test? What does a negative result of this test
indicate?
2. What reaction allows you to identify the state of increased sensitivity of the
organism to an infectious agent?

Immunoprophylaxis and immunotherapy of

infectious diseases

Attempts to prevent the severe course of a deadly disease by causing a mild form of
the disease have been made for centuries in different countries of the world.
The scientific justification and practical implementation of immunoprophylaxis was
first given by L. Pasteur, who created the principles for the use of weakened
(attenuated) microorganisms and prepared preparations (vaccines) to prevent
certain infectious diseases in humans and animals.
More than a hundred years have passed and now the artificial creation of immunity
is the basis of the fight against infectious diseases.
Immunization - the introduction of drugs to create artificial active immunity - is
carried out in certain years throughout a person's life. In the very first days after
birth, the child receives the BCG vaccine against tuberculosis. In the 1st year of life,
he is vaccinated to prevent diphtheria, whooping cough and tetanus, vaccinated
against poliomyelitis, measles, etc. Thus, specific prevention of infectious diseases is
carried out, for which vaccines are used.
Vaccines - preparations for active immunization can be:
1. Corpuscular (from microbial cells) - living and dead.
2. Chemical (antigens and antigenic fractions).
3. Anatoxins.
Live attenuated vaccines are prepared from living microorganisms, the virulence of
which is weakened (from the Latin attenuer - to weaken, soften), and the
immunogenic properties (the ability to cause immunity) are preserved.
There are different ways to obtain such microorganisms:
1) cultivation on nutrient media unfavorable for the growth and reproduction of the
pathogen; under the action of physical and chemical factors (this is how the BCG
vaccine was obtained for the prevention of tuberculosis); 2) passage of the pathogen
through the body of an animal that is not very susceptible to a reproducible infection
(this is how L. Pasteur received the rabies vaccine); 3) selection of natural cultures of
microorganisms that are slightly virulent for humans (this is how the plague vaccine
was obtained), etc.
Live vaccines create intense immunity, as they cause a process similar to a natural
infectious disease, only mildly pronounced, with almost no clinical manifestations. In
this case, the entire mechanism of immunogenesis is activated - immunity is created.
Killed vaccines are cultures of microorganisms inactivated by the action of high
temperature, chemicals (phenol, formalin, alcohol, acetone), UV rays, etc. At the
same time, such influence factors are selected that fully preserve the immunogenic
properties of microbial cells.
Chemical vaccines are individual components of a microbial cell (antigens) obtained
by special treatment of a microbial suspension.
Chemical vaccines are usually rapidly absorbed after introduction into the body,
which does not allow the desired immunogenic stimulation to be achieved,
therefore, substances are added to the vaccines that prolong the absorption time:
aluminum hydroxide, aluminum-potassium alum, mineral oils, etc. This is called the
creation of a "depot".
Chemical vaccines are used to prevent typhoid fever, meningitis, etc.
Anatoxins (from Latin ana - back) are exotoxins of bacteria, neutralized by exposure
to formalin (0.3-0.4%) and exposure at a temperature of 37 ° C for 3-4 weeks. In this
case, there is a loss of toxic properties, but the preservation of immunogenic ones.
At present, toxoids have been obtained and used from the toxins of pathogens of
diphtheria, tetanus, etc.
Anatoxins are purified from impurities of nutrient media (ballast proteins) and
sorbed on substances that are slowly absorbed from the injection site.
According to the number of antigens that make up the vaccine, they distinguish:
monovaccines (from one type of antigens), divaccines (from two antigens), three
vaccines (from three antigens), etc.
Associated vaccines are prepared from antigens of various bacteria and toxoids. For
example, the associated pertussis-diphtheria-tetanus vaccine (DPT) contains killed
pertussis microbes and toxoids: diphtheria and tetanus.
Vaccines are administered intramuscularly, subcutaneously, cutaneously,
intradermally, orally. Immunize either once, or twice and three times at intervals of
1-2 weeks or more. The frequency of administration, the intervals between
vaccinations depend on the nature of the vaccine - for each, administration schemes
have been developed.
After the introduction of the vaccine, general and local reactions may
occur. Common include fever (up to 39 ° C), headache, malaise. These phenomena
usually disappear in 2-3 days. Local reactions - redness and infiltration at the
injection site may appear 1-2 days after vaccination. With the cutaneous
administration of a vaccine (against tularemia, BCG, etc.), the appearance of a local
reaction indicates the effectiveness of the vaccination.
There are contraindications for vaccination: fever, acute infectious diseases,
allergies, etc. Do not vaccinate women in the second half of pregnancy.
Vaccines and toxoids are prepared at enterprises producing bacterial
preparations. Large quantities of microbial suspension (biomass) or material
containing viruses are needed for their manufacture.
Finished preparations are poured into ampoules or vials and mostly dried. Dry
preparations retain activity and other properties longer.
Some vaccines, such as polio, are available as tablets or dragees.
Labels are attached to each ampoule, vial and box with drugs indicating the name of
the drug, its volume, expiration date, batch number and control number.
Instructions for use are included in each box.
Store preparations mainly at a temperature of 4 ° C. Do not expose preparations to
freezing and thawing, high temperatures. During transportation, special conditions
are observed. Do not use drugs that have cracks in the ampoules and a changed
appearance.
In the USSR, there is a system of state control over the quality of medical
immunobiological preparations, which ensures their effectiveness and
standardization.
A special type of vaccine - and then the vaccine. They are prepared in bacteriological
laboratories from microbes isolated from the patient. Autovaccine is used to treat
only this patient. Most often, autovaccines are used to treat chronic infections
(staphylococcal, etc.). The autovaccine is administered repeatedly, in small doses,
according to the scheme developed for each vaccine. Autovaccines stimulate the
body's defenses, which contribute to recovery.
Serum preparations are used to create artificial passive immunity. These include
specific immune sera and immunoglobulins.
These preparations contain ready-made antibodies. They are obtained from the
blood of donors - specially immunized people or animals (against measles, influenza,
tetanus). In addition, the serum of recovered and even healthy people is used if it
contains a sufficient amount of antibodies. Placental and abortive blood is also used
as a raw material for the preparation of immune preparations.
There are antibacterial and antitoxic serums. The former are of more limited
use. Antitoxic sera are used to treat diphtheria, tetanus, botulism, etc. These sera are
produced with a certain content of antitoxin, which is measured in international
units (IU).
Immune serum preparations are obtained from the blood of animals, mainly horses,
repeatedly immunized. At the end of immunization, the level of antibodies in the
blood is determined and bloodletting is done. The resulting serum is preserved, its
sterility, activity and physical properties are controlled.
Preparations derived from the blood of horses contain proteins that are foreign to
humans, which, if repeatedly administered, can cause allergic reactions: serum
sickness and anaphylactic shock. To prevent complications, serum preparations
should be administered with caution (according to Bezredka) (see Chapter
13). Various methods are used to free animal sera from ballast proteins and to
concentrate antibodies, the main of which is the Diaferm-3 method, developed in
our country and including enzymatic hydrolysis of ballast proteins.
In addition, for the concentration of antibodies in a smaller volume of the drug,
methods have been developed for isolating gamma globulins containing antibodies
from blood serum. These drugs are called immunoglobulins. They are prepared from
human (homologous) and animal (heterologous) serum.
The effectiveness of immunoglobulins is much higher than that of immune sera, and
there are disproportionately fewer complications. Currently, immunoglobulins are
used much more widely than sera.
In our country, immunoglobulins are used to prevent measles, hepatitis, rubella, etc.
Prophylactic administration of immunoglobulins is carried out if infection is
suspected or if infection occurs. It is advisable to administer these drugs in the first
days after infection (the beginning of the incubation period), while the pathological
process has not yet developed.
In the therapeutic use of the drug, its early administration gives a greater effect.
Serum and immunoglobulins are administered intramuscularly and intravenously.
Timely and correct use of serum preparations can reduce the incidence of many
infections.
Control questions
1. What types of vaccines do you know?
2. What drugs create passive immunity?
3. What is an autovaccine?

Chapter 13. Allergy - L. B. Bogoyavlenskaya

Allergy (from lat. alios - different, ergon - action) is a state of altered, increased
sensitivity of the body to various foreign substances (antigens).
Substances that cause a state of hypersensitivity (hypersensitivity) of the body are
called allergens. Allergens can be: microorganisms (bacteria, viruses, fungi), decay
products of microbial cells; animal proteins (eggs, milk, etc.); vegetable proteins
(strawberries, mushrooms, etc.); therapeutic heterologous sera, etc.
All of these substances are complete antigens. In addition, allergies can be caused by
haptens - substances that become allergens after combining with body proteins:
industrial allergens (dyes, varnishes, soaps, etc.); household allergens (dust, dog and
cat hair, pillow fluff, etc.); plant allergens (pollen of plants during flowering,
etc.); medicinal substances (antibiotics, aspirin, etc.).
Allergy is a specific hypersensitivity of the body to various agents. It is based on the
antigen-antibody reaction. When entering the body for the first time, some allergens
form antibodies, while other allergens sensitize T-lymphocytes. In either case, the
body acquires an increased sensitivity to a re-encounter with the antigen that caused
these changes. This repeated meeting can manifest itself in different ways depending
on the allergen, the nature of the body's immune restructuring.
All allergic reactions are divided into two groups: immediate-type hypersensitivity
reactions (HHT) and delayed-type hypersensitivity reactions (DTH). Depending on the
form of allergy, a hypersensitivity reaction of one type or another is manifested.
Hypersensitivity reactions
Immediate type (HIT) Delayed type (DTH)
1. Anaphylaxis 1. Infectious allergy
2. Arthus-Sakharov phenomenon 2. Contact dermatitis
3. Serum sickness 3. Drug allergy
4. Atopy (hay fever, bronchial asthma,
hives)

Immediate type hypersensitivity reactions

Anaphylaxis (from Latin ana - against, phylaxis - protection) is a hypersensitivity that

manifests itself immediately after repeated administration of a foreign antigen in the
form of shock or states close to it.
Substances that cause anaphylaxis are called anaphylactogens. These include foreign
proteins, bacterial toxins, microbial cell polysaccharides, various medicinal
substances, i.e., full-fledged antigens and haptens.
mechanism of anaphylaxis. The first injection of an anaphylactogen (for example,
horse serum into a guinea pig) causes specific sensitization. Antibodies (IgE) appear,
which accumulate in the maximum titer after 10-12 days. Circulating in the blood,
these antibodies are partially adsorbed on the cells of the body.
The first dose of a foreign protein that causes sensitization is called the sensitizing
dose. This is usually a very small dose (0.01-0.0001 ml of horse serum for a guinea
pig). Sensitization occurs when the antigen is administered parenterally. However, it
can also occur when antigens pass through the mucous membrane of the intestine or
lungs. The developed state of allergy can persist for a long time - several months and
even years.
Repeated administration of the same anaphylactogen causes an immediate type of
reaction - anaphylactic shock, from which the animal dies. The conditions for the
development of anaphylactic shock are: 1) a repeated dose (allowing) should exceed
the sensitizing dose by 10-100 times; 2) the resolving dose must be injected directly
into the blood.
In the pathogenesis of anaphylaxis, the main role is played by antibodies formed in
response to the introduction of a foreign protein or other anaphylactogens. These
antibodies are partially adsorbed on cells, which are called target cells (fat,
basophils), etc. When a permissive dose of the allergen is re-administered, it reacts
with antibodies on the surface of these cells, and the integrity of cell membranes is
disrupted. This leads to a massive release of substances such as histamine from the
cells, which cause the development of anaphylactic shock. The combination of
antigen with antibodies circulating in the blood leads to the formation of
precipitates, which also cause the activation of mediators.
If the serum of a sensitized animal is administered to a healthy animal of the same
species, then after 1-2 days (this time is necessary for fixing the introduced
antibodies on target cells), it becomes sensitized. A resolving dose of anaphylactogen
will cause shock in an animal that has received ready-made antibodies. This is passive
anaphylaxis.
The clinical picture of anaphylactic shock is different in different animals.
In a guinea pig, the reaction after intravenous administration of the second dose of
anaphylactogen occurs immediately: the animal becomes restless, scratches its nose
with its paws, sneezes, shortness of breath appears, then convulsions, involuntary
excretion of urine and feces - the animal dies. At autopsy, bronchospasm, swollen
lungs, hyperemia and hemorrhages in the digestive tract are noted.
In dogs, anaphylactic shock is accompanied by vasospasm and stagnation of blood in
the liver. The death of the animal occurs with a pronounced drop in blood pressure.
Rabbits with anaphylaxis die from respiratory arrest and a drop in blood
pressure. These phenomena are caused by spasm of the arteries of the pulmonary
circulation.
In humans, anaphylactic shock develops most often when the rules for injecting
serum preparations are violated or when penicillin and other drugs are
administered. The reaction is accompanied by a spasm of smooth muscles, a
violation of the activity of the cardiovascular system. Body temperature drops by 1-2
° C, shortness of breath, frequent pulse appear, a drop in blood pressure,
convulsions, joint pain, etc. Sometimes anaphylactic shock ends in death.
To prevent anaphylactic shock, desensitization should be carried out, that is,
hypersensitivity should be removed. For this purpose, before the introduction of the
entire amount of the anaphylactogenic substance, a small dose of it is administered,
which does not cause shock, but binds antibodies to the injected
anaphylactogen. For example, if it is necessary to administer to a person an alien
horse serum (immune tetanus toxoid, antidiphtheria), 0.5-1.0 ml is first
administered, and after 2 hours the remaining dose is administered. This is a way to
introduce serum according to Bezredka.
Serum preparations are always administered fractionally. First, determine the
sensitivity of a person to the administered drug. For this purpose, 0.1 ml of the test
serum, diluted 1:100, is injected intradermally into the flexor surface of the
forearm. If the reaction is negative (slight redness and swelling less than 1 cm), after
20-30 minutes, 0.1-0.5 ml of undiluted serum is injected subcutaneously. With a
negative reaction after 30-60 minutes, the entire dose is administered.
Local anaphylaxis - the Arthus-Sakharov phenomenon . The reaction manifests itself
in the form of a local reaction to the repeated introduction of the allergen not into
the blood, but intradermally or subcutaneously. In experiments on rabbits, it was
found that 3-4 injections of horse serum under the skin pass without a trace, and on
the 5-7th injection, an inflammatory reaction and necrosis appear on the skin. This
phenomenon is used in practice to identify the state of sensitization in humans to
various substances. A small amount of the test substance (serum, various medicinal
substances, proteins) is injected intradermally. The appearance of redness and
swelling (swelling of more than 1 cm) indicates increased sensitivity.
Serum sickness develops when a person is injected with foreign serum (for example,
horse). It can occur immediately after the administration of the drug and is severe,
similar to anaphylactic shock. This most often occurs with repeated administration of
serum, when the body already has antibodies to it. But serum sickness can also
develop with a single administration of a large dose of serum. In this case, it
manifests itself 8-12 days after administration, since during this period antibodies to
serum are synthesized in the body. A rash (urticaria), itching, joint pain, swelling of
the lymph nodes appear, the temperature rises. Gradually, all these symptoms
disappear.
To prevent serum sickness, serum should be administered according to Bezredka.
The use of immunoglobulins avoids serum sickness.
Atonic reactions (atopy) (from Latin atopos - unusual, oddity) occur in response to
the ingestion of allergens in persons with increased sensitivity to them. The
predisposition of hypersensitivity is inherited.
The mechanism of these reactions also consists in the interaction between the
allergen and the antibodies that were formed during the first meeting of the body
with this allergen. In this case, as in anaphylaxis, histamine and substances similar to
it are released, which cause spasm of smooth muscles, increase vascular
permeability, etc.
Depending on the organ and tissue on the cells of which antibodies are fixed
(attached), various conditions arise: respiratory tract damage - allergic rhinitis and
bronchial asthma; damage to the mucous membrane of the eyes - conjunctivitis; skin
- urticaria, etc.
In addition, atopy manifests itself in the form of intolerance to certain substances:
food, medicinal, vegetable. Unlike anaphylaxis, atopic conditions are not amenable
to desensitization and are observed only in humans.
Bronchial asthma. The disease proceeds in the form of asthma attacks with severe
spastic cough. It develops as a result of muscle spasm and swelling of the mucous
membrane of the bronchioles. Asthma is often caused by exposure to various
allergens - plant pollen, animal dander, drugs, etc.
Pollinosis (hay fever). It usually develops in spring and summer, during the flowering
period of plants. When inhaled and in contact with plant pollen or fungal spores,
conjunctivitis, runny nose, headache, and sometimes asthma attacks occur. The
development of the disease is associated with previous sensitization of the
organism. In the blood, antibodies to plant pollen can be detected. In this case, the
change in sensitivity can relate to one type of plant (rye, clover, dahlias, etc.) or to
many at once.
Urticaria is manifested by a rash in the form of large red "cakes" and itching. Occurs
when eating foods (strawberries, mushrooms, eggs, etc.) or in contact with
chemicals (eg, phenolphthalein).

Delayed type hypersensitivity reactions

Delayed-type hypersensitivity reactions are forms of allergy in which sensitization of

the body is associated with the activation and accumulation of T-lymphocytes
(sensitized T-lymphocytes). Unlike GNT, DTH reactions are, firstly, not associated
with circulating blood antibodies and, therefore, passive transfer of DTH to another
animal by administration of the serum of a sensitized animal cannot be carried
out. Secondly, they do not develop immediately, but 24-28 hours after contact with
the allergen.
Mechanism of delayed-type hypersensitivity reaction. The first encounter of an
allergen with a T-lymphocyte (having a receptor corresponding to this allergen) leads
to the activation and proliferation (reproduction) of the T-cell. As a result, a clone of
T-lymphocytes sensitized to this allergen accumulates in the body. Upon a second
encounter with the same allergen, T-lymphocytes are activated again and involve
macrophages in the process of destruction of target cells carrying the antigen. At the
same time, T-lymphocytes also die. Substances that are toxic to surrounding cells are
released. The clinical picture of allergy develops.
Infectious allergy is a state of hypersensitivity to repeated contact with
microorganisms or their waste products. It develops in many infectious
diseases; plays an important role in their pathogenesis and persists for a long time
after recovery. Infectious allergies are observed in tuberculosis, brucellosis, syphilis,
etc. The specificity of reactions in infectious allergies is used to diagnose many
infectious diseases (tuberculosis, brucellosis, tularemia, etc.) - skin-allergic tests are
used. Very small amounts of allergens are injected intradermally or cutaneously -
filtrates or lysates of cultures, suspensions of bacteria killed by heat or chemicals,
etc.
With increased sensitivity at the injection site of the allergen, a reaction occurs:
redness, swelling, soreness. Sometimes general reactions also develop: weakness,
malaise, exacerbation of the general process (for example, after the introduction of
tuberculin in tuberculosis).
Contact dermatitis is an allergic skin disease. They develop with prolonged contact
with various chemicals. Allergens can be: soaps, glues, dyes, rubber, drugs, cosmetics
and other usually harmless substances. They are haptens, but when combined with
body proteins, they become antigens (allergens). The manifestations of the disease
are diverse - from reddening of the skin to necrosis, contact dermatitis includes
eczema (eczematous dermatitis).
Some people develop intolerance to various food substances (strawberries, egg
curds, etc.), drugs (acetylsalicylic acid, amidopyrine, etc.). Being haptens, these
substances, when combined with body proteins, become antigens and cause
allergies.
Many allergic reactions can only be prevented by preventing re-exposure to the
allergen. However, sometimes it is difficult to identify the agent that caused the
hypersensitivity state. To do this, put intradermal tests with the alleged allergen.
In recent years, many allergens have been obtained in our country, both infectious
(from various microorganisms that cause infectious allergies) and non-infectious
(from various types of dust, food products, chemicals, etc.). But it should be
remembered that the introduction of even small doses of antigens into the body
causes its additional allergization. Therefore, in recent years, laboratory methods of
allergy diagnostics have been increasingly used.
Immunoserological and immunocytological tests . Immunoserological tests are
based on the detection of allergic antibodies in the blood serum or an increased
concentration of IgE in B-dependent allergies (mainly atony). The most accurate is
the radioallergosorbent test (PACT), based on the principle of antiglobulin reaction
using labeled antiserum to IgE.
Immunocytological methods are based on visual registration of specific allergic cell
damage. For the diagnosis of B-dependent allergies, the Shelley basophilic test, the
mast cell degranulation test, etc. are used. For T-dependent allergies, other tests are
informative: determining the indicator of neutrophil damage (according to Fradkin),
the reaction of blast transformation of lymphocytes, inhibition of macrophage
migration. A simple and informative test is the determination of the neutrophil
damage index (DIT). Allergens for its production are produced in our country. The
PPN technique consists in recording the amoeboid activity of neutrophils upon
contact with an allergen. The appearance of amoeboid protrusions upon contact
with an allergen is the initial phase of neutrophil damage and indicates an increased
sensitivity of the body to this allergen.
To register this phenomenon, the blood of the subject is mixed with an allergen (for
example, brucellin) and an anticoagulant. The mixture is incubated As a control,
blood combined with anticoagulant alone is also incubated. Both blood samples are
then swabbed and viewed under a microscope. In each smear, 100 neutrophils are
counted. The damage index is calculated by the formula:

where H O is the number of damaged neutrophils in the experimental smear, Hk is

the number of damaged neutrophils in the control preparation. In healthy people,
the PPN does not exceed an index of 0.1.
Control questions
1. What types of allergic reactions do you know?
2. How does anaphylaxis develop and manifest itself?
3. How to prevent an anaphylactic reaction of the body when administering serum
preparations'7
 Part II. Private microbiology
Pathogenic cocci - F.K. Cherkes
Cocci are an extensive group of microorganisms, including pathogenic, opportunistic
and non-pathogenic representatives.
This chapter will discuss pathogenic and opportunistic cocci.
According to Bergi's classification, pathogenic cocci belong to three families:
1. Micrococcaceae - the genus Staphylococcus (staphylococci).
2. Streptococcaceae - the genus Streptococcus (streptococci and pneumococci).
3. Neisseriaceae - the genus Neisseria (meningococci and gonococci).
A common feature for all pathogenic cocci is their ability to cause purulent
processes, therefore they are called pyogenic (pyogenic).
The degree of organotropy in cocci is not the same, it is most pronounced in
pneumococci, meningococci and gonococci.
All pathogenic cocci are immobile, do not form spores, pneumococci form a capsule.
By tinctorial properties, they are divided into gram-positive (staphylococci,
streptococci) and gram-negative (meningococci, gonococci).
Pathogenic representatives of the pyogenic group differ from each other in nutrient
requirements and biochemical activity. The least demanding on media, and
biochemically more active are staphylococci, the most demanding on media and the
least biochemically active are gonococci.
Chapter 14. Staphylococci

Staphylococci were first discovered by L. Pasteur in 1897. They were studied in

detail by A. Ogston (1882) and F. Rosenbach (1884).

Morphology . Staphylococci (from the Greek staphyle - a bunch of grapes) have

the form of round balls with a diameter of 0.5-1.5 microns. Reproducing, they form
clusters in the form of a bunch of grapes. This form is the result of the division of
microbes in different planes. However, single and paired cocci are found in
pus. Staphylococci are immobile, do not have spores, form a microcapsule under
special cultivation conditions, and are gram-positive.

Cultivation . Staphylococci are facultative anaerobes, but grow better in the

presence of oxygen. Grow and multiply on ordinary nutrient media, grow well on
media with blood, optimal conditions - temperature 37 ° C, pH 7.2-7.4.

Elective media are yolk-salt agar and salt agar. On MPA, staphylococcus
colonies are convex, round, opaque, shiny, 2-4 mm in size with smooth edges. With
the growth of staphylococci, they form a pigment: golden, lemon yellow or
white. The pigment is best formed on a milky medium at room temperature and
diffused light. The staphylococcal pigment does not dissolve in water; it dissolves
in acetone, ether, alcohol, etc. With the growth of some strains of staphylococcus
on agar with blood, a hemolysis zone is formed around the colony. Growth on the
broth is characterized by uniform turbidity and sediment at the bottom.

enzymatic properties . Staphylococci produce saccharolytic and proteolytic

enzymes. Saccharolytic enzymes break down a number of sugars: lactose, glucose,
sucrose, maltose, glycerol and others with the formation of acid.

The proteolytic properties of staphylococcus are expressed in the ability to

dissolve casein, liquefy gelatin (slowly), break down other protein substrates.

Staphylococci produce enzymes of pathogenicity: 1) coagulase (coagulates

blood plasma); 2) hyaluronidase (spread factor); 3) lecithinase (dissolves cell
membrane lecithin); 4) fibrinolysin (lyses fibrin); 5) DNase (depolymerizes
DNA); 6) phosphatase, etc.

The presence of plasmacoagulase makes it possible to differentiate

Staphylococcus aureus from staphylococci of other species. Many staphylococci
produce penicillinase, which destroys penicillin.
 Toxin formation . Staphylococci produce exotoxins. These include hemolysins
of four types, of which the α-toxin is of the greatest importance. It has the
following properties: hemolytic - causes hemolysis of erythrocytes, dermonecrotic -
causes necrosis when administered intradermally, lethal - when administered
intravenously leads to the death of animals sensitive to it.

In addition to hemolysins, staphylococci form leukocidin, which kills

leukocytes, six types of enterotoxins, which cause food poisoning, and two types of
exfoliatins, leading to exfoliation of the epidermis in newborns.

Antigenic structure . Staphylococci have protein antigen A, common to all

Staphylococcus aureus, and polysaccharide antigens: A, B, C.

Staphylococci secrete bacteriocins (staphylocins), which have an antagonistic

effect against microorganisms of this genus.

Among golden (rarely epidermal) staphylocci, about 40 fagovars are

distinguished. Determining the sensitivity of staphylococcal cultures isolated from
various objects to typical phages is of great epidemiological importance (when
establishing the source and routes of transmission of the pathogen).

Classification . Currently, staphylococci isolated from humans are divided into

3 species (Table 23): S. aureus, S. epidermidis, S. saprophyticus.

Table 23. Differentiation of staphylococcus species isolated from humans

Note. + the presence of fermentation, stability, - the absence of fermentation,

stability.
 Resistance to environmental factors . Staphylococci are quite stable, so they
are found in the air, soil, water, and on household items. At a temperature of 100 °
C, they die instantly, at a temperature of 70 ° C - after 10-15 minutes. They tolerate
low temperatures well. When frozen, they remain viable for several years. They
tolerate drying well. Direct sunlight only kills them after a few hours. Ordinary
solutions of disinfectants (for example, sublimate at a dilution of 1:1000) kill them
in 15-20 minutes. When neutralizing secretions containing pus, protein, sputum,
phenol should not be used. This disinfectant causes proteins to coagulate, which
prevents microorganisms from dying. Staphylococci are sensitive to brilliant green.

Susceptibility of animals . Large and small cattle, horses, pigs, chickens are

sensitive to staphylococcus. From experimental animals - rabbits, white mice and
kittens.

Sources of infection . A sick person and a bacteriocarrier.

transmission paths . Contact-household, air-drop, air-dust, food.

Diseases in humans . Pyoderma, boils, carbuncles, felons,

abscesses; inflammatory processes of various organs and tissues; tonsillitis, cystitis,
osteomyelitis, cholecystitis, mastitis; sepsis and septicopyemia; food poisoning and
many others. About 120 nosological forms of staphylococcal etiology have been
described.

Pathogenesis . Staphylococci penetrate through the skin and mucous

membranes.

Staphylococcus aureus (S. aureus) is of primary importance in staphylococcal

diseases. The role of S. epidermidis and S. saprophyticus in human pathology is
less pronounced. Pathogenesis is determined by the properties of the pathogen -
enzymes, exotoxins, substances of the bacterial cell and the state of the immune
system of the macroorganism.

The skin and subcutaneous tissue are more often affected - pyodermatitis, boils,
panaritiums occur. Often, staphylococci cause secondary diseases, such as
pneumonia with influenza. They also cause wound infections. The role of
staphylococci in obstetric practice is especially great, since newborns are very
sensitive to them. In the course of staphylococcal diseases, the development of
allergies is important, so the disease is characterized by relapses.
 A special place among staphylococcal diseases is occupied by food
intoxication. Clinically, they proceed as toxicosis, accompanied by vomiting,
diarrhea, headache and other phenomena.

Immunity . A person has a natural resistance associated with mechanical

factors, phagocytosis and the presence of antibodies. The inflammatory process that
occurs at the site of the introduction of the pathogen causes the delay of
staphylococci and makes it difficult for them to spread throughout the body. In the
resulting focus, staphylococci undergo phagocytosis.

The antitoxin formed during the disease is an important factor in the overall
immunity complex. However, acquired immunity is unstable, so relapses are
observed.

Prevention . It comes down to the improvement of sanitary and hygienic

conditions, the active identification of patients and bacteria carriers, the correct
mode of operation of hospitals.

specific prophylaxis . Staphylococcal toxoid and antistaphylococcal

immunoglobulin.

Treatment . Antibacterial drugs, polyvalent staphylococcal bacteriophage,

antistaphylococcal plasma and immunoglobulin. In some cases, in the chronic
course of staphylococcal infections, an autovaccine is used.

Control questions
1. On what basis are cocci united in one group?

2. What enzymes and pathogenicity factors are produced by staphylococci?

3. What diseases are caused by staphylococci?

4. What types of staphylococci do you know?

Microbiological research
The purpose of the study: isolation and identification of staphylococci.

Research material

1. Pus (boils, carbuncles, abscesses).

2. Mucus from the pharynx (tonsillitis).

3. Phlegm (pneumonia).

4. Urine (pyelitis and cystitis).

5. Duodenal contents (cholecystitis).

6. Blood (suspected sepsis).

7. Vomit, gastric lavage, food products (food poisoning).

8. Mucus from the nose (examination for bacteriocarrier).

Material collection methods

Material collection methods

Basic research methods

1. Microscopic.

2. Microbiological.

3. Biological.

Research progress
First day of research
 First day of research

All crops are placed in a thermostat for a day.

The detection of staphylococci by microscopy of pus from a closed abscess and

urine sediment taken with a catheter allows us to give a preliminary positive
answer: staphylococcus was detected.

Second day of research

Crops on dense and liquid nutrient media are removed from the thermostat and
studied. Staphylococcus-suspicious colonies grown on yolk-salt agar are screened
on agar slant to obtain and further study a pure culture. In this case, the presence of
lecithinase is taken into account, which manifests itself in the formation of an
iridescent corolla around the colony. The plates with the remaining colonies are left
for 2-3 days at room temperature to detect the pigment. View crops on cups with
agar containing blood. Colonies with a clear zone of hemolysis (clearance) around
them are isolated on agar slant. Blood cultures in sugar broth are incubated for 10
days, after 2-3 days they are seeded on agar with blood and yolk-salt medium.

In the absence of growth on dense nutrient media, inoculation is made from the
broth with glucose on agar with blood. Crops are placed in a thermostat for a day.
 Third day of research

Take out the crops from the thermostat. Smears are made from cultures isolated
on agar slant, Gram-stained, and microscoped. In the presence of gram-positive
staphylococci, a further study of the isolated culture is carried out:

a) put the reaction of plasma coagulation;

b) study hemolytic properties;

c) determine the production of DNase;

d) determine the fermentation of mannitol under anaerobic conditions;

e) determine resistance to novobiocin.

Plasma coagulation reaction . Citrate plasma obtained from rabbit blood is

diluted with isotonic sodium chloride solution in a ratio of 1:4 and poured into two
0.3-0.5 ml precipitation tubes. A loop of the studied culture is introduced into one
tube, the other tube serves as a control. Both test tubes are placed in a thermostat at
a temperature of 37 ° C. The reaction is recorded after 2-3 hours. In the absence of
plasma clotting, the crops are left at room temperature for 24 hours, after which the
reaction is taken into account. In the presence of the enzyme coagulase, the plasma
coagulates (does not pour out of an inverted tube). In the control tube, the plasma
consistency does not change.

Accelerated method for the determination of coagulase. The isolated culture is

suspended in a sterile drop of water on a glass slide, one drop of undiluted plasma
is added to it. With a positive reaction, large flakes are formed from microbial cells
within 20-60 s. This method is used in mass surveys.

Determination of hemolytic properties . Produce sowing on agar with 5%

blood (strains that produce α-hemolysin, give zones of enlightenment of the
environment and on rabbit and sheep blood, producing β-hemolysin lyse only sheep
erythrocytes).

Definition of DNase . The studied culture is seeded on a medium containing

DNA. Crops are incubated. After 18-20 hours, 5-7 ml of hydrochloric acid solution
is added to a cup with grown staphylococcus colonies. The DNA reacts with the
acid and the medium becomes cloudy. If the isolated culture produces the DNase
enzyme, it depolymerizes the DNA and no turbidity is formed.
 Degradation of mannitol under anaerobic conditions . The culture under
study is inoculated with a prick on a semi-liquid agar with mannitol. The surface of
the medium is filled with vaseline oil. Incubate for 18-24 hours at 37°C. A positive
reaction is characterized by a change in the color of the medium (there is an
indicator in the medium).

Fourth day of research

The results are recorded (Table 24).

Table 24. Properties of Staphylococcus aureus

Note. + positive reaction.

The presence of these features allows us to differentiate Staphylococcus aureus

from Staphylococcus aureus of other species and give the final answer: S. aureus
was isolated (Fig. 37).
Rice. 37. Scheme of isolation and identification of staphylococcus aureus
 To establish the epidemiological chain, the isolated culture is phage-
typed. Phage typing can confirm the identity of staphylococci isolated from
different patients and from environmental objects.

For phage typing, critical test dilutions of phages are used. The critical test
dilution is the maximum dilution of phages at which semi-confluent lysis of the
corresponding strain of staphylococcus occurs.

Phage typing technique . Pour 20 ml of 1.5% MPA into a Petri dish, allow it to

solidify and dry in a thermostat for 30-40 minutes. 1 ml of a 4-6-hour culture of
isolated staphylococcus is applied to the surface of the agar, spread over the surface
of the entire dish, the excess liquid is sucked off or allowed to evaporate in a
thermostat in an open dish. Previously, the bottom of the cup is divided into sectors
or squares. The number of squares or sectors should correspond to the number of
phages used. Then one phage is applied to each square or sector.

The cups are placed in a thermostat at a temperature of 37 ° C. The results are

determined after 6-7 hours. If the cups are left at room temperature, then phagolysis
is recorded after 18-24 hours.

biological samples . Test to determine the lethal properties of culture. To detect

the lethal effect of the toxin, the rabbit is injected intravenously (or
intraperitoneally) with the filtrate of the broth culture of staphylococcus at the rate
of 0.1-0.2 ml of the filtrate per 1 kg of rabbit weight. The death of the rabbit after
3-4 days indicates the presence of a lethal effect of the toxin.

Dermonecrotic test. The test is done on a rabbit (the most sensitive animal to this
toxin). Previously, the hair is plucked on the side or back of the animal and 0.2 ml
of a two-billionth suspension of staphylococcal culture in isotonic sodium chloride
solution is injected intradermally. If there are necrotic properties in the isolated
culture, an infiltrate is formed at the injection site, accompanied by necrosis.

The reaction is taken into account after 24-18 hours.

The resulting culture of staphylococcus is tested for sensitivity to antibiotics

using the paper disc method (see Chapter 9).

Control questions
1. What material is examined in diseases caused by staphylococci?
 2. What are the main methods of laboratory research for the detection of
staphylococci?

3. What is the procedure for setting up the plasma coagulation reaction?

4. What medium is used to detect the hemolytic properties of staphylococci?

5. What is the purpose of phage typing?

Exercise
Check to which antibiotic the isolated culture of staphylococcus aureus is
sensitive.

Nutrient media

Chistovich's yolk-salt agar . Prepare the yolk mixture (1 egg yolk per 150 ml
of sterile isotonic sodium chloride solution). To meat-peptone salt agar (8-10%
sodium chloride), melted and cooled to 45 ° C, add 20% yolk suspension (observe
sterility) and pour into cups.

Agar with blood . See chapter 7.

Salt broth, salt agar . They are prepared as usual media - MPB and MPA, only
sodium chloride is added in a larger amount (8-10%). The broth is poured into
flasks, test tubes, agar - into cups.
 Chapter 15 . Streptococci
The genus Streptococcus includes: Streptococcus pyogenes (hemolytic) and
Streptococcus pneumoniae (pneumococcus). Streptococci were first discovered by
Billroth (1874), L. Pasteur (1879). They were studied by E. Rosenbach (1884).

Streptococcus pyogenes (hemolytic)

Morphology . Streptococci are cocci that have a spherical shape. The diameter of

each coccus is on average 0.6-1 µm, however, they are characterized by
polymorphism: there are small and large cocci, strictly spherical and
oval. Streptococci are located in a chain, which is the result of their division in the
same plane. Chain lengths vary. On a dense nutrient medium, the chains are usually
short; on liquid ones, they are long. Streptococci are immobile, do not have spores
(see Fig. 4). Freshly isolated cultures sometimes form a capsule. On ultrathin
sections, a microcapsule is visible, under it there is a three-layer cell wall and a three-
layer cytoplasmic membrane. Gram-positive.
Cultivation . Streptococci are facultative anaerobes. Grow at a temperature of 37 ° C
and pH 7.6-7.8. The optimal media for their cultivation are media containing blood or
blood serum. On dense nutrient media, streptococcal colonies are small, flat, cloudy,
grayish in color. On blood agar, some varieties of streptococci form hemolysis. β-
hemolytic streptococci form a clear zone of hemolysis, α-hemolytic streptococci form
a small greenish zone (the result of the transition of hemoglobin to
methemoglobin). There are streptococci that do not give hemolysis.
On sugar broth, streptococci grow with the formation of parietal and near-bottom
fine-grained sediment, while the broth remains transparent.
enzymatic properties . Streptococci have saccharolytic properties. They break down
glucose, lactose, sucrose, mannitol (not always) and maltose to form acid. Their
proteolytic properties are poorly expressed. They coagulate milk, gelatin does not
liquefy.
Toxin formation . Streptococci form a number of exotoxins: 1) streptolysins - destroy
red blood cells (O-streptolysin has a cardiotoxic effect); 2) leukocidin - destroys
leukocytes (formed by highly virulent strains); 3) erythrogenic (scarlet fever) toxin -
causes the clinical picture of scarlet fever - intoxication, vascular reactions, rash, etc.
The synthesis of erythrogenic toxin is determined by the prophage; 4) cytotoxins -
have the ability to cause glomerulonephritis.
Antigenic structure and classification . Streptococci have various antigens. The
cytoplasm of the cell contains an antigen of a specific nucleoprotein nature - the
same for all streptococci. Protein type antigens are located on the surface of the cell
wall. A polysaccharide group antigen was found in the cell wall of streptococci.
According to the composition of the polysaccharide group-specific antigen fraction,
all streptococci are divided into groups, denoted by capital Latin letters A, B, C, D,
etc. up to S. In addition to groups, streptococci are divided into serological types,
which are indicated by Arabic numerals.
Group A includes 70 types. This group includes most streptococci that cause various
diseases in humans. Group B includes mainly opportunistic human
streptococci. Group C includes streptococci pathogenic to humans and
animals. Group D consists of streptococci that are not pathogenic to humans, but
this group includes enterococci, which are inhabitants of the intestinal tract of
humans and animals. Getting into other organs, they cause inflammatory processes:
cholecystitis, pyelitis, etc. Thus, they can be attributed to conditionally pathogenic
microbes.
The belonging of the isolated cultures to one of the serological groups is determined
using a precipitation reaction with group sera. To determine serological types, an
agglutination reaction with type-specific sera is used.
Resistance to environmental factors . Streptococci are fairly stable in the
environment. At a temperature of 60 ° C, they die after 30 minutes.
In dried pus and sputum, they persist for months. The usual concentrations of
disinfectants destroy them in 15-20 minutes. Enterococci are much more resistant,
disinfectant solutions kill them only after 50-60 minutes.
Susceptibility of animals . Cattle, horses, dogs, and birds are susceptible to
pathogenic streptococci. From laboratory animals rabbits and white mice are
sensitive. However, streptococci pathogenic for humans are not always pathogenic
for experimental animals.
Sources of infection . People (sick and carriers), less often animals or infected
products.
transmission paths . Airborne and airborne dust, sometimes food, contact-
household is possible.
Diseases can occur as a result of exogenous infection, as well as endogenously - with
the activation of opportunistic streptococci that live on the mucous membranes of
the pharynx, nasopharynx, and vagina. A decrease in the body's resistance (cooling,
starvation, overwork, etc.) can lead to autoinfections.
Of great importance in the pathogenesis of streptococcal infections is preliminary
sensitization - as a result of a previously transferred disease of streptococcal
etiology.
When penetrating into the bloodstream, streptococci cause a severe septic process.
Diseases in humans are more often caused by β-hemolytic streptococci of the
serogroup A. They produce pathogenicity enzymes: hyaluronidase, fibrinolysin
(streptokinase), deoxyribonuclease, etc. In addition, a capsule, M-protein, which
have antiphagocytic properties, are found in streptococci.
Streptococci cause various acute and chronic infections in humans, both with the
formation of pus and non-suppurative, differing in clinical picture and
pathogenesis. Suppurative - phlegmon, abscesses, wound infections, non-
suppurative - acute infections of the upper respiratory tract, erysipelas, scarlet fever,
rheumatism, etc.
Streptococci often cause secondary infections in influenza, measles, whooping cough
and other diseases and often complicate wound infections.
Immunity . By nature, immunity is antitoxic and antibacterial. Post-infectious
antimicrobial immunity is weak. This is due to the weak immunogenicity of
streptococci and a large number of serovars that do not give cross-immunity. In
addition, with streptococcal diseases, an allergization of the body is observed, which
explains the tendency to relapse.
Prevention . It comes down to sanitary and hygienic measures, strengthening the
overall resistance of the body. Specific prophylaxis has not been developed.
Treatment . Apply antibiotics. More often, penicillin is used, to which streptococci
have not acquired resistance, as well as erythromycin and tetracycline.
The importance of streptococcus in the etiology of rheumatic heart disease . The
pathogenesis of rheumatic heart disease is not well understood. But a number of
facts speak in favor of the role of streptococcus in the development of this disease:
1. In patients with rheumatic heart disease, B-hemolytic streptococcus is sown from
the pharynx.
2. Rheumatism often occurs after suffering a sore throat, tonsillitis, pharyngitis,
sensitizing the body.
3. Antistreptolysin, antistreptohyaluronidase - antibodies to streptococcal enzymes,
toxins are found in the blood serum of patients.
4. Indirect confirmation of the role of streptococcus is the successful treatment with
penicillin.
Recently, L-forms of streptococcus have been given importance in the occurrence of
chronic forms of rheumatic heart disease.
Prevention of exacerbations of rheumatic heart disease is reduced to the prevention
of streptococcal diseases (for example, in spring and autumn, a prophylactic course
of penicillin administration is carried out). Treatment is reduced to the use of
antibacterial drugs - penicillin.
The importance of streptococcus in the etiology of scarlet fever . G. N. Gabrichevsky
(1902) was the first to suggest that hemolytic streptococcus is the causative agent of
scarlet fever. But since the streptococci isolated in other diseases did not differ from
the causative agents of scarlet fever, this opinion was not shared by everyone. It is
now established that scarlet fever is caused by group A streptococci that produce
erythrogenic toxin.
In those who have been ill, immunity arises - persistent, antitoxic. Its tension is
determined by setting the Dick reaction - intradermal injection of erythrogenic
toxin. In those who are not sick around the injection site, hyperemia and edema
occur, which is characterized as a positive reaction (lack of antitoxin in the blood
serum). In those who have been ill, such a reaction is absent, since the antitoxin
formed in them neutralizes the erythrogenic toxin.
Prevention . Isolation, hospitalization. Contact, weakened children are given gamma
globulin. Specific prophylaxis has not been developed.
Treatment . Use penicillin, tetracycline. In severe cases, antitoxic serum is
administered.
Microbiological research
The purpose of the study: detection of streptococcus and determination of its
serovar.
Research material
1. Mucus from the throat (tonsillitis, scarlet fever).
2. Scraping from the affected area of the skin (erysipelas, streptoderma).
3. Pus (abscess).
4. Urine (nephritis).
5. Blood (suspected sepsis; endocarditis).
Material collection methods

Material collection methods

Basic research methods

1. Bacteriological.
2. Microscopic.
Research progress
First day of research
 First day of research

Second day of research

Take the cups out of the thermostat and inspect. In the presence of suspicious
colonies, smears are made from a part of them, stained according to Gram and
microscopically. If streptococci are found in the smear, part of the remaining colony
is subcultured into test tubes on agar with serum to isolate a pure culture and on
broth with blood in test tubes. By the end of the day, a 5-6-hour culture from broth
or agar is subcultured onto Marten's broth with 0.25% glucose to determine the
serological group in the Lensfield precipitation reaction. Test tubes and vials are
placed in a thermostat and left until the next day.
Third day of research
The cultures are removed from the oven, the purity of the culture is checked on the
agar slant, smears are made, Gram stained and microscoped. In the presence of a
pure culture of streptococcus, they are sown on Hiss media (lactose, glucose,
maltose, sucrose and mannitol), milk, gelatin, 40% bile and put in a thermostat.
Look through Martin's broth. In the presence of specific growth, a Lensfield
precipitation test is performed to determine the serological group.
Setting up the precipitation reaction according to Lensfield . The daily culture grown
on Martin's broth is poured into several centrifuge tubes, centrifuged for 10-15
minutes (3000 rpm).
The supernatant is poured into a jar with a disinfectant solution, and the precipitate
is poured into a sterile isotonic sodium chloride solution and centrifuged again. To
the precipitate collected from all centrifuge tubes, add 0.4 ml of 0.2% hydrochloric
acid. Then the tube is placed in a water bath and boiled for 15 minutes, shaking
occasionally. After boiling, the resulting suspension is again centrifuged. The antigen
is then extracted into the supernatant, which is poured into a clean test tube and
neutralized with 0.2% sodium hydroxide solution to pH 7.0-7.2. Bromothymol blue
(0.01 ml of a 0.04% solution) is added as an indicator. With this reaction, the color
changes from straw yellow to blue.
Then, 0.5 ml of antistreptococcal group sera, which are prepared by immunizing
rabbits, are poured into 5 precipitation tubes (see Chapter 19). Serum A is
introduced into the 1st tube, serum B into the 2nd, serum C into the 3rd, serum D
into the 4th, isotonic sodium chloride solution (control) into the 5th. After that, with
a Pasteur pipette, the resulting extract (antigen) is carefully layered along the wall
into all test tubes.
With a positive reaction in a test tube with homologous serum, a thin milky-white
ring is formed at the border of the extract with serum (Fig. 38).
Rice. 38. Scheme of isolation and identification of streptococcus

Fourth day of research

The results are recorded (Table 25).

Table 25. Enzymatic properties of streptococcus

Note. to - the breakdown of carbohydrates with the formation of acid.

Currently, deoxyribonuclease is being determined, as well as
antistreptohyaluronidase, antistreptolysin-O.
Control questions
1. What are the main methods of laboratory research for the detection of
streptococci do you know?
2. What is the Lensfield precipitation reaction for?
3. Why should the antigen be transparent during this reaction? Describe the
technique for staging this reaction.
Exercise
Get antistreptococcal serum A, B, C, D and isotonic sodium chloride solution from the
teacher. Set the precipitation reaction, show the results to the teacher and draw.
Nutrient media
Agar with blood (see chapter 7).
Serum agar (see chapter 7).
Hiss media (dry).
Meat peptone gelatin (MPG) . To 100 ml of MPB add 10-15 g of finely chopped
gelatin. Gelatin should swell when slowly heated in a water bath (at a temperature of
40-50 ° C). A 10% solution of sodium carbonate (baking soda) is added to the melted
gelatin and the pH is adjusted to 7.0. It is then immediately filtered through a pleated
filter. Filtration is slow. To speed up the process, filtration can be done in a hot
autoclave. The filtered medium is poured into test tubes of 6-8 ml and
sterilized. Sterilization is carried out either fractionally at a temperature of 100 ° C for
3 days in a row, or simultaneously at 110 ° C for 20 minutes in an autoclave. Cooling
of the medium is carried out in test tubes placed vertically.
Milk preparation . Fresh milk is brought to a boil, put in a cool place for a day, freed
from cream, boiled again. Leave for a day and remove the top layer. Skimmed milk is
filtered through a layer of cotton wool, then alkalized with 10% sodium carbonate
solution to pH 7.2 and poured into test tubes of 5-6 ml.
Bouillon Martin . An equal amount of peptone Marten (minced meat from pork
stomachs exposed to hydrochloric acid) is added to the meat water. The resulting
mixture is boiled for 10 minutes, alkalized with 10% sodium hydroxide solution to pH
8.0, 0.5 sodium acetate is added, boiled again and poured into sterile dishes. 0.25%
glucose is added to Martin's broth.
Wednesday Kitt - Tarozzi (see chapter 34).

Streptococcus pneumoniae (pneumococcus)

Pneumococci were first described by R. Koch (1871).

Morphology . Pneumococci are diplococci in which the sides of the cells facing each
other are flattened and the opposite sides are elongated, so they have a lanceolate
shape resembling a candle flame (see Fig. 4). The size of pneumococci is 0.75-0.5 ×
0.5-1 μm, they are arranged in pairs. In liquid nutrient media, they often form short
chains, resembling streptococci. Prevmococci are immobile, do not have spores,
form a capsule in the body that surrounds both cocci. The capsule contains a heat-
resistant substance antiphagin (which protects pneumococcus from phagocytosis
and the action of antibodies). When growing on artificial nutrient media,
pneumococci lose their capsule. Pneumococci are gram positive. Gram-negative
bacteria are found in old cultures.
Cultivation . Pneumococci are facultative anaerobes. Grow at a temperature of 36-
37 ° C and pH 7.2-7.4. They are demanding on media, since they cannot synthesize
many amino acids, therefore they grow only on media with the addition of native
protein (blood or serum). On agar with serum form small, delicate, fairly transparent
colonies. On agar with blood, moist greenish-gray colonies grow, surrounded by a
green zone, which is the result of the conversion of hemoglobin to
methemoglobin. Pneumococci grow well in broth with the addition of 0.2% glucose
and in broth with whey. Growth in liquid media is characterized by diffuse turbidity
and dusty sediment at the bottom.
enzymatic properties . Pneumococci have a fairly pronounced saccharolytic
activity. They break down: lactose, glucose, sucrose, maltose, inulin with the
formation of acid. Do not ferment mannitol. Their proteolytic properties are poorly
expressed: they coagulate milk, do not liquefy gelatin, and do not form
indole. Pneumococci dissolve in bile. Cleavage of inulin and dissolution in bile is an
important diagnostic feature that distinguishes Streptococcus pneumoniae from
Streptococcus pyogenes.
pathogenicity factors . Pneumococci produce hyaluronidase, fibrinolysin, etc.
Toxin formation . Pneumococci produce endotoxin, hemolysin, leukocidin. The
virulence of pneumococci is also associated with the presence of antiphagin in the
capsule.
Antigenic structure and classification . In the cytoplasm of pneumococci there is a
protein antigen common to the entire group, and in the capsule there is a
polysaccharide antigen. According to the polysaccharide antigen, all pneumococci
are divided into 84 serovars. Serovars I, II, III are the most common pathogens for
humans.
Resistance to environmental factors . Pneumococci belong to the group of unstable
microorganisms. A temperature of 60 ° C destroys them in 3-5 minutes. They are
quite resistant to low temperatures and drying. In dried sputum, they remain viable
for up to 2 months. On a nutrient medium, they remain no more than 5-6
days. Therefore, when cultivating, it is necessary to do reseeding every 2-3
days. Conventional solutions of disinfectants: 3% phenol, sublimate at a dilution of
1:1000 destroy them in a few minutes.
Pneumococci are especially sensitive to optochin, which kills them at a dilution of
1:100,000.
Susceptibility of animals . Humans are the natural host of pneumococci. However,
pneumococci can cause illness in calves, lambs, piglets, dogs, and monkeys. Of the
experimental animals, white mice are highly sensitive to pneumococcus.
Sources of infection . A sick person and a bacteriocarrier.
transmission paths . Airborne, may be airborne.
Entrance gates . The mucous membrane of the upper respiratory tract, eyes and ear.
Diseases in humans . Pneumococci can cause purulent-inflammatory diseases of
different localization. Specific for pneumococci are:
1) lobar pneumonia;
2) creeping ulcer of the cornea;
3) otitis.
The most common disease is croupous pneumonia, which affects one, less often two
or three lobes of the lung. The disease is acute, accompanied by high fever, cough. It
usually ends critically.
Immunity . After the illness, unstable immunity remains, since pneumonia is
characterized by relapses.
Prevention . It comes down to sanitary and preventive measures. Specific
prophylaxis has not been developed.
Treatment . Antibiotics are used - penicillin, tetracycline, etc.
Control questions
1. Morphology of pneumococci. Cultivation and enzymatic properties.
2. What factors determine the pathogenicity of pneumococci and what protects
pneumococci from phagocytosis?
3. What are the main gates of pneumococcal infection. What diseases are caused by
pneumococci?
Microbiological research
The purpose of the study: detection of pneumococcus.
Research material
1. Phlegm (pneumonia).
2. Mucus from the pharynx (tonsillitis).
3. Discharge from the ulcer (creeping ulcer of the cornea).
4. Discharge from the ear (otitis media).
5. Pus (abscess).
6. Pleural punctate (pleurisy).
7. Blood (suspected sepsis).
Material collection methods

Material collection methods

1
 ( It is better to take morning sputum (in specific pneumonia, the sputum has a rusty color).  )
Basic research methods
1. Microscopic.
2. Microbiological.
3. Biological.
Research progress
First day of research
 First day of research

biological test . A little (3-5 ml of sputum) is emulsified in a sterile broth, 0.5 ml of

this mixture is injected intraperitoneally to a white mouse. After 6-8 hours, the mice
show signs of the disease. At this time, pneumococcus can already be detected in the
exudate of the abdominal cavity. The exudate is taken with a sterile syringe. Smears
are made from it, stained according to Gram and microscoped. To isolate a pure
culture, the exudate is inoculated onto agar with serum. If the mouse dies or
becomes ill, blood is cultured from the heart on serum agar to isolate a pure
culture. Crops are placed in a thermostat.
Accelerated method for determining the type of pneumococcus (microagglutination
reaction). 4 drops of exudate from the abdominal cavity of an infected mouse are
applied to a glass slide. Type I agglutinating serum is added to the first drop, type II
serum to the second, type III to the third, isotonic sodium chloride solution (control)
to the fourth.
Type I and II sera are pre-diluted in a ratio of 1:10, and type III serum - 1:5. All drops
are stirred, dried, fixed and stained with diluted magenta. With a positive result in
one of the drops, microbial aggregation (agglutination) is noted.
 Second day of research
The cultures are removed from the thermostat, examined, and smears are made
from suspicious colonies. In the presence of gram-positive lanceolate diplococci in
smears, 2-3 colonies are isolated on a slant of agar with serum to obtain a pure
culture. Crops are placed in a thermostat. Smears are made from the broth, Gram-
stained, and microscoped.
Third day of research
Crops are removed from the thermostat. Check the purity of the culture - make
smears, Gram stain and microscope. If Gram-positive lanceolate diplococci are
present in the isolated culture, the isolated culture is identified by inoculation:
1) on the Hiss media (lactose, glucose, sucrose, maltose), sowing is carried out in the
usual way - by injection into the medium;
2) on the medium with inulin;
3) on the medium with optochin;
4) put a sample with bile.
Inulin test . The studied culture is seeded on a nutrient medium containing inulin and
litmus tincture, and placed in a thermostat. After 18-24 hours, the crops are removed
from the thermostat. In the presence of pneumococci, the medium turns red
(streptococci do not change the consistency and color of the medium).
Determination of sensitivity to optochin . The isolated culture is seeded on 10%
blood agar containing optochin 1:50,000. Pneumococci, unlike streptococci, do not
grow on media containing optochin.
Bile test . 1 ml of the studied broth culture is poured into agglutination tubes. A drop
of rabbit bile is added to one of them, the second test tube serves as a control. Both
test tubes are placed in a thermostat. After 18-24 hours, lysis of pneumococci occurs,
which is expressed in the clearing of a cloudy broth. In the control, the suspension
remains cloudy.
A sample with bile can be placed on a dense nutrient medium. To do this, a grain of
dry bile is applied to a colony of pneumococci grown in agar and serum plates - the
colony dissolves - disappears.
Fourth day of research
The results are recorded (Table 26).

Table 26. Differentiation of pneumococcus from viridescent streptococcus

Note. to - the breakdown of carbohydrates with the formation of acid.

Currently, serological research methods (RSK and RIGA) are widely used to
determine streptococcal antibodies. Determination of the group and serovar of the
isolated culture is carried out using fluorescent antibodies.
Determination of the virulence of pneumococcus . Daily broth culture of
pneumococcus is diluted with 1% peptone water from 10 -2 to 10 -8 , 0.5 ml of each
dilution is administered to two white mice. The culture that caused the death of mice
at a dilution of 10 -7 is assessed as virulent, at a dilution of 10 -4 -10 -6 it is considered
moderately virulent. The culture that did not cause the death of mice is avirulent.
Control questions
1. What methods of isolating a pure culture of pneumococci do you know?
2. Which animal is most susceptible to pneumococcus?
3. What reactions are put with the exudate of an infected mouse and for what
purpose?
4. From which representatives of pyogenic cocci should pneumococcus be
differentiated and with what test?
5. How to determine the virulence of pneumococci?
Exercise
Draw up a sputum examination scheme, indicating its stages by day.
Nutrient media
Serum agar (see chapter 7).
Whey broth (see chapter 7).
Agar with blood (see chapter 7).
Hiss media (dry).
Inulin test medium . To 200 ml of distilled water add 10 ml of inactivated bovine
serum, 18 ml of litmus tincture and 3 g of inulin. Sterilize with flowing steam at 100°C
for 3 consecutive days. Bile broth (see chapter 7).
 Chapter 16. Meningococci
The genus Neisseria includes two types of microbes pathogenic to humans: N.
meningitidis and N. gonorrhoeae. Neisseria meningitidis were isolated from the
cerebrospinal fluid of a patient by Vekselbaum (1887).
Morphology . Meningococci are paired cocci, consisting of two bean-shaped cocci,
lying with concave sides to each other, their outer walls are convex (see Fig. 4). The
size of each coccus is 0.6-0.8 × 1.2-1.5 µm. They are polymorphic. Meningococci are
non-motile, do not have spores, form a capsule. Gram-negative. In pure cultures,
they are located in tetrads and in the form of individual cocci in no particular order,
and in smears prepared from cerebrospinal fluid, they are more often arranged in
pairs. In purulent material, they are located inside the leukocyte.
Cultivation . Meningococci are aerobes. They are demanding on nutrient media,
multiply only on media containing native protein (serum, blood). Grow at a
temperature of 36-37 ° C (growth stops at 25 ° C), pH 7.4-7.6. Their reproduction
requires a moist environment and an increased amount of carbon dioxide (a factor
that stimulates their growth). Sowing should be done on a freshly prepared medium.
On dense nutrient media, meningococci form small, 2-3 mm in diameter, tender,
translucent, bluish, viscous colonies. In broth with serum, meningococci give a slight
turbidity and a small sediment. Freshly isolated strains in S-form. Old cultures can
dissociate, forming rough R-shaped colonies.
enzymatic properties . Biochemically, meningococci are not very active. They break
down glucose and maltose to form acid. Their proteolytic properties are not
expressed (they do not curdle milk, do not dilute gelatin).
The pathogenicity of meningococci is due to the presence of a capsule that prevents
phagocytosis, pili that promote the attachment of the microbe to the surface of
epithelial cells, and the formation of enzymes: hyaluronidase and neuraminidase.
Toxin formation . When bacterial cells are destroyed, a strong heat-resistant
endotoxin is released, which is a lipopolysaccharide of the cell wall. In the disease, it
is found in the blood and in the cerebrospinal fluid of patients. The severity of the
disease often depends on the amount of accumulated toxin.
Antigenic structure . According to the polysaccharide (capsular) antigen,
meningococci are divided into serogroups: A, B, C, D, X, Y U-135 29E (a total of nine
serogroups).
According to the international classification, the main groups are A, B and C. Group A
meningococci often cause generalized processes and are of the greatest
epidemiological significance. Groups B and C meningococci cause sporadic
disease. The remaining serogroups have been little studied.
Resistance to environmental factors . Meningococci are unstable. A temperature of
70 ° C destroys them after 2-3 minutes, 55 ° C - after 5 minutes. Unlike other cocci of
this group, they do not tolerate low temperatures and are especially sensitive to
temperature fluctuations.
Ordinary concentrations of disinfectant solutions destroy them quickly.
Susceptibility of animals . Under natural conditions, animals are not sensitive to
meningococci. But when meningococci are injected subdurally into monkeys, they
can get sick.
Intraperitoneal infection of guinea pigs and white mice causes their death due to the
action of endotoxin.
Sources of infection . A sick person and a bacteriocarrier.
transmission paths . The main route is airborne.
Diseases in humans :
1) nasopharyngitis;
2) meningococcemia;
3) cerebrospinal epidemic meningitis.
Pathogenesis . Once on the mucous membrane of the nasopharynx, meningococci
can be localized there, causing carriage or causing acute nasopharyngitis. If they
penetrate into the lymphatic vessels, blood and generalize, they cause profound
changes in the parenchymal organs due to the action of
endotoxin. meningococcemia develops. With the penetration of meningococci into
the meninges, purulent inflammation occurs - meningitis. In meningococcal
meningitis, the cerebrospinal fluid is cloudy (unlike tuberculous meningitis). During a
lumbar puncture, fluid flows out in a jet due to increased intracranial
pressure. Meningeal phenomena are characterized by headache, neck stiffness,
vomiting, etc. Meningitis is more common in children. In adults, infection is more
often limited to carriage or nasopharyngitis.
Immunity . Post-infection immunity is tense, it is caused by opsonins, complement-
fixing and bactericidal antibodies. The course of the disease depends on the intensity
of the formation of antibodies to polysaccharide and protein antigens.
Prevention . It comes down to early detection of carriers, isolation of patients with
nasopharyngitis. Patients are subject to hospitalization.
specific prophylaxis . A chemical vaccine has been developed, consisting of
polysaccharides of serogroups A and C. Immunoglobulin is used for emergency
prophylaxis.
Treatment . Antibacterial drugs - penicillin, chloramphenicol, ampicillin.

Control questions

1. What are the morphological properties of meningococci?

2. On what media are meningococci grown and what conditions are necessary for
their reproduction?
3. What is the biochemical activity of meningococci and their stability in the external
environment?
4. What diseases are caused by meningococci?
5. By what antigen are meningococci divided into serogroups?

Microbiological research

The purpose of the study: to identify meningococcus and determine its serogroup.
Research material
1. Cerebrospinal fluid.
2. Detachable mucous membrane of the nasopharynx.
3. Blood.
Material collection methods
Material collection methods
 Rice. 39. Taking mucus from the nasopharynx for research on meningococci. 1 -
spatula; 2 - swab for taking material

Basic research methods

1. Microscopic.
2. Microbiological.
3. Serological.
Research progress
 Research progress

Fourth day of research

Record the results (Table 27)

Table 27. Differentiation of meningococci from non-pathogenic Neisseria

Note. + growth (positive test); - lack of growth; to - acid.

Definition of the meningococcus group . After obtaining a pure culture of
meningococcus, a serological determination of the group is carried out. For this,
commercial agglutinating and precipitating sera are used.
Apply one drop of undiluted agglutinating sera of groups A, B, C, etc., a drop of
isotonic sodium chloride solution (control) onto a glass slide. One loop of isolated
culture is added to each drop. The presence of agglutination in one of the drops
determines the group of the isolated culture.
To identify serogroups, you can put the precipitation test in the gel (see chapter 32).
Currently, serological diagnostic methods are used for diagnostic purposes: the
serum of the examined individuals is examined in the RNGA with meningococcal
erythrocyte diagnosticum A, C and other serogroups.
Control questions
1. What material is used to detect meningococci in meningitis?
2. How is the test material transported?
3. What is added to the collected material to suppress the growth of gram-positive
cocci?
4. From what microorganisms it is necessary to differentiate meningococci, what
research methods are carried out for differentiation?
5. What reaction is used to determine the serogroup of meningococci?
Exercise
1. Prepare a swab, bend it at an angle of 135 ° and take each other's mucus from the
nasopharynx. Sow it on serum agar.
2. Examine a smear from a meningococcal culture under a microscope and draw it.
Nutrient media
Serum agar (see chapter 7).
Wednesday with lincomycin . To 80 ml of melted and cooled to a temperature of 50
° C 2% agar, add 20 ml of horse or bovine serum, 0.5-0.7 ml of a solution of
lincomycin in a working dilution (working dilution of 0.001 mg per 1 ml of
medium). After mixing, the medium is poured into Petri dishes. A solution of
lincomycin is prepared in sterile water and stored in a refrigerator.
 Chapter 17. Gonococci
Neisseria gonorrhoeae is a member of the Neisseriaceae family, genus
Neisseria. Gonococci were discovered by Neisser in 1879 and the whole family is
named after him.
Morphology . Gonococci are diplococci consisting of two bean-shaped cocci lying
with concave sides to each other (reminiscent of coffee beans). The size of gonococci
is 1.2-1.3 × 0.7-0.8 μm. They are polymorphic; along with large ones, there are very
small, irregularly shaped L-shaped bacteria. Gonococci are immobile and do not have
spores. A capsule-like substance is found in the pathological material (pus). Gram-
negative. Under the influence of medicinal and other substances, they quickly
change: gram-positive forms appear. In the pathological material, they are located
intracellularly (in a leukocyte), but may be outside the cell. May be in the form of
individual cocci (see Fig. 4).
Cultivation . Gonococci are aerobes. Very demanding on nutrient media. They grow
on media containing native protein (human) - blood, serum, at a temperature of 37 °
C and a pH of 7.2-7.4. Media should be freshly prepared and moist. Sowing should be
done immediately after taking the material. On serum medium, gonococci form small
colonies 1-2 mm, transparent, shiny with smooth edges, resembling dew drops. On
the blood medium, hemolysis is not given. In whey broth, they give a slight turbidity
and a film that settles to the bottom of the tube. With poor growth after 24 hours,
the crops are left in a thermostat for the second day.
enzymatic properties . Saccharolytic properties are weakly expressed. Gonococci
break down only one sugar - glucose with the formation of acid. They do not have
proteolytic properties.
Toxin formation . In the cell wall of gonococci there is a toxic substance -
lipopolysaccharide (little studied).
Antigenic structure . The antigenic structure is heterogeneous and easily changes
under the influence of environmental factors. There is no generally accepted division
of gonococci into serovars and serotypes yet.
Resistance to environmental factors . In the external environment, gonococci are
not very stable. At a temperature of 56-60 ° C, they die. At a temperature of 40 ° C,
their viability decreases sharply. Low temperatures and drying quickly destroy
them. But in pus they remain up to 24 hours. Disinfectant solutions - 1% solution of
phenol, sublimate 1:1000 kill gonococci within a few minutes. Gonococci are
especially sensitive to silver salts - a 1% solution of silver nitrate destroys them
immediately. UV rays kill them within minutes.
Susceptibility of animals . Animals are not susceptible to gonococcus. However,
intraperitoneal administration of gonococcal toxin to white mice causes their death.
Sources of infection . A person with gonorrhea.
transmission paths . Contact-household (sexual), less often through contaminated
objects (towel, sponges, etc.).
Diseases in humans . Gonorrhea and blennorrhea.
Pathogenesis . The natural host of gonococci is a sick person. Gonococci penetrate
through the mucous membranes of the urethra (in women - the urethra and
cervix). The pathogenicity factor of gonococci is the presence of pili in them, which,
connecting with the microvilli of the cylindrical epithelium, contribute to the
penetration of the gonococcus into the epithelial cell, causing an acute inflammatory
process in the mucous membrane.
Clinically, gonorrhea is manifested by pain during urination, discharge of pus from
the urethra and vagina. The disease is acute, but sometimes becomes
chronic. Gonococci can cause gonorrheal conjunctivitis - blennorrhea (purulent
inflammation of the mucous membrane of the eyes in newborns). Gonococci rarely
spread from the urethra to other organs, but sometimes they can cause arthritis,
endocarditis, etc.
Immunity . There is no natural resistance to gonococci. The transferred disease also
does not create immunity. The observed phagocytosis is incomplete.
Prevention . Health education. Increasing the cultural and hygienic level. There is no
specific prevention. For the prevention of blennorrhea, children immediately after
birth must be injected into the conjunctival sac 1-2 drops of a 30% solution of
albucid.
Treatment . Antibiotics (penicillin, bicillin, streptomycin, etc.). Sulfa drugs are also
used. In the chronic form, a gonococcal vaccine is used.

Control questions
1. Describe the morphological properties of gonococci.
2. What are the enzymatic activity and toxin formation of gonococci?
3. What is the resistance of gonococci. To what drug are gonococci particularly
sensitive?
4. What diseases are caused by gonococci and their pathogenesis.

Microbiological research

The purpose of the study: detection of gonococci and anti-gonococcal antibodies.

Research material
1. Detachable mucous membrane of the urethra in men.
2. Discharge of the mucous membrane of the urethra and cervix in women.
3. Purulent discharge from the eyes.
4. Blood to obtain serum.
Material collection methods

Material collection methods

Note. For bacterioscopic and bacteriological examination, the material is taken: 1)

before the start of antibiotic treatment: 2) not earlier than 10 days after the end of
antibiotic treatment; 3) not earlier than 2 hours after the last urination; 4) not earlier
than 2 hours after douching.
Basic research methods
1. Microscopic (mainly used in acute forms).
2. Microbiological.
3. Serological.
Research progress
First day of research

First day of research

Second day of research

Take out the crops from the thermostat and view them. Studying the colonies. They
make smears. In the presence of suspicious gram-negative diplococci, the colonies
are subcultured on a slant medium in test tubes (the medium must be freshly
prepared and contain a sufficient amount of condensate) and a sample is taken for
oxidase. To do this, a drop of 1% dimethyl paraphenylenediamine solution is applied
to the colony with a pipette, the colonies change color from dark brown to black.
Third day of research
The cultures are taken out of the thermostat, swabs are taken from the agar slant,
stained by Gram and microscoped. Inoculated on Hiss media (lactose, glucose,
mannitol and maltose). These carbohydrates should contain 30% of blood
serum. The inoculated tubes are placed in a thermostat.
Fourth day of research
Remove the test tubes from the thermostat, in the absence of growth, leave them in
the thermostat for another 1-2 days. In the presence of growth, the results are taken
into account (Table 28).

Table 28. Differentiation of gonococci from other Neisseria

Serological diagnostics
third week of illness. In the chronic course of the disease and in doubtful cases, RSK
is placed with the patient's serum (see Chapter 12). As an antigen, a killed culture of
gonococci, which is prepared under industrial conditions, is used. You can apply the
reaction of indirect hemagglutination (see Chapter 12).
Control questions
1. What material is used to detect gonococci and how is it obtained?
2. How long after urination (or douching in women) can material be taken for
research?
3. What research method is the main one for acute gonorrhea and what method for
chronic gonorrhea?
4. When and what kind of serological reaction is given for suspected gonorrhea?
5. From what microorganisms it is necessary to differentiate gonococci?
Exercise
Get the drug from the teacher. Examine it and draw the gonococci located inside and
outside the leukocyte on a Gram stain.
Nutrient media
Yolk environment . To 100 ml of MPA from rabbit meat add 15 ml of yolk (fresh
chicken egg), 6 ml of phenol red indicator, 1.5 ml of sugar dissolved in 1 ml of sterile
distilled water.
Nutrient medium ascites-agar . 2% agar, 1% peptone and 0.5% sodium chloride are
added to the filtrate of the broth prepared from rabbit meat. Heat until the agar
dissolves, set the pH to 7.4-7.5, alkalinize with 20% sodium hydroxide. The medium is
brought to a boil, filtered, poured into sterile vials and sterilized in an autoclave for
15 minutes at 115°C.
Recipes for ascitic nutrient media (MPA pH 7.4-7.5) .
1) meat water from rabbit meat or bull hearts - 100 ml
casein hydrolyzate - 2 ml
yeast autolysate - 2 ml
blood serum of cattle - 20 ml
2) meat water from rabbit meat or bull hearts - 100 ml
5% solution of hemohydrolyzate - 2 ml
yeast autolysate - 2 ml
bovine serum - 20 ml
3) meat water from rabbit meat or bull hearts - 100 ml
chicken egg yolk - 10 ml
blood serum of cattle - 20 ml
The growth of gonococci on these media is abundant. Gonococcus colonies can be
detected by an oxidase test, which turns red to black.
 family of intestinal bacteria
The Enterobacteriaceae family includes numerous microorganisms that are similar in
morphology, tinctorial and cultural properties. They live in the intestines of humans
and animals and can be found in the external environment.
Currently, all intestinal bacteria are divided into 12 genera, of which the following
will be considered: Escherichia, Shigella, Salmonella, Proteus, Klebsiella, Yersinia.
These genera, in turn, are divided into species, biological and serological variants
(biovars and serovars).
It is believed that the ancestor of this entire group of microorganisms is Escherichia
coli. In the process of evolution, varieties of Escherichia coli have adapted to a
parasitic mode of existence, acquired pathogenic properties and are currently the
causative agents of many human and animal diseases.
Pathogenic representatives of the family of intestinal bacteria include causative
agents of typhoid fever, paratyphoid fever A and B, toxic infections, dysentery. Many
intestinal bacteria live permanently in the intestines. When the conditions of
existence change (for example, the weakening of the host organism), they become
pathogens. These are the so-called opportunistic bacteria.
All intestinal bacteria are gram-negative rods. They are facultative anaerobes. They
grow well on simple nutrient media.
Enterobacteria are distinguished by enzymatic activity, which is most pronounced in
saprophytes and decreases as pathogenicity increases. This pattern can be explained
by the fact that microorganisms, adapting to a parasitic way of life, have lost
enzymes that have become unnecessary.
 Chapter 18. Escherichia - L. B. Bogoyavlenskaya, F. K.
Cherkes
This genus is represented by only one species of bacteria - E. coli, but combines
many options. Varieties of E. coli differ in biological properties, they may have
different sets of enzymes (biovars) and different antigenic structure (serovars).
Escherichia coli was first isolated in 1888 by Escherich from human feces and named
after him.
The natural habitat of E. coli is the human intestine. E. coli is a representative of the
normal intestinal microflora.
In the process of life, E. coli produces enzymes that promote digestion (for example,
breaking down fiber), synthesizes some vitamins (for example, B vitamins). In
addition, these bacteria exhibit an antagonistic effect against pathogenic
microorganisms, such as pathogens of dysentery, typhoid fever, and toxic
infections. The absence of Escherichia coli in the large intestine leads to a serious
disease - dysbacteriosis. In this case, the normal composition of the intestinal
microflora is disturbed, Proteus, coccal flora, fungi, etc. develop.
With a decrease in the body's resistance (starvation, overwork, etc.), Escherichia can
penetrate other organs and tissues and cause severe pathological processes. Thus,
we can assume that Escherichia are typical conditionally pathogenic microorganisms:
under normal conditions, they are saprophytes, and when conditions change, they
cause diseases.
Standing out with faeces, E. coli enters the external environment. The detection of E.
coli in soil, water and other objects indicates their fecal contamination, and the
determination of the amount of E. coli (coli-titer, coli-index) characterizes the
sanitary condition of the object (see "Sanitary microbiology").
Morphology . E. coli are short, averaging 0.5-3.0 × 0.5-0.8 µm rods. Gram-
negative. In most cases they are mobile, peritrichous. However, some variants of E.
coli are non-motile. Many strains form a capsule. Dispute does not form.
Cultivation . E. coli is a facultative anaerobe. It grows well on simple nutrient media
at 37 ° C and pH 7.2-7.8. Strains of E. coli isolated from the intestines of humans and
animals develop even at 43-45°C, while Escherichia coli of cold-blooded animals do
not multiply under these conditions. This difference in the properties of E. coli of
different origin is used to determine the sanitary state of the object, since only the
detection of warm-blooded E. coli indicates a sanitary problem.
On MPA, Escherichia coli forms cloudy, slightly convex, moist colonies with a smooth
edge. On the MPB gives a uniform turbidity. Capsulated cultures grow as slimy
colonies.
To identify Escherichia, differential diagnostic media are used: Endo and agar with
eosinmethylene blue (EMS). On Endo's medium, E. coli grows as crimson-red
colonies with or without a metallic sheen. On EMS medium - in the form of dark
purple colonies.
enzymatic properties . E. coli have significant enzymatic activity. They break down
lactose, glucose, mannitol, maltose, sucrose and other carbohydrates and alcohols
with the formation of acid and gas. Proteolytic properties: form indole. Gelatin is not
broken down. Some biovars do not ferment lactose and sucrose (Table 29).

Table 29. Enzymatic properties of Escherichia

Note, kg - formation of acid and gas; + the presence of a sign; - lack of sign.
Toxigenicity . Escherichia have endotoxin (liggopolysaccharide).
Antigenic structure. Escherichia differ in the antigenic structure of the microbial cell,
which is the basis for the classification of bacteria of this genus. There are three
types of Escherichia antigens: O-antigen (somatic), K-antigen (capsular) and H-
antigen (flagellate). The thermostable O-antigen is a lipopolysaccharide-protein
complex located in the bacterial cell wall. The O-antigen determines whether a
culture belongs to a serological group. More than 170 such groups have been
described. Some components of the O-antigen are common to different O-groups of
Escherichia, and sometimes other enterobacteria (Shigella, Salmonella,
etc.). Escherichia K-antigens are different: A, B, L and M. Antigens A and M are
thermostable, B and L are thermolabile. The K-antigen is located in the microbial cell
more superficially than the O-antigen, and therefore, in its presence, the
agglutination reaction of a live culture with O-serum does not occur. To identify the
O-antigen, the culture is heated for an hour at 100 ° C: the K-antigen is destroyed
during heating, and the O-antigen becomes able to interact with the serum. It has
been established that Escherichia have about 100 types of K-antigens, mainly of the
B-antigen type (thermolabile). The H-antigen is present only in motile strains, as it is
associated with flagella. More than 50 types of H-antigen are known in
Escherichia. The determination of the H-antigen makes it possible to establish the
serovariant of the isolated culture (Fig. 40). since it is associated with flagella. More
than 50 types of H-antigen are known in Escherichia. The determination of the H-
antigen makes it possible to establish the serovariant of the isolated culture (Fig.
40). since it is associated with flagella. More than 50 types of H-antigen are known in
Escherichia. The determination of the H-antigen makes it possible to establish the
serovariant of the isolated culture (Fig. 40).

Rice. 40. Antigenic structure of enteropathogenic Escherichia coli. 1 - cytoplasm; 2 -

cell wall; 3 - flagella

Characterization of the antigenic composition of the isolated culture of Escherichia is

given on the basis of the results of the agglutination reaction with sera containing O-,
K- and H-antibodies. At the same time, it is determined which antigens are present in
the culture, and their combination characterizes the antigenic formula of the isolated
culture, i.e., its serovariant. Table 30 shows examples of the antigenic structure of
some E. coli serovariants whose K antigens are B antigens.
 Table 30. Antigenic structure of Escherichia

If the culture is agglutinated by OK-serum OP1:K58 (B4) and H-serum "6", then the
serovariant E. coli O111:B4:H6 is isolated; if an agglutination reaction with OK-serum
O26:K60 (B6) and with H-serum "11" is noted, a culture of E. coli 026:B6:H11 is
isolated, etc.
In addition to determining the E. coli serovariant, it is also possible to determine the
fagovar of the isolated culture. There are sets of bacteriophages that lyse Escherichia
of individual serogroups. According to the lysis of the culture, one of the phages
establishes its fagovar. The definition of fagovars is of epidemiological significance.
The antagonistic effect of E. coli, their ability to suppress the growth of putrefactive
and pathogenic bacteria is used to create bacterial preparations for the treatment of
dysbacteriosis and various intestinal diseases (colibacterin, bifikol).
Resistance to environmental factors . E. coli are quite resistant. At 55°C they die
within an hour, at 60°C - in 15 minutes. They remain in soil and water for up to 2-3
months, in milk they not only remain, but also multiply. Solutions of disinfectants
(3% chloramine, sublimate solution 1:1000, etc.) kill them in 20-30 minutes. E. coli
are especially sensitive to brilliant green.
Susceptibility of animals . Escherichia of certain serogroups are pathogenic for
various animals and cause diseases of the gastrointestinal tract in them. From
laboratory animals guinea pigs, rabbits, white mice are most sensitive to E.
coli. Depending on the method of administration, the culture of Escherichia coli
causes various pathological processes: inflammation and abscess with subcutaneous
injections, peritonitis and sepsis with intraperitoneal and intravenous administration.
Sources of infection . A sick man. In this case, bacteria enter the body from the
external environment (exogenous infection). E. coli can also cause the development
of the pathological process "from the inside" (endogenous infection).
transmission paths . The main route of transmission in the exogenous form of
infection is contact-household (indirect contact). Pathogens can be carried on dirty
hands, through dishes, toys, underwear, food, flies.
Pathogenesis . Diseases caused by Escherichia are called Escherichiosis. The
development of escherichiosis depends on the path of introduction of the pathogen
into the body and on the serogroup to which the pathogen belongs. When bacteria
enter through the mouth, intestinal diseases can occur in children and adults. Some
O-groups of Escherichia (serovars) are most often the causative agents of human
diseases. These bacteria are called enteropathogenic Escherichia coli
(EPEC). Currently, many variants of EPKD are known, causing a different course of
escherichiosis. There are several groups of EKPC:
group I - causative agents of colienteritis in young children (serogroups O111, O26,
O55, O86, etc.);
group II - causative agents of dysentery-like diseases in children and adults (O25,
O124, O143, O144, etc.);
group III - causative agents of cholera-like diseases (O1, O5, O6, O78, etc.).
Once in food, E. coli can multiply in them. Eating such foods leads to the
development of food poisoning.
The development of endogenous infection leads to damage to various organs:
inflammation of the gallbladder (cholecystitis), bladder (cystitis), blood poisoning
(sepsis), etc.
Immunity . Immunity is developed only in relation to one serovariant of Escherichia -
the causative agent of this disease. The variety of Escherichia practically makes this
immunity ineffective. In the development of the immune state in children with
illness, the formation of IgM antibodies is of great importance, which do not pass
through the placenta, and therefore are not transmitted from the mother. IgA
antibodies to Escherichia are transmitted to the child from the mother with breast
milk.
Prevention . Compliance with personal hygiene and sanitary-hygienic regime. There
is no specific prophylaxis.
Treatment . Antibiotics: ampicillin, tetracycline, etc. Currently, coliproteus phage is
being produced, the use of which gives good results.
 Control questions

1. What are the main features of intestinal bacteria?

2. What antigens does Escherichia have?
3. What medications are prepared from Escherichia coli?

Microbiological research

The purpose of the study: isolation and identification of EPKD.

Research material
1. Bowel movements.
2. Vomit.
If necessary, examines the discharge from the nose and throat, pus from the ear,
blood, urine, pieces of the organs of the corpse.
When a focus of coli-enteritis occurs, food products, washings from the hands of
attendants, toys and other items are examined (according to epidemiological
indications).
Material collection methods

Material collection methods

Note. The earlier the feces are examined from the onset of the disease, the more
likely the possibility of isolating the pathogen.
Main research method
Bacteriological
Research progress
First day of research

First day of research

Second day of research

The cups sown the day before are removed from the thermostat and viewed in
incident or transmitted light. In the presence of raspberry-red colonies on Endo's
medium (with or without a metallic sheen) or purple on EMS medium, a trial
agglutination reaction on glass is performed to differentiate EPP from other varieties
of Escherichia.
To set up a trial agglutination reaction, at least 10 isolated colonies are selected,
marking or numbering them on the back of the dish; a part of each target colony is
removed with a loop and agglutinated in a drop of polyvalent serum or
immunoglobulin. Only part of the colony is tested so that in the case of a positive
agglutination reaction, a pure culture can be isolated from the remaining part of the
colony.
Typical or polyvalent escherichial sera (or immunoglobulins) are produced under
production conditions. Polyvalent Escherichia OK sera (or OK immunoglobulins)
contain antibodies to several Escherichia O and K antigens. With their help, it is
tentatively determined that the isolated culture belongs to EPKP. For example,
polyvalent serum O26, O55, O111 makes it possible to identify cultures of
Escherichia of the same name. Serums are diluted as directed on the label.
In the laboratory, a mixture of individual OK sera can be prepared by combining no
more than 5 sera so that the dilution of each is no higher than 1:10.
Setting up a trial agglutination reaction . 10 drops of polyvalent serum (or
immunoglobulin) are applied to one or two well-defatted glass slides. A part of the
intended colony is added to each drop and triturated. Colonies that gave an
agglutination reaction are sifted into test tubes with slant agar and placed in a
thermostat for 18-20 hours. If none of the 10 colonies gave an agglutination reaction,
give a negative answer.
Third day of research
The crops are taken out of the thermostat and viewed. On MPA, enteropathogenic
Escherichia coli usually form a moist, shiny, grayish coating, less often it is
cloudy. The culture grown on slant agar is checked again in the agglutination reaction
on glass with polyvalent Escherichia sera (or immunoglobulins). If the isolated culture
gives an agglutination reaction with a polyvalent serum (immunoglobulin), then it is
agglutinated with each typical serum (immunoglobulin) separately at a dilution of 1:5
- 1:10. Agglutination with live culture is indicative.
Next, it is necessary to confirm the belonging of the isolated culture to the genus
Escherichia by biological tests. To do this, the culture is sown on semi-liquid Giss
media with lactose, glucose, mannitol, sucrose, maltose and other sugars, as well as
on broth or peptone water to determine the formation of indole and hydrogen
sulfide. To do this, two indicator papers moistened with reagents that detect the
formation of these substances are lowered into test tubes under the cork. One piece
of paper turns red in the presence of indole, the other turns black in the presence of
hydrogen sulfide.
During the fermentation of sugars, the reaction of the medium becomes acidic and
the color of the indicator changes. If, in addition to the acid, a gas is formed, bubbles
appear in the medium. At the same time, the mobility of bacteria is determined:
inoculation is made in semi-liquid (0.2%) agar by injection. Motile bacteria give
clouding of the entire environment, immobile - grow only by injection.
For the final identification of the isolated culture, a detailed agglutination reaction is
performed with live and heated cultures: with live - to determine the K-antigen, with
heated - to determine the O-antigen. To set up a detailed agglutination reaction, the
antigen is prepared as follows: 3-5 ml of isotonic sodium chloride solution is washed
off the culture from the slant agar. The resulting suspension is poured into two test
tubes. One of them is heated in a water bath at 100 ° C for an hour.
An extended agglutination reaction is placed in two rows of test tubes. The serum in
both rows is diluted in a ratio of 1:50 - 1:100 (in the 1st test tube) to the titer
indicated on the label of the serum ampoule. 2 drops of live culture are added to the
first row, 2 drops of heated culture are added to the second row.
The tubes are shaken and placed in a thermostat for 18-24 hours.
Fourth day of research
The changes in the Hiss media are recorded, the formation of indole and hydrogen
sulfide is recorded.
Most representatives of Escherichia ferment carbohydrates with the formation of
acid and gas, break down the protein nutrient substrate to form indole.
Accounting for the test-tube agglutination reaction is carried out using a magnifying
glass or an agglutinoscope. Agglutination with live culture is coarse-grained, with
dead culture - fine-grained. The reaction is considered positive if agglutination with a
heated culture is noted in a serum dilution of at least half a serum titer, and a live
culture is agglutinated with serum diluted at least 1:200. The ratio of antibodies to
heated and live culture also plays a role. The dilution of serum, in which
agglutination with a heated culture is noted, must exceed the dilution of serum, in
which a live culture is agglutinated, by at least 2 times. In table. 31 shows various
options for the result of the agglutination reaction.

Table 31. Results of the agglutination reaction with Escherichia cultures

Note. Three reaction options are possible: 1) the heated culture is agglutinated with
serum in higher dilutions than the live one, the reaction is positive; 2) live and heated
culture give agglutination in the same serum dilutions. Such a result may indicate the
absence of the K-antigen in the culture; agglutination of live and heated cultures is
caused by O-antigen. In these cases, it is necessary to repeat the agglutination
reaction; 3) agglutination of a live culture in the absence of agglutination with heat
allows you to give a negative answer. Obviously, in culture there is no O-antigen
corresponding to O-antibodies in serum (Fig. 41).
Rice. 41. Scheme for the isolation and identification of enteropathogenic Escherichia
coli
 Control questions
1. What material is examined to isolate Escherichia?
2. What sera can be used to differentiate EPKD?
3. Why is an extended agglutination reaction performed with live and heated
Escherichia cultures?
Exercise
1. Get from the teacher Petri dishes with culture inoculated on Endo medium and
inoculate on a test tube with slant agar.
2. Take the culture of EPP on agar slant from the teacher, wash it with isotonic
sodium chloride solution. Warm part of the wash in a water bath at 100°C. Dilute the
serum in two rows of test tubes and set up the agglutination reaction as described
above.
Nutrient media
Endo and EMS differential media are used to grow intestinal bacteria. Available in dry
powder form. According to the directions on the label, a certain amount of dry
medium is weighed, dissolved in the appropriate amount of water, boiled with
stirring, and poured into sterile Petri dishes.
 Chapter 19. Salmonella - L. B. Bogoyavlenskaya, F. K.
Cherkes
More than 2,000 different bacteria that cause diseases in humans and animals
belong to this genus of the Enterobacteriaceae family. These diseases are called
salmonellosis. Salmonella are similar in morphological, cultural and enzymatic
properties, but differ in antigenic structure.
Salmonella is divided into monopathogenic and polypathogenic. The former include
causative agents of typhoid, paratyphoid A and paratyphoid B. Only humans are
affected by these diseases. The second group includes pathogens that affect humans
and animals.
S. typhi were first discovered by Ebert (1880) in the organs of a person who died of
typhoid fever. Ashar and Bansod (1886), in diseases similar to typhoid fever, isolated
from the pus and urine of patients with bacteria that differ in biochemical and
serological properties from the causative agents of typhoid fever. They were called S.
paratyphi A and S. paratyphi B. Almost simultaneously, the American scientist D.
Salmon (1885) first described the causative agents of swine cholera (S.
choleraesuis). Subsequently, many similar bacteria were described, united in the
genus Salmonella, named after the scientist who described them.
Morphology . All Salmonella are small, 1.0-3.0 × 0.6-0.8 µm sticks with rounded
ends. Gram-negative. Mobile, peritrichous. Spores and capsules do not form.
Cultivation . Salmonella are facultative anaerobes. They are not demanding on
nutrient media. They grow well on MPA and MPB at 37°C (from 20 to 40°C) and pH
7.2-7.4 (from 5.0 to 8.0). On MPA they form tender, translucent, slightly convex,
shiny colonies, on MPA - uniform turbidity.
During the initial sowing of material from patients (feces, urine, vomit, blood, bile), a
slow growth of Salmonella is often noted. For their accumulation, seeding is carried
out on enrichment media: selenite broth, Muller's medium, Kaufman's
medium. Elective (selective) media are also used: bile (10-20%) and Rappoport
medium.
On the differential diagnostic media of Endo, EMS, Ploskirev, Salmonella grow in the
form of colorless colonies, since they do not break down lactose, which is part of the
medium. On bismuth-sulfite agar, after 48 hours they form black colonies, leaving a
trace after they are removed with a loop (except for Salmonella paratyphoid A).
In freshly isolated cultures of S. paratyphi B, after incubation in a thermostat for 18-
20 hours and keeping at room temperature for 1-2 days, a mucous wall forms on the
periphery of the colony.
enzymatic properties . Salmonella break down glucose, mannitol, maltose with the
formation of acid and gas. An exception is the causative agents of typhoid fever (S.
typhi), which break down these sugars only to acid. Salmonella does not ferment
lactose and sucrose. Proteolytic properties: most Salmonella breaks down protein
media with the formation of hydrogen sulfide (the causative agents of paratyphoid A
are distinguished by the absence of this property). Indole does not form. Gelatin is
not liquefied.
Toxigenicity . Salmonella contains endotoxin - a lipopolysaccharide-protein complex.
Antigenic structure and classification . As early as the beginning of the 20th century,
scientists noticed the different nature of Salmonella antigens. Kaufman (1934), based
on the results of the agglutination reaction of various Salmonella with a set of sera,
divided all Salmonella into groups and types and proposed a diagnostic scheme for
their antigenic structure. In accordance with this scheme, Salmonella is currently
being identified.
Salmonella contains two antigenic complexes: O and H, O-antigen - a
lipopolysaccharide-protein complex; it is thermostable, inactivated by the action of
formalin. The H-antigen is associated with flagella, has a protein nature; it is
thermolabile, inactivated by alcohol and phenol, but resistant to formalin.
All Salmonella are divided into O-groups, each of which is characterized by the
presence of certain O-antigens: the main one, indicated by an Arabic numeral (2, 4,
7, 8, 9, etc.), and additional ones common to several O-groups ( 1, 12). Currently,
more than 60 O-groups are known, denoted by capital letters of the Latin alphabet
(A, B, C, D, E, etc.).
S. typhi also contains the Vi-antigen, which is located in the microbial cell more
superficially than the O-antigen, and prevents agglutination of the culture with O-
serum. This antigen is thermolabile. Its presence was associated with the virulence of
the pathogen. The Vi antigen is also contained in the cells of S. paratyphi C.
Salmonella H-antigens have two phases. Salmonella of different serovariants of the
same O-group have a different first phase of the H-antigen, which is denoted by
lowercase letters of the Latin alphabet: a, b, c, d, eh ... u, z, etc. The second phase of
the H-antigen is usually denoted in Arabic numbers: 1, 2, 5, 6, 7 and lowercase Latin
letters. The combination of various O- and H-antigens determines the antigenic
structure of cultures and their name.
In practical work, to determine the antigenic structure of Salmonella, adsorbed
monoreceptor agglutinating sera are used, which contain antibodies to one
antigen. Put the agglutination reaction on the glass and the presence of agglutination
with certain sera characterize the antigenic structure of the selected culture. For
example, the culture is agglutinated with O-sera "9" and "12" and H-serum "d"; find
in the scheme a serovar with such an antigenic composition (S. typhi), put an
additional reaction with Vi-serum
There are sets of specific Salmonella phages that lyse only the Salmonella of the
corresponding phage. To determine the fagovar of S. typhi cultures containing the Vi
antigen, 45 phages are produced in our country; for S. paratyphi B-11 phages; S.
paratyphi A - 6, etc. These studies are carried out to determine the source and route
of transmission of the infection.
Resistance to environmental factors. Salmonella are quite resistant. At a
temperature of 100 ° C, they die instantly, at 60-70 ° C - in 10-15 minutes. They
tolerate low temperatures well, can be stored in clean water and ice for several
months; in smoked and salted meat - up to 2 months. Resistant to drying, long-
lasting in dust.
Under the action of disinfectants, they die within a few minutes (2-5% phenol
solution, 1:1000 sublimate solution, 3-10% chloramine solution).
Susceptibility of animals . Most salmonella causes disease in humans and many
species of animals and birds (polypathogenic).

Typhoid fever, paratyphoid A and B

source of infection . A sick person and a bacteriocarrier.

transmission paths . Infectious agents are transmitted through objects contaminated
with human secretions, through hands, water, food. Often pathogens are carried by
flies. Depending on the routes of transmission, household, water, food outbreaks of
typhoid fever and paratyphoid fever are distinguished.
Pathogenesis. Infection occurs through the mouth. From the oral cavity,
microorganisms enter the stomach, where they are partially destroyed under the
influence of gastric juice and enzymes. The remaining Salmonella enter the small
intestine, penetrate into the lymphoid tissue of the small intestine (group lymphatic
and solitary follicles), in which they multiply during the incubation period (10-14
days). By the end of this period, pathogens enter the lymph and blood (bacteremia)
and spread throughout the body. During this period, they are localized in the
lymphoid tissue of internal organs, the macrophage system, the liver, spleen, and
bone marrow. Salmonella accumulate in the gallbladder, where they find favorable
conditions for reproduction, since bile is a good breeding ground for these
bacteria. At the same time, they again enter the small intestine and,
 Rice. 42. Salmonella circulation in the human body. 1 - small intestine; 2 - group
lymphatic follicles; 3 - blood vessels; 4 - gallbladder

During the period of bacteremia, part of the microorganisms is destroyed, while

endotoxin is released and intoxication occurs: the temperature rises, general
malaise, weakness, headache, etc. appear. , urine, saliva, etc.
The period of convalescence (recovery) is characterized by the cleansing of the body
from the pathogen, increased phagocytic activity of cells, and the accumulation of
antibodies in the blood.
However, in typhoid fever and paratyphoids, bacterial excretion often does not end
with the patient's recovery - a bacteriocarrier is formed. Chronic inflammatory
phenomena in the gallbladder contribute to the survival of Salmonella in bile and
their long-term excretion from the body (sometimes up to several years).
Immunity . Post-infectious immunity is quite tense and long. Recurrences are
rare. During the course of the disease, antibodies are produced: at the end of the 1st
week, agglutinins, precipitins and other types of antibodies appear. Their number
increases, reaching a maximum on the 14-15th day of illness. Antibodies remain in
the blood serum of the ill person for a long time.
The activity of phagocytes and other cellular protective factors are also important in
the formation of the body's immune state.
Prevention . Compliance with personal hygiene and carrying out all sanitary and
hygienic measures: supervision of water sources, control of food and catering
establishments.
specific prophylaxis . Chemical vaccine containing full antigens of causative agents of
typhoid, paratyphoid A and B and tetanus toxoid (TAB'te). There is also a typhoid
alcohol vaccine enriched with Vi-antigen, the introduction of which for prophylactic
purposes gives a good effect. In the focus of the disease, persons who have been in
contact with the patient are given a typhoid bacteriophage.
Treatment . Antibiotics: chloramphenicol, tetracycline, etc.

Food poisoning

When eating foods contaminated with Salmonella of various serovars (except for S.
typhi, S. paratyphi A and B), food toxic infections occur.
Sources of infection . Animals and birds sick with salmonellosis, or healthy, in the
body of which, without harming them, there are salmonella.
transmission paths . Infection occurs when eating meat, meat products, eggs, milk,
dairy products infected with salmonella. The most dangerous is the use of food in
which the reproduction and death of Salmonella and the accumulation of endotoxin
occur.
Pathogenesis . Once in the body through the mouth, salmonella penetrate the
digestive tract. At the same time, a significant part of the bacteria dies and endotoxin
is released, which can penetrate into the blood. There are symptoms of damage to
the gastrointestinal tract and general toxicosis. The disease lasts no more than 4-5
days; sometimes those who have been ill become carriers of salmonella.
Immunity is short lived. Various antibodies accumulate in the blood of patients and
convalescents: agglutinins, precipitins, etc. There are a lot of Salmonella serovars,
and immunity is specific, that is, directed against only one pathogen, so a person can
get sick with salmonellosis again.
Prevention . Constant strict veterinary and sanitary control over livestock, slaughter
and cutting of carcasses, storage and processing of meat and meat products. It is
necessary to strictly observe the sanitary and hygienic regime and personal hygiene
in catering establishments.
specific prophylaxis . People who are in the foci of food poisoning should be given a
salmonella polyvalent bacteriophage.
Treatment . The main therapeutic agent is detoxification of the body - the
introduction of a large amount of liquid, gastric lavage. Antibiotics are also used.

Nosocomial Salmonella Infection

The causative agent of nosocomial Salmonella infection is most often S.

typhimurim. There are also "hospital" outbreaks caused by S. heidelberg, S. derby,
etc. Although the morphological and cultural properties of these pathogens do not
differ from those of other Salmonella, there are some biological features
characteristic of them. So, for example, pathogens of nosocomial infections belong
to certain biovars, they are more pathogenic for white mice, etc.
Sources of infection . More often a bacteriocarrier, less often a patient.
transmission paths . Indirect contact predominates (toys, underwear, patient care
items). Less common are airborne and foodborne transmission routes.
Pathogenesis . The disease develops against the background of a weakening of the
body and a decrease in its immune activity. The pathogen enters the body orally or
through the respiratory tract, which determines the development of the pathological
process: dysfunction of the gastrointestinal tract with dehydration or damage to the
respiratory system, bacteremia, septic complications. First of all, young children get
sick.
Immunity . It is produced only in relation to one serovar of Salmonella.
Prevention . Strict observance of the sanitary and hygienic regime in medical
institutions.
specific prophylaxis . If nosocomial Salmonella infection occurs, children who have
been in contact with the patient should be given a Salmonella polyvalent
bacteriophage.
Treatment . Symptomatic.

Control questions

1. What are the morphological, cultural and enzymatic properties of Salmonella?

2. What is the classification of Salmonella based on?
3. What diseases are caused by salmonella?
Microbiological research
The purpose of the study: the isolation of pathogens and the determination of the
serovar of Salmonella.
Research material
1. Blood.
2. Bowel movements.
3. Urine.
4. Duodenal contents.
Depending on the stage of the disease, different materials are examined.
The contents of roseola, bone marrow, sputum and the material obtained at the
autopsy - pieces of organs can also be subjected to research.
In case of toxic infections, gastric lavage, vomit, food residues can serve as research
material.
Material collection methods
 Material collection methods

Regardless of the nature of the material taken for the study, from the moment the
pure culture was isolated, the study is carried out according to the general scheme.
Basic research methods
1. Bacteriological (Fig. 43).
2. Serological.
Rice. 43. Scheme of microbiological research in typhoid fever and paratyphoid fever
 in different periods of the disease. I - 1st period of the study (hemoculture); II - 2nd
period of the study (Vidal reaction); III - 3rd period of the study (coproculture)

Research progress
First day of research

First day of research

Second day of research

The cups are removed from the thermostat (incubation 18-24 hours) and the grown
colonies are viewed with the naked eye and with a magnifying glass. Several (5-6)
suspicious colonies are isolated on Olkenitsky's or Russell's medium. Sowing is
carried out as follows: the removed colony is carefully, without touching the edges of
the test tube, introduced into the condensation liquid, then the entire sloping
surface of the medium is inoculated with strokes and an injection is made into the
depth of the column to detect gas formation. The injection should be made in the
center of the agar column.
Test tubes with crops are placed in a thermostat. If the test material was sown on
the enrichment medium, then after 18-24 hours, the enrichment medium is sown on
plates with differential media. Further research is carried out according to the
general scheme.
 Table 32. Growth of Salmonella on differential diagnostic media
1
 ( A black trace remains at the site of the removed colonies (the color of the medium changes).  )
Third day of research
Take out test tubes with crops from the thermostat and view the nature of growth.
Combination media contains lactose, glucose, sometimes urea, and an indicator. The
breakdown of glucose occurs only under conditions of anaerobiosis. Therefore, the
sloping surface of the medium does not change during the breakdown of glucose,
and the column turns into a color corresponding to the indicator. Bacteria that break
down lactose and urea change the color of the entire medium.
If isolated cultures ferment lactose or break down urea, changing the color of the
entire medium, then they are not Salmonella and a negative answer can be given.
A culture that degrades only glucose is subjected to further study: smears are made,
stained by Gram and microscopically. If there are gram-negative rods in the smears,
their mobility and enzymatic properties are studied.
Motility can be determined in a hanging drop or a crushed drop, and by growth
pattern in semi-liquid Hiss medium or 0.2% agar. In the presence of mobility during
sowing by injection, growth on the medium is diffuse, the medium becomes cloudy.
To detect enzymatic activity, inoculation is carried out on Hiss media, MPB, peptone
water. Indicator papers are lowered (under the cork) into test tubes with the last
media for the determination of indole and hydrogen sulfide. Do also sowing on
litmus milk.
Fourth day of research
Biochemical activity is taken into account according to the result of fermentation of
carbohydrate and other media (see Table 33).

Table 33. Enzymatic properties of Salmonella

Note. to - the formation of acid; kg - formation of acid and gas; u - alkalization; +

presence of property; - lack of property.
Having determined the morphological, cultural and enzymatic properties of the
isolated culture, it is necessary to analyze the antigenic structure (Table 34).
 Table 34

Serological identification of Salmonella begins with an agglutination test on glass

with polyvalent O-serum A, B, C, D, E. In the absence of agglutination, the isolated
culture is tested with polyvalent O-serum to rare groups of Salmonella. In case of a
positive reaction with one of the sera, the culture is tested with each O-serum, which
is part of the polyvalent one, to determine the O-serogroup. Having established that
the culture belongs to the O-group, its H-antigens are determined with the sera of
the first, and then the second phase (Table 35).

Table 35. Antigenic structure of typhoid and paratyphoid pathogens

Salmonella typhoid culture is also tested with Vi-serum. The causative agents of
typhoid fever containing the Vi-antigen are tested with Vi-phages (there are 86 of
them). Determination of the phage type is of great epidemiological importance (see
Fig. 43).
Phage typing technique . 1st method. 20-25 ml of agar are poured into Petri dishes
and dried with open lids in a thermostat. The bottom of the cup is divided into
sectors. The name of the phage is written on each sector. A 4-6 hour broth culture is
studied as it contains more Vi antigen. 8-10 drops of broth culture are applied to the
surface of the agar and rubbed over the surface of the agar with a glass spatula. Cups
with crops are dried with open lids in a thermostat. A drop of the corresponding
typical phage is applied to each sector. After the drops have dried, the cups are
placed in a thermostat for 18-24 hours. The result is taken into account with the
naked eye or with a magnifying glass through the bottom of the cup.
The presence of culture lysis by one or several typical phages makes it possible to
determine whether the isolated strain belongs to a particular phage type.
2nd method. The culture is applied dropwise to the nutrient medium. After drying
the culture in a thermostat, a drop of a typical phage is applied to each drop. Put in a
thermostat.
The degree of lysis is expressed by the four-cross system.
Control questions
1. What material is examined for typhoid, paratyphoid and toxic infections?
2. In what period of the disease is the blood culture isolation method used?
3. In what period of the disease with typhoid fever and paratyphoid fever are feces
and urine examined?
4. On what differential diagnostic media is the test material inoculated?
5. What media are used for the accumulation of Salmonella?
6. What is determined by monoreceptor O-sera and what by monoreceptor H-sera?
Exercise
1. Study according to the table. 32 the nature of the growth of Salmonella on
differential media. Look at the teacher's cups with the inoculation of typhoid
Salmonella on the Endo, Ploskirev media, bismuth-sulfite agar. Sketch the colonies
with colored pencils and show the teacher.
2. Take Salmonella culture, O- and H-monoreptor sera from the teacher. Put the
agglutination reaction on the glass. Consider the reaction and show the teacher.
Task
The isolated culture gave a positive agglutination test with O-serum 4. What H-sera
should be tested with agglutination if you think it is a culture of Salmonella
paratyphoid B?
Serological diagnosis of typhoid and paratyphoid fever
Vidal reaction . From the second week of the disease, antibodies against the
infectious agent accumulate in the blood of patients. To identify them, the patient's
blood serum is examined in the agglutination reaction. Killed Salmonella cultures -
diagnosticums - are used as an antigen.
To set up the Vidal reaction, the patient's serum, a set of diagnostic kits, and isotonic
sodium chloride solution are used.
Blood (2-3 ml) from the pulp of the finger or cubital vein is collected in a sterile tube
and delivered to the laboratory. In the laboratory, the test tube is placed in a
thermostat for 20-30 minutes to form a clot, then the clot is circled with a Pasteur
pipette to separate it from the wall of the test tube, and placed in the cold for 30-40
minutes. The separated serum is sucked off and used to set up an agglutination
reaction with diagnosticums from salmonella typhoid and paratyphoid. To obtain
serum, blood can be centrifuged.
When an infectious process occurs - typhoid fever or paratyphoid fever - O- and H-
antibodies to the pathogen antigens of the same name are produced in the body.
O-antibodies appear first and disappear rather quickly. H-antibodies persist for a long
time. The same thing happens during vaccination, therefore, a positive Vidal reaction
with O- and H-antigens indicates the presence of a disease, and a reaction with only
H-antigens can be both in those who have been ill (anamnestic reaction) and in those
who have been vaccinated (vaccination). Based on this, the Vidal reaction is placed
separately with O- and H-antigens (diagnosticums).
Since clinically typhoid fever and paratyphoid fever A and B are similar, to identify
the nature of the disease, the patient's serum is tested simultaneously with
diagnosticums from salmonella typhoid and paratyphoid A and B.
The Vidal reaction is widely used because it is simple and does not require special
conditions.
There are two ways to set up the reaction: drop and volume (see Chapter 12). In
practice, the volumetric method is more often used. When setting up a linear
agglutination reaction, the number of rows should correspond to the number of
antigens (diagnosticum). The causative agent of the disease is considered to be a
microorganism, the diagnosticum from which was agglutinated by the patient's
serum. Sometimes group agglutination is noted, since the causative agents of
typhoid and paratyphoid fever have common group antigens. In this case, the result
of the reaction in the series in which agglutination is noted in a larger dilution of
serum is considered positive (Table 36).

Table 36. Possible result of the agglutination reaction

Note. In practice, the Vidal reaction is put with four diagnosticums: typhoid fever "O"
and "H", and paratyphoid A and B - with diagnosticums "OH".
If agglutination occurs only in small dilutions of serum - 1:100, 1:200, then to
distinguish the reaction in case of a disease from a vaccination or anamnestic, they
resort to re-staging the agglutination reaction after 5-7 days. In a patient, the
antibody titer rises, but in a vaccinated or recovered patient it does not
change. Thus, an increase in the titer of antibodies in the blood serum serves as an
indicator of the disease.
Vi-agglutinins appear in the patient's blood in response to the introduction of
typhoid pathogens possessing the Vi-antigen into the body. They are determined
from the 2nd week of illness, but their titer usually does not exceed 1:10. The
detection of Vi-antibodies is associated with the presence of typhoid pathogens in
the body; therefore, the determination of these antibodies is of great
epidemiological importance, since it makes it possible to identify bacterial carriers.
Vi-hemagglutination reaction . This is the most sensitive reaction for detecting
antibodies.
The principle of the reaction is that human (group I) or sheep erythrocytes, after
special treatment, can adsorb the Vi-antigen on their surface and acquire the ability
to agglutinate with the corresponding Vi-antibodies.
Erythrocytes with antigens adsorbed on the surface are called erythrocyte
diagnosticums.
To set up the Vi-hemagglutination reaction, take:
1) patient's blood serum (1-2 ml); 2) erythrocyte Salmonella Vi diagnosticum; H) Vi-
serum; 4) O-serum; 5) isotonic sodium chloride solution.
The reaction is placed in agglutination test tubes or in plastic plates with wells.
Blood is taken from the patient in the same way as for the Vidal reaction. Get the
serum. Two-fold serial dilutions are prepared from serum, starting from 1:10 to
1:160.
0.5 ml of each dilution is added to the well and 0.25 ml of erythrocyte diagnosticum
is added. The reaction is put in a volume of 0.75 ml.
The controls are: 1) standard agglutinating monoreceptor serum + diagnosticum - the
reaction must be positive up to the serum titer; 2) diagnosticum in isotonic sodium
chloride solution (control) - the reaction should be negative.
The contents of the wells are thoroughly mixed, placed in a thermostat for 2 hours
and left at room temperature until the next day (for 18-24 hours).
Accounting begins with control. The reaction is evaluated depending on the degree
of diagnosticum agglutination.
The results are taken into account according to the four-cross system:
++++ erythrocytes are completely agglutinated - sediment at the bottom of the well
in the form of an "umbrella";
+++ "umbrella" is smaller, not all erythrocytes were agglutinated;
++ "umbrella" is small, at the bottom of the hole there is a sediment of non-
agglutinated erythrocytes;
- the reaction is negative; erythrocytes did not agglutinate and settled on the bottom
of the well in the form of a button.
Control questions
1. In what period of the disease is the Vidal reaction diagnosed?
2. What ingredients are needed to perform the Vidal reaction?
3. What diagnosticums are used for Vidal's reaction?
4. Which of the serological reactions is the most sensitive in diagnosing typhoid and
paratyphoid infections?
5. What diagnosticum is used in the formulation of the Vi-hemagglutination
reaction?
6. What serum is used to determine the presence of Vi-antigen in the studied
culture?
7. What is the significance of the definition of the Vi-phage type?
Exercise
Take from the teacher O- and H-diagnosticums from salmonella typhoid, paratyphoid
A and paratyphoid B and the patient's serum. Put Vidal's reaction.
Nutrient media
Environments EMS, Ploskirev, bismuth-sulfite agar are produced by the medical
industry in the form of a dry powder. They are prepared according to the instructions
on the label: a certain amount of powder is weighed, the appropriate amount of
water is poured, boiled and poured into sterile Petri dishes.
Russell Wednesday . In 950 ml of distilled water add 40 g of dry nutrient medium
and add 5 g of nutrient agar. Heat to boil and dissolve the powders. Dissolve 1 g x in
50 ml of distilled water. hours of glucose and added to the prepared mixture. The
medium is poured into sterile test tubes of 5-7 ml, sterilized with flowing steam (2
days for 2 minutes) and beveled so that a column remains. Russell's medium with
mannitol and sucrose is prepared in the same way.
Olkenitsky's medium from dry agar . 2.5 g of dry nutrient agar is melted in 100 ml of
distilled water. All the ingredients indicated in the recipe (label) are added to the
agar cooled to 50 ° C. The medium poured into test tubes is sterilized with flowing
steam (3 days for 20 minutes) and then bevelled. The finished medium should be a
pale pink color.
 Chapter 20. Shigella - F.K. Cherkes
The first causative agent of dysentery was discovered by A. V. Grigoriev (1891), and
in 1898 the Japanese scientist Shiga studied and described it. In subsequent years,
other representatives of this genus were isolated and described: Flexner (1900),
Sonne (1915), Stutzer-Schmitz (1917), Large-Sachs (1934).
According to the International Classification, all bacteria that cause dysentery are
combined in honor of Shiga into one genus - Shigella.
Morphology . Shigella are small (2-3 × 0.4-0.6 µm) rods with rounded ends. They
differ from other members of the Enterobacteriaceae family in the absence of
flagella. They do not have spores or capsules. Gram-negative.
Cultivation . Shigella are facultative anaerobes. Unpretentious to nutrient
media. They multiply on MPA and MPB at a temperature of 37 ° C and pH 7.2-
7.4. Elective and differential diagnostic environments for them are Ploskirev, Endo,
EMS environments. Grow in the form of small, translucent, grayish, round colonies,
15-2 mm in size in an S-shape. An exception is Sonne's Shigella, which often
dissociate, forming large, flat, cloudy, R-shaped colonies with jagged edges (Fig.
44). In liquid nutrient media, Shigella give a uniform turbidity, R-forms form a
precipitate.
 Rice. 44. Scheme of bacteriological research in dysentery

enzymatic properties . The enzymatic properties of Shigella are less pronounced

than those of other representatives of Enterobacteriaceae: they break down
carbohydrates without gas formation, do not break down lactose and sucrose. The
exception is Sonne shigella, which break down these carbohydrates on the 2nd-3rd
day.
The proteolytic properties of Shigella are not very pronounced - the formation of
indole and hydrogen sulfide is inconsistent, they coagulate milk, and do not liquefy
gelatin.
In relation to mannitol, all shigella are divided into splitting and non-cleaving
mannitol (Table 37).

Table 37. Enzymatic properties of Shigella

Note. to - splitting with the formation of acid.

Sonne shigella are currently divided into four enzymatic types. They differ in their
ability to break down rhamnose and xylose (Table 38).

Table 38. Biovariants of Shigella Sonne

Note. + splitting; (+) splitting after 3-5 days; - does not change.

Toxin formation . Shigella have endotoxin. An exception is Shigella Shigi, which, in
addition to endotoxin, secrete exotoxin, which has a neurotoxic effect.
Antigenic structure and classification . Shigella contain somatic antigens, which
include group and type antigens. According to the International Classification,
Shigella is divided into four groups, denoted by Latin capital letters A, B, C, D.
Group A S. dysenteriae: 1 - Grigorieva - Shigi; 2 - Stutzer - Schmitz; 3-7 - Large - Saks
and 8-10 - provisional. Representatives of this group have only typical antigens,
denoted by Arabic numerals.
Group B S. flexneri. Microbes of this group have a more complex antigenic structure -
they contain typical antigens, denoted by Roman numerals, and group antigens,
denoted by Arabic numerals. Shigella Flexner has 6 serotypes. Shigella Flexner 6 was
formerly designated as a subspecies of S. newcastle.
Group C S. boydii. Has only typical antigens. There are 15 serological types in this
group.
Group D S. sonnei has its own species antigen (Table 39).
 Table 39. Classification of bacteria of the genus Shigella

In a microbiological study, the answers indicate the serovariant and subserovariant

of the isolated culture. For example, a culture of Shigella Flexner 1a was isolated.
Resistance to environmental factors . A temperature of 100°C kills Shigella
instantly. A temperature of 60 ° C kills them in 20-30 minutes. Shigella are resistant
to low temperatures - they remain in river water for up to 3 months, on vegetables
and fruits - up to 10-15 months. Sunlight kills them in 2-3 hours, and Shigella Shigi -
in 20 minutes. Commonly used concentrations of disinfectant solutions destroy them
in 20-30 minutes. Group A shigella are the least resistant to external factors, and
Sonne shigella are the most resistant.
Susceptibility of animals . Animals are not sensitive to pathogens of dysentery, with
the exception of monkeys. Experimental infection of rabbits and white mice causes
intoxication and death in them.
Sources of infection . A person suffering from acute and chronic forms of dysentery,
and a bacteriocarrier.
transmission paths . Food. Of great importance is the waterway, vegetables, fruits,
various items contaminated with shigella, and flies.
Pathogenesis . Once in the intestine with food, Shigella penetrate into the epithelial
cells of the mucous membrane of the large intestine, where they multiply. Some of
them die. The endotoxin formed during the destruction of bacteria sensitizes the
mucous membrane, the permeability of blood vessels increases, and the endotoxin is
absorbed into the blood, causing intoxication. The defeat of the mucous membrane
is accompanied by swelling, necrosis, hemorrhage. In addition, the toxin affects the
central nervous system, which leads to trophic disorders. Especially severe is the
disease caused by Shigella Shigi, which penetrate deep into the mucous membrane
of the colon, causing severe hyperemia, swelling and bloody diarrhea. The exotoxin
formed by them causes severe intoxication.
The magnitude of the infectious dose is important for the occurrence of the disease.
Immunity . A person has a natural resistance to dysentery infection. After the illness,
immunity is unstable, and after Sonne's dysentery, it is practically absent. With a
disease caused by Shigella dysentery 1 (Grigorieva - Shigi), a more stable antitoxic
immunity is developed.
Prevention . General sanitary and anti-epidemic measures: isolation, early diagnosis,
disinfection.
Specific prophylaxis has not found wide application. Persons who have been in
contact with patients are given a polyvalent dysenteric bacteriophage.
Treatment . Complex, sulfonamides with antibiotics. There is no specific treatment.

Control questions

1. Name the representatives of the genus Shigella - the causative agents of

dysentery.
2. The relation to which carbohydrate is the basis for dividing Shigella into two
groups? What types of Shigella are included in each of these groups?
3. What are the routes of entry of Shigella and what part of the intestine do they
affect?
4. What type of Shigella often grows in an R-shape?
5. Which Shigella have type and group antigens?

Microbiological research

The purpose of the study: detection and identification of Shigella for

diagnosis; detection of bacteria carriers; detection of shigella in foods.
Research material
1. Bowel movements.
2. Sectional material.
3. Food products.
Material collection methods
 Material collection methods

Basic research methods

1. Microbiological.
2. Serological.
Research progress
First day of research
 First day of research

Second day of research

The seeded cups are removed from the thermostat, viewed with the naked eye or
through a magnifying glass. Suspicious colonies (colorless) in the amount of 4-6 are
screened on Russell's medium and mannitol. Sowing is done by strokes on a sloping
surface and an injection into an agar column. The inoculated Russell medium is
placed in a thermostat for 18-24 hours (in parallel, reseeding from the selenite
medium to differential media is done).
Third day of research
Take out the cultures made on Russell's medium from the thermostat. Cultures that
have not broken down lactose are subjected to further study: smears are made,
stained according to Gram and microscopically. In the presence of gram-negative
rods, inoculation is carried out on Hiss media, broth with indicator papers (to detect
indole and hydrogen sulfide) and litmus milk. The inoculated media are placed in a
thermostat for 18-24 hours.
Fourth day of research
Take out the crops from the thermostat and take into account the result. Cultures
suspicious for their enzymatic and cultural properties against Shigella (see Table. 37)
are subjected to serological identification. In the absence of such cultures give a
negative answer.
Serological identification
The species, serovar, subserovar of the isolated culture is determined using adsorbed
sera. Analysis of the antigenic structure begins with an agglutination test on glass
with mixture No. 1. This mixture includes sera with antibodies to Sonne, Newcastle
shigella and polyvalent serum to Flexner's shigella. With a positive agglutination
reaction with the mixture, the isolated culture is agglutinated separately with each
serum included in the mixture.
A positive agglutination test with adsorbed serum against Shigella Sonne and
Newcastle gives the right to answer. To establish the serovar and subserovar of
Shigella Flexner, it is necessary to additionally perform agglutination reactions with
typical (I, II, III, IV, V) and group (1-3, 4-6-7, 8) sera. For example, the isolated culture
gave a positive reaction with type II serum and group serum 3, 4. As can be seen
from the table, the Flexner Shigella culture, serovar 2, podserovar 1a, was
isolated. Answer: Shigella Flexner 2a are isolated.
In the absence of agglutination with mixture No. 1, an agglutination reaction is
performed with other polyvalent sera.
When setting up the agglutination reaction, one should take into account the ratio of
the studied culture to mannitol and, depending on this, use one or another
serum. So cultures that do not break down mannitol are tested with polyvalent sera
to Shigella dysentery Grigoriev-Shiga and Stutzer-Schmitz (1, 2), Large-Sachs (3-7),
provisional types (8-10).
Mannitol degrading cultures are tested with Mix No. 1 and Boyd's Shigella polyvalent
sera.
In the presence of agglutination of the isolated culture of one of these sera, a culture
test is carried out with each of the sera included in the polyvalent one. A positive
result with one of the sera determines the serovariant of the isolated culture.
When using serum for Boyd's shigella, agglutination is started with the serum of the
serovar that is most common in the area. In our country, Boyd's shigella of
serovariants 4, 5, 7, 9 and 12 are more often isolated (see Fig. 44).
As accelerated methods of microbiological research in dysentery, fluorescent
microscopy and a biological test on guinea pigs are used. With the introduction of
virulent strains of Shigella into the conjunctival sac (under the lower eyelid), by the
end of the 1st day, the animals develop conjunctivitis.
Control questions
1. What material is used for bacteriological examination in the diagnosis of dysentery
and how is it collected?
2. The breakdown of what carbohydrate gives the right to give a negative answer?
3. What sera can be used to determine the type, subspecies, serovar and subserovar
of Shigella Flexner?
4. What serums are included in Blend #1?
Exercise
Study the dysentery study chart by day.
Nutrient media
Environments Ploskirev, Endo, EMS (see Chapter 19).
 Conditionally pathogenic bacteria - L. B.
Bogoyavlenskaya
The development of microbiology and the study of infectious diseases has led to the
idea that certain pathogenic microorganisms are the causative agents of infectious
diseases with their characteristic clinical picture, transmission routes and distribution
patterns (epidemiology). However, since the second half of the 20th century,
diseases caused by microorganisms that are usually associated with a person and
harmless to him have become widespread: E. coli, which constantly lives in the
intestines, staphylococcus a saprophyte of the skin, hemophilic microflora of the
upper respiratory tract, etc. These microorganisms were called conditionally
pathogenic , since their pathogenic effect is manifested only under certain
conditions: the weakening of the host organism or the acquisition of special
properties by the pathogen (resistance to antibiotics,
All over the world, there is a sharp increase in the frequency of purulent-
inflammatory diseases that complicate the course of the postoperative period, the
healing of burns, and the postpartum period. Such diseases are called clinical, since
they occur, as a rule, in medical institutions of various profiles.
The reasons for the development of such diseases include a change in the immune
reactivity of a person due to the receipt of various medicines during his life, including
antibiotics, hormones, and vaccine preparations. Allergization of the body and the
development of dysbacteriosis also play a role - violations of the usual ratio of
various microflora of the body. It is no coincidence that diseases caused by
opportunistic bacteria often affect newborns, especially premature ones, whose
body does not yet have a sufficient level of immune protection.
The spread of clinical infections also depends on the expansion of reservoirs of
infection: modern methods of examination and treatment are often associated with
instrumental intervention (insertion of catheters, endo- and bronchoscopes,
inhalers), which requires especially careful observance of asepsis and antisepsis.
And, finally, one of the main reasons for the manifestation of the pathogenic effect
of commensals is a change in their properties: the emergence of resistance to drugs,
an increase in resistance in the external environment, and the acquisition of toxic
properties.
Currently, the range of opportunistic pathogens is very wide. These include many
representatives of the intestinal family (Klebsiella, Proteus, Providencia, Serration),
coccal (Staphylococcus, group B streptococcus, enterococcus), Pseudomonas
aeruginosa, non-spore-forming anaerobes, etc.
The occurrence of clinical infections is usually associated with a violation of the
nosocomial regimen. The source of infection can be medical staff, patients,
visitors. The infection is transmitted through linen, tools, household items, air. This is
an exogenous infection, as microbes enter the body from the environment. In some
cases, the occurrence of infection occurs due to the acquisition of pathogenic
properties by the microflora of the organism itself. So, for example, after surgery,
microorganisms in the respiratory tract, due to congestion and insufficient oxygen
supply, cause pneumonia.
Manifestations of clinical infections, despite the multiplicity of pathogens, are
similar, which makes it difficult to recognize and treat them. Therefore,
microbiological diagnostics is of particular importance, which makes it possible to
determine the pathogen, prescribe rational treatment and effective measures to
limit the spread of infection.
 Chapter 21. Klebsiella
The genus Klebsiella, belonging to the family of enterobacteria, combines capsular
bacteria that cause various diseases: pneumonia and purulent-inflammatory
processes - K. pneumoniae, rhinoscleroma - K. rinoscleromatis, ozena (fetid rhinitis) -
K. ozaenae.
Morphology . Klebsiella are short thick sticks, 0.6-6.0 × 0.3-1.5 µm in size with
rounded ends. Motionless. Form a capsule. In smears, they are located singly, in pairs
or in short chains.
Cultivation . Klebsiella are facultative anaerobes. They grow well on simple nutrient
media at 35-37 ° C. On dense media they form dome-shaped mucous colonies, on
broth - intense turbidity.
enzymatic properties . They ferment lactose, break down glucose and mannitol with
the formation of acid and gas, decompose urea, do not form indole and hydrogen
sulfide.
Toxin formation . They have endotoxin. Their virulence depends on the presence of
a capsule - non-capsular forms are less virulent.
Antigenic structure . Klebsiella contains capsular K- and somatic O-antigens. The
combination of these antigens determines the belonging of cultures to certain
serovars. Currently, 80 K- and 11 O-antigens are known.
Resistance to environmental factors . Due to the presence of Klebsiella capsules,
they are stable and persist for a long time in soil, water, and on household items. At
65°C they die within an hour. Sensitive to the action of solutions of disinfectants
(chloramine, phenol, etc.). There is a high resistance to antibiotics.
Susceptibility of animals . Under natural conditions, they cause diseases of various
animals: cows, pigs, horses (mastitis, pneumonia, septicemia).
Sources of infection . With exogenous infection, the source of infection is a sick
person and a healthy carrier.
transmission paths . Contact-household (dirty hands, household items). In children's
institutions and hospitals, the infection is often transmitted through linen, tools, and
toys.
Pathogenesis . Klebsiella develops mostly as a secondary infection in individuals with
reduced resistance and in newborns (premature). Bacteria from the upper
respiratory tract and intestines penetrate into various organs and blood and cause
purulent-inflammatory processes, sepsis, meningitis.
Immunity . Post-infection immunity is short-lived and develops only in relation to
one specific pathogen (serovar).
Prevention . Compliance with the sanitary and hygienic regime in maternity
hospitals, hospitals, children's institutions. There is no specific prophylaxis .
Treatment is difficult due to the high resistance of Klebsiella to antibiotics. The most
effective use of gentamicin, kanamycin, sometimes ampicillin.

Microbiological research

The purpose of the study: isolation and identification of Klebsiella from pathological
material and environmental objects.
Research material
1. Phlegm.
2. Mucus from the pharynx, pus from the ear, wound discharge.
3. Bowel movements.
4. Washouts from environmental objects.
Material collection methods
 Material collection methods

Basic research methods

1. Microbiological.
2. Serological.
Research progress
First day of research

First day of research

Second day of research

They make smears, stain according to Gram. In the presence of amnegative rods,
mucous colonies (4-5) are selected and subcultured onto slant agar and Worfel-
Ferguson medium (to isolate a pure culture) and Russell's combined medium (or
medium with urea) to determine enzymatic properties and mobility. Strips of paper
impregnated with reagents for the determination of indole formation and hydrogen
sulfide are lowered into a test tube under the stopper.
Do a seeding from glucose agar on dense nutrient media for (if necessary) additional
research.
 Third day of research
With the growth of an immobile culture that ferments lactose, glucose, urea, which
does not form indole and hydrogen sulfide, inoculation is done on media with citrate
and malonate and smears to determine the presence of a capsule. In the presence of
a capsule, an agglutination reaction is put on glass with agglutinating K-sera. View
additional sowing on dense nutrient media. You can give an approximate answer:
"Klebsiella isolated."
Fourth day of research
The results of inoculation on a medium with citrate, malonate (growth) and other
carbohydrates (such as Russell or Olkenitsky) are recorded. They give the final
answer: "Klebsiella (K11) have been isolated."
Serological diagnostics
On the 7th-8th day of illness, if a disease with rhinoscleroma is suspected, RSC is put
with the patient's serum at a dilution of 1:100 - 1:1600 and a scleroma diagnosticum
from killed scleroma Klebsiella. The increase in antibody titer in the dynamics of the
disease is a confirmation of the diagnosis.

Control questions

1. What are the features of opportunistic bacteria?

2. How do Klebsiella differ from other enterobacteria?
 Chapter 22
The Proteus genus of the Enterobacteria family unites several species distinguished
by the ability to ferment many nutrient substrates.
Morphology . Bacteria of all species of this genus are small, polymorphic gram-
negative rods. The average size is 0.4-0.6 × 1.0-3.0 µm. Mobile, peritrichous. Spores
and capsules do not form.
Cultivation . Proteas are facultative anaerobes, they grow well on simple nutrient
media at 20-37 ° C. Some species give creeping growth on a dense nutrient medium,
and when sown in condensate water of slant agar, they grow over the entire surface
of the medium (the method of isolating a pure culture according to Shukevich) .
enzymatic properties . They have saccharolytic and proteolytic enzymes.
Toxin formation . Contains endotoxin.
Antigenic structure . Proteas contain O- and H-antigens. Currently, more than 150 O-
antigens and about 80 H-antigens are known. The combination of O- and H-antigens
in a microbial cell determines whether pathogens belong to one or another O-
serogroup or serovar.
Resistance to environmental factors . Relatively stable in the environment. They
tolerate heating up to 60 ° C for an hour and exposure to weak solutions of
disinfectants. They are resistant to many antibiotics.
Susceptibility of animals . Animal diseases caused by Proteus have not been
described.
Sources of infection . Proteas usually enter the external environment with human
feces. Thus, a person is a source of infection.
transmission paths . Food, contact and household (dirty hands, underwear,
household items, surgical instruments).
Pathogenesis . Proteus causes various forms of infection. When it enters the body
through the mouth, especially with food products in which the pathogen has
multiplied, Proteus causes food poisoning. When introduced into the body through
the wound and burn surface, purulent-inflammatory processes develop in various
organs.
Prevention . Compliance with the sanitary and hygienic regime at food enterprises,
in hospitals and children's institutions. There is no specific prophylaxis.
Treatment . Gentamicin, carbenicillin, kanamycin, etc. When the genitourinary
region is affected, drugs of the nitrofuran series are used: furagin, 5-NOC, etc. There
is a protein phage, the use of which gives a good effect.

Microbiological research

Purpose of the study: isolation and identification of the proteus from pathological
material and environmental objects.
Research material
1. Bowel movements.
2. Vomit.
3. Urine.
3. Mucus from the throat, pus from the ear, wound discharge.
5. Sectional material.
6. Washouts from environmental objects.
Material collection methods
 Material collection methods

Basic research methods

1. Microbiological.
2. Serological.
Research progress
First day of research

First day of research

Second day of research

The nature of growth on nutrient media is noted (swarming - a veil-like coating).
Separate colonies or part of continuous growth are isolated on Russell's combined
medium (with urea) or Olkenitsky's medium, test tubes with slant agar (according to
Shukevich) are inoculated into condensation water.
 Third day of research
A smear is made and stained according to Gram. In the presence of gram-negative
small rods, the nature of growth on Russell's or Olkenitsky's medium and the
presence of growth in a test tube with inoculation according to Shukevich are taken
into account. Proteus does not ferment lactose, ferments glucose with the formation
of gas, mostly hydrolyzes urea.
In the sample according to Shukevich - growth over the entire surface of the slant
agar.
Inoculation is carried out on additional media of the "variegated series": mannitol,
broth (to determine indole formation and the formation of hydrogen sulfide, papers
moistened with appropriate reagents are put into a test tube), semi-liquid agar,
gelatin. Do sowing on the medium with the amino acid phenylalanine.
Fourth day of research
The results of inoculation are taken into account: Proteus does not ferment mannitol
(most strains), forms indole and hydrogen sulfide, is mobile, liquefies gelatin and
forms the enzyme phenylalanine deaminase, which changes color in a test tube with
the amino acid phenylalanine. With these results, the isolated culture can be
attributed to the genus Proteus.
The final stage of the study is the formulation of the agglutination reaction on glass
with agglutinating sera to bacteria of the genus Proteus. First, they put an
agglutination reaction with polyvalent O-sera. With a positive reaction with one of
them, the agglutination reaction is repeated with each of the typical O-sera included
in the polyvalent one. After determining the O-group, a reaction is carried out with
H-sera and the serovar is determined. They give the answer: "Proteus 09:H 1.2 are
selected".

Control questions

1. What diseases does Proteus cause?

 Chapter 23
The genus Yersinia belongs to the Enterobacteria family and includes several species:
Yersinia pestis - the causative agent of plague; Yersinia pseudotuberculosis - the
causative agent of pseudotuberculosis, Yersinia enterocolitica, etc.
Yersinia enterocolitica is widely distributed in nature. Usually they live in the body of
rodents, often found in domestic animals.
Morphology . Small Gram-negative rods with rounded ends. The average size is 0.8-
1.2 × 0.3-0.7 µm, but in older cultures it can be longer and look like
threads. Mobile. Dispute does not form.
Cultivation . facultative anaerobes. They grow well on simple nutrient media. The
most favorable temperature for growth is 22-28 ° C.
On MPA they form small shiny colorless colonies (dewdrops), which increase with
the lengthening of the growing time (at 22-25 ° C). When cultivated at 37°C, the
colonies are opaque, have an uneven scalloped edge and a convex center.
They can grow at a high content of sodium chloride in the medium (up to 4%).
enzymatic properties . They break down glucose without the formation of gas, do
not ferment sucrose. Hydrogen sulfide is not formed, the formation of indole is not
constant.
Toxin formation . Contains endotoxin. Some strains produce an exotoxin.
Antigenic structure . Y. enterocolitica have O-, K- and H-antigens. The O-somatic
antigen is a lipopolysaccharide. The causative agents of human diseases most often
belong to the O9, O3, O5 serovars.
Resistance to environmental factors . Not resistant to high temperatures. At 100 ° C,
they die instantly, when heated to 60-80 ° C - after 15-20 minutes. They are well
preserved at low temperatures (-15 -20 ° C), at 4-14 ° C they not only persist, but also
multiply. Direct sunlight kills them within 30 minutes, scattered - after 6-8 hours.
They die quickly when dried. In food products, they can persist for a long time and
even multiply.
Solutions of disinfectants (mercuric chloride, chloramine, phenol) kill Yersinia in a
few minutes.
Susceptibility of animals . Laboratory animals are practically insensitive to Y.
enterocolitica. Under natural conditions, they can cause severe diseases in rodents,
pigs, cats, dogs, often fatal.
Sources of infection . More often sick animals, less often people.
transmission paths . Food.
Pathogenesis . Getting through the mouth into the gastrointestinal tract, Yersinia
multiply. Sometimes they penetrate into the epithelial cells of the intestine and
multiply inside them. Endotoxin and toxic substances produced by Yersinia cause
acute gastroenterocolitis. With the penetration of pathogens into the blood,
bacteremia and a generalized process develop, in which various organs are affected:
the liver, spleen, etc.
Prevention . Sanitary control over the storage and processing of food
products. Compliance with the sanitary and hygienic regime at catering
establishments and the rules of personal hygiene.
There is no specific prophylaxis .
Treatment . Antibiotics and symptomatic treatment.

Microbiological research

The purpose of the study: isolation and identification of Yersinia from pathological
material.
Research material
1. Bowel movements.
2. Vomit and gastric lavage.
3. Blood.
4. Urine.
5. Mucus from throat and nose, wound discharge.
6. Sectional material.
Material collection methods
 Material collection methods

Main research method

Microbiological
Research progress
First day of research

First day of research

Second day of research

View crops on Endo medium, EMS. Choose small round shiny colonies. Allocate to
the combined environment of Russell or Olkenitsky. The plates are left at 20-28°C.
The enrichment medium is inoculated onto Endo or EMS media.
Third day of research
Re-view plates with crops. Choose larger colonies (0.1-0.2 mm), round with a smooth
edge, shiny with a pinkish tint. Allocate to Russell's or Olkenitsky's medium. The
dishes with cultures from the enrichment medium are viewed, the colonies
described above are selected and isolated on Russell or Olkenitsky medium.
View crops on the environment of Russell and Olkenitsky. Make smears and stain
them according to Gram. In the presence of small gram-negative rods (sometimes
polymorphic) that do not ferment lactose, ferment glucose and urea, and do not
form hydrogen sulfide, re-seeding is done to determine mobility (at 18-20 ° C and 37
° C) and in Hiss media (mannitol, maltose, sucrose , rhamnose, gelatin, citrate).
Fourth day of research
Repeat viewing plates and combination media. The results of growth on Hiss media,
mobility agar, and gelatin are taken into account.
When isolating gram-negative rods that do not break down lactose, rhamnose, do
not form hydrogen sulfide, ferment glucose, mannitol, sucrose, mobile at 22 ° C and
immobile at 37 ° C, they give the answer: "Yersinia enterocolitica has been isolated."

Control questions

1. What are the features of Yersinia enterocolitica?

 Chapter 24
Pseudomonas aeruginosa belongs to the family Pseudomonadoceae, genus
Pseudomonas.
These bacteria are typical opportunistic pathogens. They are widely distributed in
the external environment, constantly inhabiting the body of humans and animals.
Morphology . Small Gram-negative rods. The average size is 1.5-3.0 × 0.5-0.8
µm. Motile, lophotrichous. Dispute does not form. Sometimes they form a capsule-
like extracellular mucus.
Cultivation . Strict aerobes. They grow well on simple nutrient media. The optimum
growth temperature is 37°C, but they can grow at 5-42°C. On MPA they form
colonies 2-5 mm in size, round, translucent, bluish-gray with a pearly tint; on the BCH
they give a haze and form a film.
A characteristic feature of P. aeruginosa is pigmentation and aroma formation. Most
strains form a blue-green pigment - pyocyanin, which stains the nutrient
medium. Pyocyanin is soluble in water. It has antagonistic properties against many
bacteria, but is toxic and therefore not used for therapeutic purposes. Almost all
strains of P. aeruginosa have a characteristic jasmine odor.
enzymatic properties . It ferments only one carbohydrate - glucose. Proteolytic
activity is well expressed: it liquefies gelatin and curdled whey, coagulates milk. Gives
a positive reaction to cytochrome oxidase.
Toxin formation . It forms toxins with hemolytic and cytotoxic effects and leukocidin,
which lyses human leukocytes. Has endotoxin.
Antigenic structure . P. aeruginosa has O- and H-antigens. The belonging of the
isolated culture to a certain O-serogroup is established in the agglutination test using
O-sera produced in our country.
Resistance to environmental factors . At 60°C, it dies within 15 minutes. Withstands
UV exposure. A 2% solution of phenol destroys Pseudomonas aeruginosa. It persists
for a long time in burn crusts and room dust (up to 2 weeks). Resistant to most
antibiotics.
Susceptibility of animals . Rabbits, white mice and guinea pigs are susceptible to P.
aeruginosa.
Sources of infection . Human (patient and carrier).
transmission paths . Mostly indirect contact. The air-dust path is also important.
Pathogenesis . Pseudomonas aeruginosa causes purulent-inflammatory processes,
the localization of which depends on the entrance gate of the infection: burn,
wound, respiratory tract, etc.
Diseases usually develop in persons with reduced resistance, mostly as a result of
nosocomial infection: cholecystitis, cystitis, pyelonephritis, purulent-inflammatory
complications after surgery and burns, sepsis, etc.
Immunity . Low stress.
Prevention . Compliance with the sanitary and hygienic regime in hospitals.
Treatment . Antibiotics (carbenicillin, polymyxin, gentamicin, etc.). A good effect is
the introduction of plasma from donors vaccinated against P. aeruginosa and the use
of a bacteriophage (pyophage).

Microbiological research

The purpose of the study: detection and identification of Pseudomonas aeruginosa

from pathological material and environmental objects.
Research material
1. Mucus from throat and nose, wound discharge.
2. Blood.
3. Urine.
4. Sectional material.
5. Washes from environmental objects and personnel hands.
Material collection methods
 Material collection methods

Main research method

Microbiological.
Research progress
First day of research

First day of research

Second day of research

View cups and test tubes with crops. Select dishes in which the medium is bluish-
greenish in color and has the smell of jasmine (strawberry soap). They give an
approximate answer: "The culture of P. aeruginosa has been isolated."
Colonies are isolated on lactose tubes and on agar slant tubes. Fill with vaseline oil
(create anaerobic conditions).
If there is no growth on the plates or the result is questionable, take tubes of broth
with signs of growth and plate them on nutrient agar plates. The vials are examined,
if there are signs of growth, they are plated on nutrient agar plates.
 Third day of research
Select test tubes in which lactose is not split. A smear is made from the culture in a
test tube with agar slant, Gram-stained - the presence of gram-negative rods
confirms the isolation of P. aeruginosa. Test for cytochrome oxidase. The test must
be positive.
Based on the totality of all signs: the presence of a blue-green pigment, the smell of
jasmine, gram-negative rods, the absence of lactose cleavage under anaerobic
conditions, a positive test for cytochrome oxidase give the answer: "P. aeruginosa
culture has been isolated."
If the study is not completed, continue in the same way.

Control questions

1. What are the features of Pseudomonas aeruginosa?

 The causative agents of especially dangerous infections
- F. K. Cherkes
Especially dangerous infections include cholera and a number of zoonotic diseases:
plague, tularemia, brucellosis and anthrax. Work with especially dangerous infections
is regulated by a special instruction of the USSR Ministry of Health. The work is
carried out in special laboratories.
Material sampling and laboratory tests are performed in special protective suits. The
costume consists of overalls, a dressing gown, rubber boots, canned goggles, rubber
gloves, a towel soaked in a disinfectant solution, a scarf, and a cotton-gauze mask
that covers the mouth, nose, and chin (Fig. 45).

Rice. 45. Type I anti-plague suit

Putting on and taking off the elements of the anti-plague suit is carried out in a
certain order. When undressing the suit, all removed items are immersed in a 5%
Lysol solution, and glasses are immersed in a container with 70% ethyl alcohol, after
which the clothes are autoclaved.
The utensils intended for taking the material must be preliminarily checked for
integrity and labeled before the material is added to it (the label contains all the data
about the patient, the time of sampling and the signature of the medical worker who
made the sampling). After adding the material, the dishes are closed with stoppers
and waxed.
The glass slide is inscribed before the smear is applied.
The dishes with the collected material should be wrapped with a napkin moistened
with a disinfectant solution (5% solution of Lysol or carbolic acid). Test tubes with
inoculated material are placed in metal cases, and Petri dishes - in metal cans. On
pencil cases and banks mark "top".
The collected material is sent by special transport or with a special courier.
 Chapter 25. Cholera
The species Vibrio cholerae belongs to the family Vibrionaceae, genus Vibrio.
The causative agents of cholera are represented by two biovars. The V. cholerae
biovar was isolated and studied by R. Koch (1883) and the eltor biovar was isolated
by F. Gottschlicht (1906). For a long time, the biovar El Tor was not considered the
causative agent of cholera. In 1962, by decision of the WHO, he was recognized as a
biovar of cholera vibrio.
Recently, NAG-vibrios have been isolated from water and other environmental
objects, which have not yet received a final name, but their role in acute intestinal
diseases has been established. The nature of NAG vibrios is being studied. According
to morphological, cultural and enzymatic properties, they do not differ from cholera
vibrios, they have a common H-antigen with them. They have different O
antigens. According to the O-antigen, 60 O-groups of NAG vibrios were established.
Morphology . Vibrio cholerae are small (1-3 × 0.2-0.4 microns) slightly curved rods,
comma-shaped, very polymorphic. On artificial nutrient media, especially in old
cultures, they can look like balls, grains, threads, spirals. Vibrio cholerae are very
mobile. Monotrichous - flagellum several times the length of the cell. Spores and
capsules do not form. Gram-negative. In stained smears, they are arranged in the
form of a flock of fish (see Fig. 51). Electron microscopy revealed that there are
vacuoles between the wall and the cytoplasm. It is believed that exotoxin is
synthesized in these vacuoles.
Cultivation . Vibrio cholerae are facultative anaerobes. To nutrient media
unpretentious. Alkali-loving. They reproduce at a temperature of 37-39 ° C and pH 8-
9. Grow well on MPA and MPB. The elective medium is alkaline 1% peptone
water. On the surface of this medium, they form a delicate bluish film. On solid TBRS
medium (thiosulfate citrate sucrose agar with the addition of bile salts), yellow
colonies are formed against a bluish medium. They multiply quickly: in liquid nutrient
media 6-8 hours, on dense media - 12-14 hours (on alkaline media, the growth of
cholera vibrios outstrips the growth of other bacteria). Vibrio cholerae dissociate
from S- to R-form. This process is accompanied by a change in the antigenic
structure.
enzymatic properties . Vibrio cholerae are biochemically active. They have
saccharolytic, proteolytic and diastatic properties. Saccharolytic properties are
expressed in the breakdown of sugars to form an acid. Fermentation of glucose,
sucrose, mannitol, mannose and absence of arabinose fermentation are important
diagnostic features. Proteolytic properties: Vibrio cholerae liquefy gelatin,
decompose tryptophan to form indole, produce oxidase, reduce nitrates to nitrites,
curdle milk. Hydrogen sulfide does not form. Diastatic properties are expressed in
the breakdown of soluble starch.
Vibrio cholerae produces pathogenicity enzymes: fibrinolysin, plasmacoagulase,
hyaluronidase, lecithinase, collagenase, etc.
Toxin formation . Vibrio cholerae produce three types of toxins. Type I toxin -
endotoxin, is released during the destruction of the microbial cell, thermostable
(lipopolysaccharide). It is believed that it contributes to the development of
antibacterial immunity. Type I toxin - exotoxin (cholerogen) is thermolabile, has an
enterotoxic effect and plays an important role in the pathogenesis of cholera (it
enhances the function of the secretory cells of the small intestine, which leads to
dehydration of the body). Type III toxin is thermostable, it is believed that it inhibits
the active transport of sodium through the intestinal epithelium.
Antigenic structure . Vibrio cholerae have a thermostable somatic O-antigen and a
thermolabile H-antigen. The H-antigen is not specific and is common to the entire
genus Vibrio. O-antigen has species and type specificity. According to the O-antigen,
cholera vibrios are divided into 54 groups. Vibrio cholerae and Vibrio eltor belong to
the O1 group. Within the O1 group, three components are distinguished - A, B, C,
according to the combination of which three serovars are distinguished. AB
combination - Ogawa serovar, AC combination - Inaba serovar, ABC combination -
Gikokshima serovar.
Resistance to environmental factors . At a temperature of 60 ° C, cholera vibrios die
within 5 minutes, and when boiled - instantly. They tolerate low temperatures
well. They remain in ice for several months, in sea and river water - for several
weeks, in the intestines of flies - for 4-5 days. Vibrio cholerae are very sensitive to
drying and sunlight. Common concentrations of disinfectants kill them
quickly. However, when working with the causative agent of cholera, disinfectant
solutions of a higher concentration are used. Vibrio cholerae are sensitive to acids
(hydrochloric acid, etc.). Vibrio cholera El Tor is more stable.
Susceptibility of animals . Under natural conditions, animals do not get
cholera. Under experimental conditions, intraperitoneal administration of cholera
vibrios to rabbits and guinea pigs is accompanied by severe toxicosis, which leads to
their death.
Sources of infection and routes of transmission . The only source of cholera is a
person who releases vibrio cholera into the external environment during the period
of illness or carriage. With cholera caused by vibrio El Tor, there is a long-term
carriage. Human infection occurs through food (vegetables, fruits), water and other
environmental objects.
Cholera is a long-known infection that periodically spread to many countries and
continents and caused the death of millions of people. Several pandemics of cholera
are known. In 1917-1926. There have been 6 cholera pandemics. These pandemics
were caused by classical Koch's vibrio. In the 60s of the XX century, the causative
agent of cholera, Vibrio El Tor, began to spread. In the 1970s, cases of cholera caused
by vibrio El Tor were registered in some cities of the Soviet Union.
Pathogenesis . Infection occurs through the mouth. Once in the stomach, some of
the cholera bacteria die in the acidic environment of the stomach, and some
penetrate into the intestines, where the alkaline environment and the abundance of
protein breakdown products (in particular, peptone) contribute to their
reproduction.
On the mucous membrane of the small intestine, a large number of cholera vibrios
and the toxin formed during their destruction accumulate. The toxin disrupts the
function of the mucosa, it becomes hyperemic, the permeability of the intestinal
epithelium increases, and its secretory and absorption functions are disturbed. There
are profuse diarrhea, repeated vomiting, which remove a large amount of water and
salts (potassium and sodium) from the body. The loss of a large amount of fluid and
salts leads to drying of the tissue, thickening of the blood, impaired mineral
metabolism, damage to the central and autonomic nervous systems, and other
phenomena of intoxication. The form of cholera, which proceeds in the form of
cholera enteritis, gastroenteritis, algid and dry forms, depends on the degree of
intoxication (see the textbook of infectious diseases).
Immunity . Persistent, has an antimicrobial and antitoxic nature associated with the
presence of agglutinins, vibriolysins, antitoxins and other antibodies. In addition,
local defense factors are of great importance in immunity.
Prevention . Carrying out general anti-epidemic measures: early detection of
patients, isolation and hospitalization, disinfection, observation; protection of water
sources, supervision of food products, protection of borders during epidemic
outbreaks, etc. For specific prophylaxis, a killed cholera vaccine is used (cholerogen-
toxoid in combination with the O-antigen of vibrio cholerae).
Treatment . Antibiotics of the tetracycline series, as well as the introduction of fluids
and electrolytes (potassium and sodium salts).

Control questions

1. What is the morphological characteristic of Vibrio cholerae? What biovars of

Vibrio cholerae do you know?
2. What is the elective and accumulation environment?
3. What are the growing conditions and cultural properties of cholera vibrios?
4. What are the enzymatic properties of cholera vibrios?
5. What toxins form cholera vibrios?

Microbiological research

Vibrio cholerae are very sensitive to disinfectants, so the container where the test
material is placed should not contain even traces of disinfectants. The time from
taking the material to inoculation should not exceed 3 hours. It is better to take the
material immediately in 1% peptone water, which is the accumulation medium
(other accumulation media can be used - alkaline canned liquid with sea salt, etc.).
The purpose of the study: the identification of cholera vibrio and the determination
of its serovar.
Research material
1. Bowel movements.
2. Vomit.
3. Sectional material.
In addition, be sure to examine water, food and washouts from environmental
objects.
 Material collection methods

Material collection methods

In mass surveys according to epidemiological indications, group crops can be done to

identify carriage. Material from 4-5 subjects is inoculated into one flask with 100 ml
of 1% peptone water (the study is carried out as one analysis). If the answer is
positive, the material is taken again and sown individually.
When sending the test material, the jars with the material are tightly placed in a
metal container (bottle bottle, saucepan), an accompanying document is attached
listing the materials to be sent, indicating the expected diagnosis, time and place of
taking the material, and the name of the person who collected the material.
Basic research methods
1. Bacteriological.
2. Microscopic.
3. Serological.
The study is carried out in stages. The intervals between stages should be as short as
possible. Transfers to liquid media are carried out after 6-8 hours, and on dense ones
- after 12-18 hours (a phased study requires round-the-clock work).
Research progress
Stage I

Stage I

Stage II
After 6-8 hours, the inoculations in 1% peptone water are removed from the
thermostat. A film or material from the surface of the medium is inoculated into the
second peptone water and a smear is made, fixed, stained with carbol fuchsin and
Gram. Microscopic. If mobile bacteria similar to cholera vibrio are found in smears,
then an approximate agglutination reaction with serum is put. To do this, one drop
of O-serum, diluted 100 times, is applied to a fat-free glass slide. The control is a
drop of isotonic sodium chloride solution. Both drops are filled with material from
the film and emulsified thoroughly. In the presence of agglutination in a drop of
serum and its absence in the control, the film is seeded on alkaline agar and
incubated in a thermostat.
Stage III
After 12-14 hours, the crops are removed from the thermostat. Study the growth of
crops on a dense nutrient medium. In the presence of suspicious colonies, at least
five are selected and an agglutination test is performed with O-serum diluted 1:100;
in the presence of a positive agglutination test, an approximate agglutination test
can be performed with typical Ogawa and Inaba sera (diluted 1:50). The presence of
positive agglutination with typical morphology and cultural properties gives the right
to give a preliminary positive answer.
In addition, material from typical colonies (which gave a positive agglutination
reaction) is sown on a polycarbohydrate medium (contains 2-3 sugars) to isolate a
pure culture, as well as in a broth.
The broth is incubated for 3-4 hours at 37°C. An extensive agglutination reaction is
set up with a young broth culture.
Stage IV
After 12-14 hours, the crops are removed from the thermostat. On a
polycarbohydrate medium, the color of the column changes (sucrose
breakdown). The beveled part does not change. The resulting culture is identified.
Culture Identification
Culture is identified by the following tests.
1. Study of morphological properties, mobility of a hanging and crushed drop).
2. Study of cultural properties.
3. The study of antigenic properties (in the agglutination reaction).
4. Study of saccharolytic properties.
5. Determination of proteolytic properties.
6. Determination of hemolytic properties.
7. Determination of urease activity.
8. Study of restorative properties (cholera-mouth reaction).
9. Study of diastatic activity.
10. Setting up the Voges-Proskauer reaction.
11. Test for oxidase.
12. Determination of sensitivity to phages.
13. Determination of sensitivity to polymyxin.
The study of morphological properties, mobility and cultural properties is carried
out from the first stages of the study.
Determination of antigenic properties . An extended agglutination reaction is
performed with species-specific cholera O-serum and type-specific Ogawa and Inaba
sera.
The reaction is put in a volume of 1 ml. Serums are diluted from 1:50 to titer. 2 drops
of isolated culture are added to each dilution, the reaction is followed by a serum
control and a culture control. Take into account the reaction after 18-20 hours. A
positive reaction should be at least half the serum titer.
Carbohydrate fermentation is determined on Hiss media. The results are taken into
account after 6-14 hours. The breakdown of glucose, sucrose, mannitol, maltose and
manose in the absence of arabinose fermentation is a diagnostic indicator.
Proteolytic properties are determined by seeding the isolated culture in a column of
gelatin; incubated at 22°C for 2-3 days. A positive reaction is expressed in a funnel-
shaped liquefaction of gelatin.
Hemolytic properties are determined by adding to 1 ml of a 24-hour broth culture 1
ml of a 1% suspension of sheep erythrocytes. The control is 1 ml of broth + 1 ml of
1% erythrocyte suspension. Both test tubes are incubated for 2 hours at 37°C. Then
they are transferred to the cold.
Accounting is made after 16 hours. If the result is positive in the test tube (V. eltor),
erythrocytes are hemolyzed (lacquer blood), in the control - an unchanged
suspension of erythrocytes. V. cholerae does not cause hemolysis.
Urease activity is determined by inoculation of the isolated culture on a medium
with urea. A positive result is characterized by a change in the yellow color of the
medium to red.
Study of restorative properties (cholera-mouth reaction). Vibrios cholerae reduce
nitrates to nitrites. When concentrated sulfuric acid is added to a 24-hour culture at
the rate of 2-3 drops per 1 ml of culture, nitrous acid is released, which, binding to
indole, forms a new compound (nitrosoindole). The medium turns ruby red.
Diastatic activity is determined on Kodama medium containing soluble starch. The
indicator is Lugol's solution. Vibrio cholerae break down starch, so the color of the
medium after the addition of Lugol's solution does not change.
Voges-Proskauer reaction . The culture under study is inoculated on Clark's
medium. Sowing is placed in a thermostat. After 2-3 days of incubation, the crops are
removed from the thermostat and 0.6 ml of α-naphthol and 0.2 ml of 40% sodium
hydroxide solution are added to 1 ml of the grown culture. A positive reaction is
characterized by the appearance of a red color (due to acetylmethylcarbinol).
Oxidase test . A drop of 1% aqueous solution of paraamino-dimethylaniline
(hydrochloride or oxalate) is applied to the surface of an 18-hour agar culture, a drop
of 1% alcohol solution of α-naphthol is added. With a positive reaction to oxidase,
after 2-3 minutes, the culture turns bright blue.
Determination of sensitivity to phages . The studied culture is inoculated on alkaline
agar and bacteriophage C Mukherjee type IV and bacteriophage El Tor are
applied. The reaction is taken into account after 14-16 hours (see Chapter 8; Table
40).

Table 40. Susceptibility to cholera phages

Note. + lyses; - does not lyse.

Determination of sensitivity to polymyxin . Polymyxin is added to the melted and
cooled agar (at the rate of 50 units in 1 ml of the medium). The medium is stirred
and poured into Petri dishes. The isolated culture is seeded with a loop on the frozen
medium and incubated in a thermostat. After 8-10 hours, the crops are removed
from the thermostat. Vibrios cholerae do not grow in the presence of
polymyxin. Vibrios El Tor grow (tab. 41).
 Table 41. Differential properties of V. cholerae, V. eltor and non-cholera vibrios

Accelerated Research Methods

immobilization reaction . 2 drops of feces or material from the surface of peptone
water are applied to a glass slide. One drop of O-serum (1:100) is added to the 1st
drop, and a drop of isotonic sodium chloride solution is added to the 2nd. Each drop
is emulsified with a Pasteur pipette or loop, covered with a coverslip and viewed
under a microscope. With a positive result, the movement of vibrios stops in the first
drop (the action of a specific serum), and movement is observed in the second.
Luminescent-serological method . See chapter 2.
Control questions
1. What are the main methods for identifying Vibrio cholerae?
2. The results of what studies give the right to give a preliminary answer?
3. What methods are used to differentiate the classical cholera vibrio from the vibrio
El Tor?
4. What is the basis of the accelerated method of immobilization of cholera vibrios?
Nutrient media
1% peptone water . 10 g of peptone, 5 g of sodium chloride, 0.1 g of potassium
nitrate and sodium carbonate are taken per 1 liter of distilled water until the pH
reaches 9.0. The resulting medium is poured into vials and test tubes. Sterilize in an
autoclave for 20 minutes at 120°C.
Alkaline agar . To 1 liter of MP A add 30 ml of 10% sodium carbonate solution, boil
for 45 minutes, set the pH to 8.0-9.0. Poured into vials and test tubes. Sterilize in an
autoclave for 20 minutes at 120°C.
Wednesday Clark . 5 g of dibasic potassium phosphate, 5 g of peptone, 5 g of glucose
are dissolved in 1 liter of distilled water. The medium is brought to a boil, filtered,
poured into test tubes. Sterilize at 0.5 atm for 30 minutes.
Wednesday TSV . Dry medium (pH 8.6) is produced by Difco (USA).
 Chapter 26
Plague bacteria were discovered by Yersen in Hong Kong in 1894, and the entire
genus was named Yersinia after him. A great contribution to the study of plague was
made by Russian scientists D.K. Zabolotny, N.K. Klodnitsky, I.A. Lebedinsky, N.F.
Gamaleya and Indian scientists who proposed streptomycin for the treatment of
plague.
The genus Yersinia includes three types of bacteria:
1. Yersiniae pestis - plague pathogens.
2. Yersiniae pseudotuberculosis - causative agents of pseudotuberculosis.
3. Yersiniae enterocolitica - causative agents of intestinal infections.
All representatives of this genus are gram-negative rods, often having an ovoid shape
and a size of 0.4-0.7 × 1-2 microns. Dispute does not form. The causative agents of
pseudotuberculosis and Yersinia enterocolitica have flagella. All Yersinia are
unpretentious to nutrient media. Enzymatically they are active: they break down a
number of carbohydrates with the formation of acid.
Morphology . The causative agent of the plague is a bvoid bacillus, the average size is
0.3-0.6 × 1-2 microns. They are very polymorphic. In smears from a dense nutrient
medium, the rods are elongated, filamentous, filtering forms are also
described. Plague bacteria do not have spores, flagella, and form a delicate
capsule. Gram-negative. Due to the uneven distribution of the cytoplasm, the ends of
the rods stain more intensely. Such bipolarity is clearly visible when stained with
methylene blue (Fig. 46).

Rice. 46. Morphological and cultural properties of the plague pathogen (Jersinia

pestis). a - plague bacteria (stained with Leffler's blue); b - growth per MPA: 1 - after
 24 hours in the form of broken glass; 2 - after 48 hours in the form of a lace
handkerchief; c - growth on the BCH - 'stalactite'

Cultivation . The causative agents of plague are facultative anaerobes. Not

whimsical, grow on ordinary nutrient media at a temperature of 28-30 ° C, pH 7.0-
7.2. Growth appears after 12-14 hours. Stimulants are used to accelerate growth
(extracts of some bacteria, such as sarcin, freshly hemolyzed blood, sodium sulfite,
etc.). Casein media and hydrolysates of blood clots are selective media for the
cultivation of plague pathogens. Growing colonies after 18-24 hours of incubation
look like small clumps with uneven edges, after 48 hours the edges of the colonies
become scalloped and resemble a "lace handkerchief" (see Fig. 46).
On agar slant, the culture grows as a viscous coating; on airbag - in the form of loose
flakes suspended in a clear liquid. With longer growth, loose threads descend from
the surface of the medium: "stalactite growth". Plague bacteria grow in an R-form,
which is virulent. However, they easily dissociate under the influence of a number of
factors, for example, a bacteriophage, and pass through the O-form to the S-
avirulent form.
enzymatic properties . Plague bacteria have a pronounced saccharolytic activity -
they break down sucrose, maltose, arabinose, rhamnose, glucose (not always) and
mannitol with the formation of acid. There are two variants of plague bacteria -
decomposing and not decomposing glycerol. Proteolytic properties are weakly
expressed: they do not liquefy gelatin, do not coagulate milk, and form hydrogen
sulfide.
Plague bacteria produce fibrinolysin, hemolysin, hyaluronidase, coagulase.
Toxin formation . Plague bacillus toxin is a special protein that combines the
properties of exo- and endotoxin, it consists of two protein fractions (A and B), which
differ in amino acid composition and antigenic properties. It is highly toxic to
humans. Plague toxin is called mouse poison, as mice are highly sensitive to its
action.
The antigenic structure of plague bacteria is complex. Plague microbes contain
about ten different antigens: fractions F, V, W, etc. Fraction F is the main component
associated with the capsule; V and W components prevent cell phagocytosis. Plague
bacteria have common antigens with the causative agent of pseudotuberculosis,
Escherichia, Shigella and O-group human erythrocytes.
Resistance to environmental factors . High temperatures (100°C) destroy plague
bacteria instantly, 80°C - after 5 minutes. Plague bacteria tolerate low temperatures
well: at 0 ° C they persist for 6 months, in frozen corpses - a year or more. Direct
sunlight kills them after 2-3 hours. Plague bacteria are very sensitive to
desiccation. In foodstuffs they remain from 2 to 6 months. In fleas - up to a year.
Normal concentrations of disinfectant solutions kill them in 5-10 minutes. They are
especially sensitive to sublimate and carbolic acid.
Susceptibility of animals . The main carriers of the plague are rodents: marmots,
ground squirrels, tarabagans; they determine the natural focality of the plague. Gray
and black rats and mice are very sensitive to the plague; camels, foxes, cats are also
susceptible. Mice, rats, guinea pigs, etc. are susceptible to experimental infection.
Sources of infection . Sick animals, mostly rodents. Epidemics in humans often
precede epizootics in rodents.
Transmission routes and vectors . 1. The main transmission route is
transmissive. Carriers - fleas (rodents → fleas → humans).
2. Airborne route (infection of a person from a person with pneumonic plague).
3. Food - when eating poorly cooked infected meat (this path is rare).
Pathogenesis and forms of the disease . The entrance gates are the skin and mucous
membranes of the respiratory tract and the digestive tract. Plague pathogens are
highly invasive. At the site of penetration of the pathogen, papules are formed,
turning into a pustule with bloody-purulent contents. Regional lymph nodes are
involved in the pathological process, through which microbes enter the bloodstream,
causing bacterhymia. With blood, they enter the internal organs.
Depending on the place of localization, a person may experience different forms of
the disease: skin, skin-bubonic, intestinal, pulmonary, primary septic; each form can
end in sepsis (secondary septicemia). The most common form is the bubonic
form. Bubo is painful. When a large dose of the pathogen and a small resistance of
the organism enter, a primary septic form may occur. The disease begins acutely and
proceeds with symptoms of intoxication - high fever, headache, etc.
Immunity . Intense and prolonged (in past centuries, during the period of large
epidemics, those who had been ill were used to care for the sick). Immunity is
determined by the macrophage system. The phagocytic factor is of great importance.
Prevention . General measures are early diagnosis, isolation of patients. Quarantine
for people who have been in contact with sick people. Carrying out in the centers of
disinsection and deratization. The protection of medical personnel located in the
outbreaks is carried out by the introduction of streptomycin and the anti-plague
vaccine. Implementation of international conventions for the prevention of plague
(deratization and disinfection of ships in ports). Protection of state borders.
specific prophylaxis . In the USSR, a live EV vaccine is used. This strain was obtained
from a virulent culture by successive subcultures of the pathogen on nutrient media
for 5 years. The strain lost its virulence, while retaining its immunogenic
properties. The immunity lasts for about a year. Vaccinate only people who are at
risk of infection.
Treatment . Streptomycin, tetracycline, specific phage and anti-plague
immunoglobulin.
A great contribution to the study of the prevention and treatment of plague was
made by Soviet scientists M. P. Pokrovskaya and N. N. Zhukov-Verezhnikov.

Control questions

1. What group of infections does plague belong to?

2. How does the plague pathogen grow on solid and liquid nutrient media? Which
form is virulent - R or S?
3. What toxin forms the causative agent of plague and what pathogenicity enzymes
do you know?
4. Who is the source and carrier of the plague?
5. What forms of disease does the plague bacillus cause?

Microbiological research

The purpose of the study: to identify the causative agent of plague.

Research material
1. Detachable ulcers or punctate from the carbuncle - skin form.
2. The content of the bubo is the bubonic form.
3. Sputum - pulmonary form.
4. Defecation - intestinal form.
5. Blood - in all forms.
6. At the autopsy, they take pieces of the organs of the corpse, blood, bone marrow.
7. Fleas - intestinal contents.
8. Rats, mice and other dead rodents (and sick) - open, examine organs and blood.
Material collection methods

Material collection methods

Basic research methods

1. Microscopic.
2. Bacteriological.
3. Biological.
4. Luminescent-serological method (see chapter 2).
Methods of serodiagnosis are not widely used.
Research progress
First day of research
 First day of research

Sowing . The material uncontaminated by extraneous flora is sown on dense and

liquid nutrient media (MPA and MPB) with the addition of stimulants to them: blood,
sodium sulfite, etc. Growth stimulation is necessary, since the inoculum dose may be
insufficient. Material containing extraneous flora (sputum, contents of open ulcers) is
inoculated on Tumansky's medium or Korobkova's medium. These media contain
gentian violet (1:50,000) to inhibit the growth of extraneous flora. The cultures are
incubated in a thermostat at 28°C.
biological test . The bioassay is carried out on guinea pigs and white mice. The
method of introducing the test material depends on the nature of the
material. Sputum, pus from an open abscess is injected by rubbing into the skin of
the abdominal wall (the skin is first epilated, treated with a sterile isotonic sodium
chloride solution and scarified). The test material is applied to the scarified area by
rubbing it with the flat part of the scalpel, under the cover of a special funnel or a
glass lid from a Petri dish. Uncontaminated material (blood, contents of a closed
bubo) is administered to animals subcutaneously or intraperitoneally. Depending on
the method of administration, the animal dies on the 3rd-9th day.
Second day of research
Crops are removed from the thermostat. Growth is studied on a dense and liquid
nutrient medium.
From the broth culture at typical growth, smears are made, stained by Gram and
methylene blue. Microscopic. From a dense nutrient medium in the presence of
typical colonies, a pure culture is isolated and placed in a thermostat. Plague
bacteriophage is applied to 2-3 colonies suspicious of the plague
pathogen. Incubated in a thermostat. After 10-12 hours, the colonies change - they
lyse. The lysis of colonies under the action of a plague bacteriophage is of diagnostic
value.
Third day of research
The tubes with the culture on the slant agar are removed from the incubator. On the
surface of the agar, the plague bacillus forms a viscous grayish-white coating. The
isolated culture is examined microscopically. In the presence of typical rods, the
saccharolytic properties are checked by inoculation for sugars: glucose, maltose,
sucrose, rhamnose, mannitol. Put a sample with a bacteriophage.
Fourth day of research
The results are recorded: 1. Enzymatic properties (Table 42).

Table 42. Enzymatic properties of plague pathogens

Note. to - acid; - lack of splitting; ± does not always split; + splitting.

Sample with bacteriophage - lysis of colonies.
Accelerated test method with bacteriophage . The test material is applied to 3 cups
with Tumansky's medium.
1st cup - inoculated with plague bacteriophage.
2nd cup - inoculate with a uniform distribution of the material over the surface of
the medium (with a spatula), after which a path is made from the plague
bacteriophage.
3rd cup (control) - inoculate only with the test material. Crops are incubated at 28°C.
After 12-14 hours, the cups are removed from the thermostat.
In the presence of a plague pathogen in the test material, the following is noted:
in the 1st cup - negative colonies (lysis of plague colonies), in the 2nd cup - a sterile
path, in the 3rd cup - typical colonies of plague bacteria.
Plague bacteria are differentiated from pseudotuberculosis bacteria (Table 43).

Table 43. Differentiation of plague pathogens and pseudotuberculosis bacteria

Continue monitoring animals infected on the first day of the study. Dead or dead

animals are opened. Study changes in organs. Usually, in animals that died from the
plague, regional nodes are enlarged, in the organs - hemorrhagic and necrotic
areas. The liver and spleen are enlarged. At autopsy, smears-imprints are made from
organs and blood on special media. Further research is carried out in the manner
described above.

Control questions

1. What mode of operation must be observed when working with plague pathogens?
2. What methods are leading? When should gentian violet be added to the medium?
3. What animals are subjected to a bioassay? What changes are found in dead
animals?
4. How are plague pathogens differentiated from pseudotuberculosis bacteria?
 Chapter 27
The causative agents of pseudotuberculosis are similar in morphological, cultural and
enzymatic properties to the plague pathogens. However, there are
differences. Pseudotuberculosis bacteria are motile, peritrichous. Biochemically
more active (see table. 43).
antigenic properties . Pseudotuberculosis bacteria have a flagellar H-antigen and two
somatic antigens: smooth - typical and rough - group, common with the antigen of
the plague pathogen.
Animals are mainly sensitive to the causative agent of pseudotuberculosis: rodents,
domestic and predatory. A person becomes infected through food.
Pathogenesis . The causative agents of pseudotuberculosis penetrate the
gastrointestinal tract, affect the lymphoid tissue, multiply and penetrate the blood
through the lymphatic vessels, causing bacteremia and toxicosis.

Microbiological research

Microbiological research
 Chapter 28
The causative agent of tularemia was first isolated in 1911 by McCoy and Chapin
while studying the disease of ground squirrels in the USA (California, Tulare
County). In 1921, the American researcher E. Francis found out that this disease is
also characteristic of people and described it. Therefore, the pathogen was named
Francisella tularensis.
Morphology . The causative agents of tularemia are small coccobacteria. Their
average value is 0.3-0.6 × 0.1-0.2 µm. They are very polymorphic: spherical, filiform
and other forms are found in smears. There are cultures that pass through bacterial
filters. Tularemia bacteria are immobile and do not form spores. Possess a delicate
capsule, gram-negative. In smears-imprints made from organs and stained according
to Romanovsky, the bacteria have a soft purple color.
Cultivation . The causative agents of tularemia are facultative anaerobes. They grow
on media rich in nutrients: folded yolk media, agar meat or fish media with the
addition of cystine, glucose and blood. They reproduce better on dense nutrient
media, but growth can also be on liquid and semi-liquid media. On dense nutrient
media, tularemia bacteria grow slowly, 4-14 days at a temperature of 36-37 ° C and a
pH of 6.8-7.2. They form small, whitish, convex, shiny colonies with smooth edges, 1-
3 mm in diameter. Virulent strains in S-form. Vaccine strains in SR form. The R-form
of bacteria is avirulent (with prolonged cultivation in the laboratory, they turn into
the R-form).
enzymatic properties . In tularemia bacteria, enzymatic properties are poorly
expressed and are detected only on special media. They can ferment glucose,
maltose, mannose, levulose with the formation of acid without gas. Some strains
break down glycerol, sometimes produce hydrogen sulfide.
Toxin formation . No exotoxin was found in tularemia bacteria. Pathogenic action of
microbes is connected, apparently, with endotoxin.
Antigenic structure . The S-form of tularemia bacteria contains two antigenic
complexes: O- and Vi-antigens. The Vi antigen is associated with virulence and
immunogenicity. R-forms lose the Vi antigen. The O antigen shares a common
antigen with Brucella bacteria.
Resistance to environmental factors . At a temperature of 100 ° C, tularemia
bacteria die instantly, at a temperature of 60 ° C, they persist for 20 minutes. At low
temperatures and in moist soil, pathogens persist for up to 4-5 months. At 1 ° C in
water, they persist for up to 9 months, grain and straw at 0 ° C - up to 150 days,
bread - up to 14 days, meat - up to 30 days, etc. Ordinary solutions of disinfectants
kill them within 10 - 15 minutes. Tularemia bacteria are sensitive to many antibiotics.
Susceptibility of animals . The causative agents of tularemia are pathogenic for many
animal species. Natural infection with tularemia is known in 145 species of
vertebrates and more than 100 species of invertebrates. Most susceptible to
tularemia are many species of rodents and some insectivores.
Of the experimental animals, guinea pigs and white mice are sensitive.
The sources of infection are rodents, mainly water rats, voles, house mice, muskrats,
hamsters and hares. The source of infection can be water, food, straw and other
substrates contaminated with secretions from sick animals.
transmission paths . Transmissive, air-dust, food, contact household.
Pathogenesis . Tularemia bacteria have a high invasive capacity. They penetrate
through damaged and intact skin and mucous membranes.
Depending on the route of entry into the body, pathogens can be localized in the
skin, mucous membranes of the intestinal tract, respiratory tract, eyes and other
organs. From the entrance gate along the lymphatic pathways, they enter the
nearest lymph nodes, where they multiply and enter the bloodstream. In the foci of
accumulation of pathogens of tularemia, specific tularemia granulomas are formed -
primary buboes. With further spread of microbes, secondary buboes may
occur. Buboes range in size from a hazelnut to a chicken egg.
There are the following clinical forms of the disease: bubonic, anginal-bubonic,
oculobubonic, pulmonary, abdominal and generalized. According to the severity of
the course - light and severe forms. According to the duration of the course - acute
and protracted forms.
Immunity . Tense and lengthy. Determined by humoral and cellular
factors. Characteristic of tularemia is an allergic condition that occurs from the first
days of the disease.
Prevention . Fight against rodents and insects. Public health activities.
specific prophylaxis . Immunize people living in the area of natural
foci. Immunization is carried out with a live Gaisky-Elbert vaccine. Vaccinated once,
skin. The duration of immunity is 3-6 years.
Treatment . Tularemia bacteria are sensitive to many antibiotics: streptomycin,
biomycin, tetracycline, monomycin, kanamycin. They are not sensitive to penicillin
and sulfamides.

Control questions

1. What are the morphological and cultural features of tularemia pathogens?

2. Antigenic structure. Which antigen is associated with virulence and which S or R
form is more virulent?
3. What is the resistance of tularemia bacteria in the external environment?
4. What animals are susceptible to tularemia? main sources of infection.
5. Entrance gate of infection. Pathogenesis. The main forms of the disease.
6. Specific prevention.

Microbiological research

The purpose of the study: to identify the causative agent of tularemia.

The collection of material and the study is carried out in strictly regime conditions !
Research material
1. The contents of the bubo (bubonic, ulcerative-bubonic and anginal-bubonic
forms).
2. Discharge of the mucous membrane of the eye (oculobubonic form).
3. Phlegm (pulmonary form).
4. Defecation (abdominal form).
5. Blood (generalized form).
In special laboratories, rodents and their secretions, arthropods (ticks, fleas,
mosquitoes, horseflies), water, food products, etc. are examined.
 Material collection methods

Material collection methods

Basic research methods

1. Allergic.
2. Serological.
3. Biological.
4. Luminescent-microscopic.
5. Bacteriological (did not find wide application, since direct inoculation of the test
material on artificial nutrient media does not give bacteria growth).
Research progress
 Research progress

Control questions
1. List what material for research is used in different clinical forms of tularemia.
2. List the main research methods.
3. What serological methods are used to diagnose tularemia?
4. What animals are subjected to a biological test?
5. Why is the bacteriological method not widely used?
Exercise
Make a table of morphological, cultural and enzymatic properties of tularemia
bacteria.
Nutrient media
Yolk environment. The yolks of fresh eggs are mixed with an isotonic sodium chloride
solution in a ratio of 3:2. The mixture is poured into test tubes of 4-5 ml and
sterilized in an oblique position at a temperature of 80 ° C for an hour. The medium
is checked for sterility (in a thermostat at 37 ° C) and stored in the cold for up to 1
month.
 Chapter 29
In 1886, D. Bruce found a small coccobacterium in the spleen of a patient who died
from Maltese fever, which he isolated in pure culture and named Micrococcus.
In 1896, B. Bang also isolated coccobacteria from the amniotic fluid during the
abortion of cows. In 1914, J. Traum isolated a similar rod from sick pigs. And in 1916,
Ivens, having studied all the isolated microorganisms, determined their similarity,
and in honor of Bruce they were named brucella. Later (1953, 1957, 1966) other
types of Brucella were discovered. All of them are united in the genus Brucella.
Currently, brucellas are divided into species according to their main host: B.
melitensis - small cattle (sheep, goats) are sick; B. abortus - sick cattle; B. suis - pigs
get sick, etc.
Each type of Brucella is divided into biovars: B. melitensis includes 3 biovars; B.
abortus - 9 biovars; B. suis - 5 biovars. The most pathogenic for humans is B.
melitensis. B. abortus rarely cause clinical disease in humans.
Morphology . The causative agents of brucellosis are small 0.6-0.8 × 0.3-0.5 microns
bacteria of rod-shaped or ovoid shape. Motionless. They don't have a dispute. Form
a delicate capsule. Gram-negative. In the smear are arranged randomly.
Cultivation . Brucella are aerobes. Tolerant of nutrient media. Characterized by slow
growth (2-3 weeks). They are grown on special nutrient media: whey-dextrose agar,
potato infusion agar with serum and blood agar (5% sheep blood), medium "D",
hepatic agar MPA and MPB. They grow at a temperature of 37 ° C and a pH of 6.8-
7.2. Some strains require 5-10% CO 2 for growth, especially at the initial selection. On
dense nutrient media, delicate, small, colorless, convex S-shaped colonies with a
pearly sheen grow. Under the influence of some factors, they can dissociate into the
R-form. Under the action of antibiotics, they develop L-forms. In liquid nutrient
media, brucella gives a uniform turbidity. Brucella can be cultured in the yolk sac of a
chick embryo.
Brucella species are differentiated based on their ability to form hydrogen sulfide
and grow on media with dyes - basic fuchsin and thionine (Table 44).
 Table 44. Biological properties of brucella

enzymatic properties . Brucella break down D-ribose, D-galactose, alanine,

asparagine. Some strains hydrolyze amino acids to form ammonia.
Brucella form hyaluronidase, catalase, peroxidase, lipase, phosphatase, etc. Brucella
have pronounced invasive and aggressive properties.
Toxin formation . The pathogenic effect of Brucella is determined, apparently, by the
presence of endotoxin. In addition, they have allergenic properties.
Antigenic structure . Brucella contain two somatic antigens A and M. These antigens
are species-specific. They are always part of the microbial cell, but in different
proportions. In B. melitensis, antigen M predominates, in B. abortus and B. suis,
antigen A predominates. In addition, they have a heat-labile Vi antigen.
Resistance to environmental factors . At 100°C, brucella die instantly. At a
temperature of 80-85 ° C - after 5 minutes, at 60 ° C - after 30 minutes. They are very
resistant to low temperatures. Direct sunlight is detrimental to them. In a humid
environment, brucella persist for a long time - 3-4 months. In dairy products - up to
40-45 days, frozen meat - up to 5 months, soil and water - up to 3-5 months.
Susceptibility of animals . Brucellosis mainly affects farm animals: small and large
cattle, pigs, deer, etc. Each type of Brucella affects a certain type of animal. But
brucella can migrate, that is, move from one animal species to another. For example,
B. abortus can infect small cattle.
The main signs of the disease are: in females - abortions, in males - orchitis. In
addition, they have joint damage, weight loss, hair loss, etc. But brucellosis in
animals can occur in a latent form, which contributes to the spread of infection.
Of the experimental animals, white mice and guinea pigs are sensitive to
Brucella. After infection, they abort, lose weight dramatically, their hair falls
out. Mice occasionally develop septicemia.
Sources of infection . The main source of human brucellosis is small and large
cattle. The role of a person in the transmission of brucellosis infection has no
epidemiological significance.
transmission paths . Food, contact-household, airborne.
Contact way - when working with animals: in the process of caring for animals, at
enterprises processing raw materials and products of animal origin; in contact with
the secretions of sick animals, the fetus, in the process of slaughter, carcass cutting,
etc.
By aerogenic way, brucella penetrate the skin and intact mucous membranes.
The food route is the consumption of contaminated food. Dairy products are the
most dangerous - milk, cheese, etc.
Pathogenesis . Once in the body, Brucella penetrate the lymphatic pathways into the
lymph nodes, blood, bone marrow, parenchymal organs and are localized inside the
cells. With an exacerbation of the process, brucella from the cells again enter the
bloodstream and a relapse occurs. The disease is characterized by inflammation of
the joints, neuralgia and natural abortions.
Immunity - is determined by cellular (phagocytosis) and humoral factors -
agglutinins, complement-fixing antibodies, etc. Immunity is combined with an allergy
state. In experiments on guinea pigs, it was shown that their resistance to re-
infection was combined with a positive reaction to brucellin.
Prevention . Scheduled surveys in livestock farms, pastures, slaughterhouses, meat
and dairy plants.
specific prophylaxis . Vaccination with a live vaccine B. abortus (strain 19-
BA). Vaccinations are carried out by the cutaneous method once, revaccinated after
8-12 months.
Treatment . Antibiotics: chloramphenicol, erythromycin. Brucella immunoglobulin is
also used to prevent relapse.

Control questions
1. What species of Brucella do you know and which one is pathogenic for humans?
2. On what media are brucella cultivated and what characterizes their growth on the
media?
3. What is the pathogenic effect of Brucella?

Microbiological research

The purpose of the study: to identify the causative agent of brucellosis.

Work with brucella is carried out under strictly controlled conditions .
Research material
1. Blood.
2. Cerebrospinal fluid.
3. Bone marrow.
4. Urine.
5. Breast milk.
6. Sectional material.
Material collection methods

Material collection methods

Basic research methods

1. Serological
2. Allergic.
3. Biological.
4. Bacteriological.
Research progress
Research progress

Research progress
 Control questions
1. Under what conditions are they working with the material obtained from a patient
with brucellosis?
2. What material is used for diagnostic testing?
3. List the main research methods.
4. What animals are subjected to a biological test?
Nutrient media
BCH (see chapter 7).
MPA (see chapter 7).
Serum Dextrose Agar. The basis of this medium is nutrient agar, which is prepared as
follows: 15 g of agar-agar, 10 g of peptone, 5 g x. hours of sodium chloride and 165
ml of meat water. All ingredients are introduced into the vessel and subjected to
flowing steam for an hour. The pH is adjusted to 7.8. Then the vessel is placed in an
autoclave, at 2 atm, a temperature of 120 ° C, phosphates precipitate. The medium is
filtered through paper filters, adjusted to pH 7.4, poured into volumetric dishes and
sterilized at a temperature of 116°C for 15 minutes. The agar thus prepared is melted
as needed in a water bath and cooled to 50 ° C. Then normal inactivated (at 56 ° C for
30 minutes) horse or bovine serum and a dextrose solution sterilized by filtration
through a Seitz filter are added to it.
Blood agar . See chapter 7.
Medium "D" : a) broth "D" - 2.5 g of powder of standard broth "D" is added to 100
ml of cold distilled water, heated and thoroughly stirred until it is completely
dissolved; then the broth is filtered, poured into the necessary dishes and sterilized
at 120 ° C for 20 minutes (pH of the medium 7.1-7.2); b) agar "D" - add 20 g of
powder of standard agar "D" to 100 ml of cold distilled water, heat with stirring until
the powder is completely dissolved, preventing it from burning, then filtered, poured
into the necessary dishes and sterilized at 120 ° C in for 20 min (pH 7.2). The media
are prepared by the Institute of Epidemiology and Microbiology named after N. F.
Gamaleya of the USSR Academy of Medical Sciences.
 Chapter 30
The causative agent of anthrax Bacillus anthracis is included in the family Bacillaceae,
genus Bacillus. The name of the disease - "coal" was given by the Russian doctor
Andrievsky, who at the end of the 18th century studied this disease in Siberia during
a great epizootic among cows.
The causative agent of anthrax was discovered by Pallender in 1849. R. Koch, L.
Pasteur and L. S. Tsenkovsky made a great contribution to the study of this disease.
Morphology . The causative agents of anthrax are large sticks 6-8 × 1-1.5 microns
with chopped off or somewhat concave ends. Gram-positive. In the body, they are
arranged in pairs or in the form of short chains. Long chains are found on nutrient
media. Anthrax bacilli are non-motile. In the body they form a capsule surrounding
one, two individuals or the entire chain. Anthrax bacilli form oval-shaped spores
located in the center and not exceeding the diameter of the microbial
cell. Sporulation occurs best with access to oxygen and a temperature of 30-40 ° C.
At temperatures above 43 ° C and below 15 ° C, sporulation stops. During the period
of spore formation, the cytoplasm of the cell is almost completely lysed, the cell wall
is torn and the spore comes out (Fig. 47).

Rice. 47. Morphological and cultural properties of the anthrax pathogen (Bacillus

 anthracis). a - B. anthracis (in mouse blood); b - formation of capsules; in -
disputes; d - colony growth; e - culture growth on the MPB; e - culture growth on
gelatin

Cultivation . The causative agents of anthrax are facultative

anaerobes. Unpretentious. They grow at a temperature of 35-38 ° C and a pH of 7.2-
7.6. On MPA they form large colonies with uneven fringed edges (R-form). Bundles of
threads extend from the edge of the colony. The appearance of the colonies
resembles the head of a jellyfish or a lion's mane. The R-form is characteristic of
virulent strains of anthrax bacilli. In old cultures, smooth S-shaped colonies appear -
not virulent.
In broth, the growth of anthrax bacilli is characterized by benthic growth. At the
bottom of the tube, a precipitate is formed in the form of a lump of cotton wool,
while the medium remains transparent.
When sowing on 10-12% gelatin, after 2-3 days of incubation, growth appears along
the injection in the form of white strands, decreasing downwards (view of an
overturned Christmas tree).
When sowing pathogens on MPA with penicillin (on plate agar), bacilli disintegrate
into balls, a chain of which resembles a pearl necklace. The nature of growth on
media is of diagnostic importance (see Fig. 47).
enzymatic properties . Anthrax bacilli have a pronounced enzymatic
activity. Saccharolytic properties: break down glucose, lactose, maltose, levulose and
other sugars to form acid.
Proteolytic properties are expressed in milk peptonization, gelatin liquefaction, milk
coagulation (slowly). They form hydrogen sulfide and ammonia, convert nitrates into
nitrites, hydrolyze starch, etc. They do not hemolyze erythrocytes, which is how they
differ from Vanthracoid. Lysed by anti-anthrax phage. Anthrax bacilli form enzymes:
diastase, peroxidase, lipase.
Toxin formation . B. anthracis forms a toxin - a protein complex containing
edematous and lethal factors. This toxin is called "mouse toxin" (due to the high
sensitivity of mice). A large role in the virulence of anthrax bacilli belongs to the
capsule, which is associated with a toxic substance.
Antigenic structure . Anthrax bacilli contain two antigens: 1) somatic
(polysaccharide), which is located in the cell wall of the microbe. Heat resistant. No
antibodies are produced against this antigen. This antigen is stored for a long time in
cultures and cadaveric material. Ascoli's precipitation reaction is based on its
discovery; 2) capsular (protein) antigen, which causes antiphagocytic action.
Being in the body or on media containing tissue extracts, anthrax bacilli produce a
protective heat-labile antigen, which is atoxic, but has an immunizing ability.
Anthrax bacilli have a common antigen with anthracoid and other spore-forming
saprophytes (B subtilis, B. cereus, etc.).
Resistance to environmental factors . Vegetative forms of anthrax pathogens are
unstable. At 100°C, they die instantly; temperatures of 55-60°C destroy them in 30-
40 minutes. Ordinary concentrations of disinfectant solutions kill them in a few
minutes. Capsules of anthrax bacilli are highly resistant. When examining the corpses
of animals exposed to putrefactive microflora, empty capsules (shadows) can be
found. Spores are resistant: they can withstand boiling for 15-20
minutes. Autoclaving (120°C) kills them after 20 minutes. Not sensitive to low
temperatures. In a dry state, they remain up to 30 years, in the soil - decades.
Ordinary solutions of disinfectants destroy them in 2-3 days (Table 46).

Table 46. Properties of anthrax pathogens

Note. B. anthracoides have poor mobility, cause hemolysis of erythrocytes, and are
non-pathogenic for guinea pigs.
Susceptibility of animals . Cows, sheep, horses, deer, pigs are sensitive to anthrax
bacilli. They become infected from each other by food, absorbing spores of the
pathogen with food.
Of the laboratory animals, white mice, guinea pigs, and rabbits are the most
susceptible. These animals after infection die in 2-4 days from septicemia. Edema
and hyperemia are observed at the injection site. The blood of dead animals is thick
and dark red in color, since anthrax bacilli have an anticoagulant effect.
Sources of the disease . Sick animals.
transmission paths . Contact-household, air-dust, food (when using products
contaminated with anthrax bacilli).
Man from man usually does not become infected, however, when a person becomes
ill with anthrax, all necessary precautions are taken.
Pathogenesis . The entrance gates are the skin and mucous membranes of the
respiratory tract and the digestive tract. Depending on localization, skin, pulmonary
and intestinal forms are distinguished. Each form can be generalized.
Skin form - redness appears at the site of penetration, turning into a papule (itchy). A
copper-red papule turns into a vesicle with serous-hemorrhagic contents, after
drying, a black scab (carbon) is formed.
Pulmonary form - specific pneumonia develops, proceeding according to the type of
pulmonary edema. Usually ends in death.
Intestinal form - all of the above phenomena develop in the intestinal
mucosa. Usually ends in death.
Immunity . Quite resistant, antimicrobial and antitoxic. Depends on the formation of
protective antibodies. A large role belongs to the phagocytic reaction. In the serum
of those who have recovered from anthrax, antibodies are found that destroy the
capsular substance of bacilli.
With anthrax, hypersensitivity develops, which is recorded in an allergic test with
anthraxin.
Prevention . All measures to prevent anthrax are carried out jointly with the
veterinary service. They provide for the timely detection, isolation of sick animals,
and thorough disinfection of the territory.
specific prophylaxis . Currently, the STI vaccine is used, which was made in 1942 by
N. N. Ginsburg from a capsuleless culture. People who, by the nature of their work,
are usually associated with farm animals are usually vaccinated. For emergency
prophylaxis (people who have been in contact with patients), anthrax
immunoglobulin and antibiotics are administered.
Treatment . Anthrax immunoglobulin, antibiotics: penicillin, streptomycin,
tetracycline.

Control questions

1. Describe the morphology of the anthrax pathogen.

2. Describe the cultural properties and which form: S or R is virulent?
3. What are the properties of anthrax toxin?
4. Ways of transmission and forms of anthrax disease.
5. What factors determine immunity in anthrax.

Microbiological research

The purpose of the study: to identify the causative agents of anthrax and
differentiate it from anthracoid, to identify the pathogen's antigens.
Work with the causative agent of anthrax is carried out under strictly regime
conditions !
Research material
1. Contents of vesicles, carbuncle, sloughed off scab (skin form).
2. Phlegm (pulmonary form).
3. Excrements (intestinal form).
4. Blood (septic form).
5. Soil, animal hair (for setting the Ascoli reaction).
Material collection methods
 Material collection methods

Basic research methods

1. Microscopic.
2. Bacteriological.
3. Biological.
4. Allergic.
5. Ascoli precipitation reaction.
Research progress
First day of research
 First day of research

Second day of research

1. Crops are removed from the thermostat. Growth is studied on a dense and liquid
nutrient medium. Colonies on a dense nutrient medium are examined under a
microscope at low magnification. In the presence of suspicious colonies, a pure
culture is isolated on a slanted MPA. Crops are incubated in a thermostat.
2. From the broth culture (growth in the form of a lump of cotton wool at the
bottom, the broth is transparent) make a hanging drop (to establish immobility -
differentiation from anthracoid).
3. They put the "pearl necklace" test (accelerated research method). For this
purpose, 30% inactivated horse serum and penicillin are added to Hottinger's broth
at the rate of 0.5 IU per 1 ml of broth. The prepared medium is poured into test
tubes of 2-3 ml and 2 drops of the studied broth culture are added to each. Crops are
incubated in a thermostat for 3 hours at a temperature of 37 ° C. Then they are
removed from the thermostat. 2-3 smears are made from each tube, dried in air and
fixed in Carnoy's liquid (6 parts of ethyl alcohol + 3 parts of chloroform + 1 part of
glacial acetic acid). Fixation is carried out until complete evaporation of the
liquid. The resulting smears are stained with methylene blue and microscoped.
In smears, anthrax bacilli are found in the form of a chain of balls resembling a pearl
necklace - the result of the action of penicillin.
4. Examine infected animals. Dead animals are opened, swabs and smears-imprints
are made, which are fixed, stained and studied under a microscope. In the presence
of suspicious sticks, crops are made for MPA and MPB.
Third day of research
Take out the crops from the thermostat, make smears, microscopic. On MPA and
MPB, anthrax bacilli grow as non-encapsulated individuals. Produce crops for sugar,
litmus milk, gelatin, cups with 2% blood and put a test with anthrax
bacteriophage. Crops are incubated in a thermostat.
Fourth day of research
The crops are removed from the thermostat and the results obtained are taken into
account (see Table 46).
The study of sputum, feces and blood after special treatment is carried out in the
same way.
Allergy test
For the diagnosis of anthrax, an allergic test with anthrax antigen (anthraxin) is
used. To do this, anthraxin is injected intradermally on the inner surface of the
forearm. The reaction is taken into account after 24-48 hours. A positive reaction is
manifested from the first days of the disease.
Ascoli reaction
The reaction is set to detect the specific antigen of anthrax bacilli in animal hair, skin,
corpses, soil, etc.
Preparation of the antigen: the test material is crushed in a mortar, poured with 25-
50-fold volume of isotonic sodium chloride solution and boiled (the antigen is heat-
resistant). The extract obtained is filtered through filter paper moistened with the
same solution. The filtrate-thermoextract is a transparent liquid. For the reaction,
precipitating anthrax serum is used and anthrax antigen is used for control (Fig. 48).

Rice. 48. Ascoli reaction. 1 - precipitating serum + investigated thermal extract; 2 -

precipitating serum + standard anthrax antigen (control); 3 - precipitating serum +
antigen from the hair of a healthy animal (foreign antigen); 4 - precipitating serum +
isotonic sodium chloride solution; 5 - normal serum + tested antigen

Statement of the reaction: 1st test tube - precipitating serum + test thermal extract;
2nd tube - precipitating serum + standard anthrax antigen (control).
3rd tube - precipitating serum + thermal extract from the hair of a healthy animal
(control).
With a positive reaction, a precipitation ring is formed in the first two test tubes, and
the ring is absent in the third.
This reaction is very sensitive (see Fig. 48).
Nutrient media
MPA, MPB, gelatin medium (see Chapter 7).

Control questions

1. What material is used for bacteriological research?

2. Basic methods of microbiological research.
3. What are the accelerated methods of research? How to get the "pearl necklace"
test?
4. What method can be used to detect anthrax bacilli in the external environment?

Exercise

Draw a diagram of a laboratory study of anthrax by day.

 Bordetella - F.K. Cherkes
Chapter 31
The causative agents of these diseases belong to the genus Bordetella.
1. Bordetella pertussis - the causative agent of whooping cough, described by Bordet
and Zhangu in 1906.
2. Bordetella parapertussis - the causative agent of parapertussis, described by
Eldering and Kondrik in 1937.
3. Bordetella bronchiseptica - causes disease in animals. In humans, these bacteria
cause bronchopneumonia with whooping cough. For the first time in humans, this
disease was described by Brown in 1926 (rare).
Morphology . Whooping cough bacteria are small, ovoid-shaped rods, 0.3-0.5 × 1-1.5
µm. The causative agent of para-whooping cough is slightly larger. Both microbes do
not have spores and are immobile. With special coloring, the capsule is visible. Gram-
negative. More intensively stained at the poles.
Ultrasections show a capsule-like shell, currency grains, and vacuoles in the nuclide.
Cultivation . The causative agents of whooping cough and parapertussis are
aerobes. Tolerant of nutrient media. For their cultivation, the Borde-Zhang medium
(glycerin-potato agar with blood) is used. Currently, the AMC medium (casein-coal
agar) is used - this is a semi-synthetic medium without blood. The source of amino
acids here is casein hydrolyzate. The AMC environment differs from the Bordet-
Zhangu environment in a simpler and more affordable manufacturing method.
To inhibit the growth of foreign flora, penicillin is added to the medium at 0.25 - 0.5
ME per 1 ml of medium or methicillin - 2.5-4 μg per 1 ml. Penicillin can be applied to
the surface of the media in the plates.
The seeded media are incubated in a thermostat at a temperature of 35-36°C, pH
6.8-7.4. Crops must be protected from drying out; for this, a vessel with water is
placed in a thermostat.
B. pertussis colonies appear after 48-72 hours, and B. parapertussis - after 24-48
hours.
On the AMC medium, colonies of B. pertussis are small, 1-2 mm in diameter, B.
parapertussis are somewhat larger. The colonies of both microbes are shiny, grayish-
cream in color (on casein-charcoal agar, they resemble droplets of mercury). When
removing the colonies, a viscous, creamy trace remains. When studying colonies in a
stereoscopic microscope, a light cone is visible (the colonies cast a shadow). When
the position of the light source (light bulb) changes, the shadow changes position.
The presence of a light cone (tail) is of diagnostic value.
B. parapertussis forms the enzyme tyrosinase, therefore, in media containing
tyrosine, it is cleaved and the medium turns brown. A change in the color of the
medium is a differential diagnostic sign.
In a liquid medium, pertussis and parapertussis bacteria form uniform turbidity and
bottom sediment. On agar with blood, they give a zone of hemolysis.
Freshly isolated cultures most often have a smooth S-shape (I phase). When
cultivating under unfavorable conditions or in the material taken in the late stages of
the disease, dissociated forms (II-IV phases) may appear.
enzymatic properties . Whooping cough pathogens do not break down
carbohydrates and do not ferment proteins. Parapertussis bacteria produce the
enzymes urease and tyrosinase.
Whooping cough and parapertussis bacteria produce pathogenicity enzymes:
hyaluronidase, plasmacoagulase and lecithinase.
Toxin formation . In animal experiments, four types of protein toxin were identified
in pertussis bacillus: 1) thermolabile dermonecrotic toxin; 2) thermostable
endotoxin; 3) leukocytosis-stimulating factor (stimulating leukocytosis); its parenteral
administration caused the death of experimental animals; 4) histamine-sensitizing
factor - when administered to mice, they increased their sensitivity to histamine.
The first two types of toxin are also characteristic of the causative agent of
parapertussis.
 Table 47. Differential characters of bacteria of the genus Bordetella

Antigenic structure . Bacteria of the genus Bordetella have a complex antirenal

structure. The most important antigens for laboratory diagnosis are
agglutinogens. The generic agglutinogen is 7. The species-specific agglutinogen for
whooping cough bordetella is 1, for parapertussis bordetella - 14, for bronchoseptic
bordetella - 12.
Monospecific sera 1, 14, 12 are used for species differentiation (the sera are
produced by the Institute of Epidemiology and Microbiology named after N. F.
Gamaleya).
In addition to species-specific antigens, representatives of Bordetella also have other
agglutinogens, a different combination of which determines the serovar (Table 48.).

Table 48. Diagram of the composition of the agglutinogenic genus Bordetell

According to the combination of the three main agglutinogens 1,2,3, determined in

the agglutination reaction with monospecific sera, B. pertussis distinguish three
serovars: 1,2,3; 1,2,0; 1,0,3.
Resistance to environmental factors . The causative agents of whooping cough and
parapertussis are not very resistant. At a temperature of 56 ° C, they die in 20-30
minutes. Low temperatures also have a detrimental effect on them. Direct sunlight
kills them in 1-2 hours; UV rays - after a few minutes. In dry sputum, these bacteria
persist for several hours. Ordinary disinfectant solutions destroy them quickly.
Both types of microbes are not very sensitive to antibiotics, they are not sensitive to
penicillin.
Susceptibility of animals . Under natural conditions, animals are not susceptible to
pathogens of this genus. Under experimental conditions, it is possible to reproduce
whooping cough in monkeys and young dogs, and cause the death of mice.
Sources of infection . A sick man. Patients in the catarrhal period are especially
contagious.
transmission paths . Airborne way. The role of various objects is unlikely due to the
instability of pertussis bacteria in the external environment.
Pathogenesis . The causative agents of whooping cough and parapertussis cause an
acute illness accompanied by a convulsive cough. Once on the mucous membrane of
the upper respiratory tract, bacteria multiply there and are partially destroyed. The
released toxin acts on the central nervous system, irritates the nerve receptors of
the mucous membrane of the upper respiratory tract, which activates the cough
reflex. As a result, there are bouts of convulsive coughing. In the process of the
disease, several periods are observed: catarrhal, spasmodic cough and resolution of
the process.
Immunity . After the disease, a stable immunity is developed, which is determined by
humoral and cellular factors.
Prevention . Identification and isolation of patients. Weakened children who have
been in contact with sick whooping cough are given immunoglobulin. The main
measures of specific prevention are immunization of children with DTP (pertussis-
diphtheria-tetanus vaccine). The vaccine is administered three times at the age of up
to 6 months, followed by revaccination.
Treatment . In the early stages of the disease, antipertussis immunoglobulin is
used. Treatment is with erythromycin and ampicillin.
 Control questions

1. Describe the morphological properties of the causative agent of whooping cough

and parapertussis.
2. On what media and what is the nature of the growth of pertussis and
parapertussis microbes?
3. Stability of pertussis and parapertussis pathogens in the external environment.
4. Differential signs of pertussis and parapertussis pathogens.
5. Sources of infection, transmission routes, pathogenesis of whooping cough.

Microbiological research

The purpose of the study: identification of the pathogen and differentiation of

pertussis pathogens from parapertussis.
Research material
Detachable mucous membrane of the nasopharynx.
Material collection methods
 Material collection methods

Main research method

Microbiological
Research progress
First day of research

First day of research

Second - third days of the study

Crops are removed from the thermostat and viewed using a magnifying glass or a
stereoscopic binocular microscope. In the presence of suspicious colonies, they are
isolated for AMC: in Petri dishes divided into sectors, or in test tubes. Crops are
placed in a thermostat. If there are many colonies, smears can be made from part of
them, painted and viewed under a microscope. In the presence of small gram-
negative rods, a test agglutination reaction is performed with monospecific generic
serum 7. A positive agglutination reaction indicates that the isolated culture belongs
to the genus Bordetella. To determine the type of bordetella, an agglutination
reaction is performed with monospecific species sera 1 and 14. The reactions are
placed on a glass slide. A positive result of the agglutination test allows you to give a
preliminary answer.
Fourth day of research
The crops are removed from the thermostat and viewed: first with the naked eye,
paying attention to the color of the medium (if there is any brown staining), then
growth is studied using a stereoscopic microscope.
In the presence of suspicious colonies, smears are made from the isolated culture,
Gram-stained and examined under a microscope. Then again (from a pure culture)
an agglutination reaction is performed on glass with monospecific sera 1,2,3 and 14.
The results of agglutination make it possible to differentiate B. pertussis from B.
parapertussis, and if it is B. pertussis, then determine the serovar: 1- serovar 2 -
(1,2,3), serovar 2 - (1,2,0), serovar 3 - (1,0,3). The determination of the serovar is of
epidemiological significance.
For the final identification of the isolated culture (with positive agglutination with
monospecific sera), a sample is put for the presence of urease and inoculated on a
slant agar containing 0.1% tyrosine (see Fig. 49).
Rice. 49. Scheme for the isolation and identification of pertussis and parapertussis
 pathogens (Bordetella pertussis and Bordetella parapertussis)

Urease test . 0.3-0.4 ml of a 2% urea solution is poured into a small test tube, a

culture loop is introduced and 2-3 drops of phenolphthalein are added. The tube is
shaken and placed in a thermostat. Take into account the reaction after 2 and 24
hours. Pertussis bacteria do not change the color of the medium. Parapertussis
bacteria have the enzyme urease, which breaks down urea to form
ammonia. Ammonia changes the indicator and the medium turns red.
Test with tyrosine . On slanted MPA in test tubes with 0.1% tyrosine, the isolated
culture is inoculated and placed in a thermostat. The next day, take the tube out of
the thermostat and inspect it. The presence of growth in the test tube and the brown
coloration of the medium indicate the growth of parapertussis pathogens. Whooping
cough pathogens do not grow on this medium.
Fifth day of the study
In the absence of suspicious colonies give a negative answer.
Accelerated Diagnostics
With the bacteriological method of research, the answer can be obtained in 3-4 days.
1. The use of the immunoluminescent method makes it possible to give an answer a
few hours after taking the material by directly detecting microbes in swab swabs.
2. From crops on the AMC medium, in the absence of visible growth, a smear-imprint
can be made: for this, a sterile rubber stopper is touched to the seeding site and the
imprint is transferred to a glass slide. The imprint smear is studied by
immunofluorescence. Bacteria B. pertussis or B. parapertussis are found in smears.
Control questions
1. What is the material for research in suspected whooping cough?
2. What methods of material collection are used to identify the pathogen in case of
suspected whooping cough?
3. What is added to the medium to suppress the growth of foreign microflora?
Exercise
1. Take 10 cups of AMC medium, a vial of penicillin containing 300,000 units. Make a
dilution of penicillin so that 0.1 ml contains 7.5 units. Calculate the total amount of
the medium.
2. Collect nasopharyngeal discharge from each other and inoculate on AMC medium.
3. Take a dish with a culture of whooping cough or parapertussis bacteria from the
teacher, study the nature of the colonies using a stereoscopic microscope. Suspicious
colonies sow on the AMC medium sector (isolation of pure culture).
4. Take from the teacher a pure culture of whooping cough or parapertussis
pathogens grown on the AMC medium sector and perform an agglutination test with
diagnostic pertussis serum.
In the presence of a positive agglutination test, test for urease and inoculate on a
medium with tyrosine (0.1%).
Nutrient media
AMC. Wednesday prepared by the Institute of Epidemiology and Microbiology. N. F.
Gamaleya. The AMC ready medium is black, the condensate water must not contain
carbon particles. In finished form, the medium can be stored for a long time (up to a
month or more), protecting it from drying out
 Pathogenic corynebacteria - F. K. Cherkes
Chapter 32
The causative agent of diphtheria belongs to the genus Corynebacterium (from Latin
coryna - mace, diphthera - film). Bacteria have club-shaped thickenings at the
ends. This genus includes diphtheria bacilli pathogenic for humans and non-
pathogenic species - false diphtheria bacilli and diphtheroids found on mucous
membranes and skin.
The causative agents of diphtheria - Corynebacterium diphtheriae - were discovered
by T. Klebs (1883) and isolated in pure form by F. Leffler (1884).
Morphology. The causative agents of diphtheria are slightly curved, thin sticks, 3-6 ×
0.3-0.5 microns in size, with thickenings at the ends. These thickenings contain
volutin grains (Babesh-Ernst grains). Diphtheria bacteria are immobile, do not have
spores and capsules. Gram-positive. They stain well with basic aniline dyes, while
volutin grains stain more intensely. For coloring, alkaline methylene blue or crystal
violet is usually used. A feature of diphtheria corynebacteria is their
polymorphism; in the same culture there are sticks of various shapes and sizes:
curved, straight, long, short, thick, sometimes coccobacteria. The location of bacteria
in smears is characteristic - they are usually arranged in pairs at an acute or obtuse
angle, in the form of spread fingers, etc. The location in smears and the presence of
volutin grains is a differential diagnostic sign during microscopic examination. Non-
pathogenic representatives of the genus Corynebacteria - false diphtheria bacilli and
diphtheriodes are more often located in the form of a palisade, they may have no
volutin grains or be at one end (see Fig. 4).
Cultivation . Corynebacterium diphtheria are facultative anaerobes. Grow at a
temperature of 35-37 ° C, pH 7.4-7.8. They do not reproduce on conventional
nutrient media. Cultivate them on media containing blood or serum.
At the end of the 19th century, the French scientist E. Roux suggested using curdled
bovine or horse serum for the cultivation of diphtheria bacteria, and F. Leffler
recommended adding broth (25%) and 1% glucose to it. Corynebacteria grow rapidly
on these media; within 14-18 hours they form non-merging convex cream-colored
colonies (growth on a slanted medium resembles shagreen leather). However, it is
impossible to differentiate diphtheria bacilli from false diphtheria on these media.
Currently, the main growing media are Clauberg's medium (containing blood serum
and potassium tellurite), Bunin's quinosol medium, Tynsdal's medium, etc. Based on
the cultural and enzymatic properties of corynebacteria, diphtheria is divided into
three biovars: gravis (gravis), mi tis (mitis ), intermediate (intermedins). Biovar gravis
is usually in the R-form. On Clauberg's medium, the bacteria of this biovar grow in
the form of large colonies 2-3 mm, grayish-black in color (since they reduce tellurite
to tellurium), have jagged edges, which gives them the appearance of a
rosette. When you touch the colony with a loop, it seems to crumble. On the broth,
the bacteria of this biovar form a crumbling film and a granular sediment.
Corynebacteria biovar mitis (mitis) grow in Clauberg's medium in the form of small,
smooth colonies (S-form) of black color. On the broth, they give a uniform turbidity.
Corynebacteria biovara intermedius (intermedins) are intermediate. On Clauberg's
medium, the bacteria of this biovar often grow in the form of shiny, small, black
colonies (this biovar is rare).
enzymatic properties . All three biovars of diphtheria bacteria have the enzyme
cystinase, which breaks down cystine to form hydrogen sulfide. These properties are
used to differentiate diphtheria pathogens from non-pathogenic representatives of
this genus (Table 49).

Table 49

Note. + positive reaction (splits); - negative reaction (does not split).

The causative agents of all three biovars break down glucose and maltose to form
acid. C. gravis break down starch. This property distinguishes it from the other two
biovars. Corynebacterium diphtheria reduce nitrates to nitrites, do not form indole,
do not decompose urea.
Diphtheria corynebacteria produce neuraminidase, hyaluronidase and other
pathogenicity enzymes.
Toxin formation . Virulent strains of diphtheria pathogens produce
exotoxin. Chemically, it is a thermolabile protein consisting of two fractions. Fraction
B fixes the toxin on sensitive tissues of the body. Fraction A is responsible for the
toxic effect. The strength of diphtheria toxin cultures can be established "in vivo" in
guinea pigs sensitive to this toxin. Dim diphtheria exotoxin - minimum lethal dose,
this is the minimum amount of poison that kills a guinea pig weighing 250 g on the
4th day.
The presence of exotoxin can also be detected "in vitro" - on a dense nutrient
medium. This method is widely used in practical work. Diphtheria exotoxin is
unstable. It is rapidly destroyed under the influence of temperature, light and
atmospheric oxygen. After adding formalin (0.3-0.4%) to the toxin and keeping it at a
temperature of 37-38 ° C for several weeks, it turns into an anatoxin, which loses its
toxicity, but retains the antigenic properties of the toxin. The toxins produced by the
various strains do not differ and can be neutralized with diphtheria antitoxin * .
*
 ( It is now established that all biovars of corynebacteria can be toxigenic and non-toxigenic.  )
Antigenic structure . Diphtheria bacteria have a surface thermolabile protein antigen
and a type-specific polysaccharide O-antigen. In addition, 19 fagovars are
distinguished among Corynebacteria, which are taken into account when identifying
cultures. With the help of fagovars, the source of the disease is identified.
Resistance to environmental factors . The causative agents of diphtheria are
relatively stable. A temperature of 60 ° C kills them in 10-15 minutes, 100 ° C - in a
minute. In a film, they can withstand heating up to 90 ° C. On curdled whey at room
temperature, they remain for up to 2 months, on children's toys - for several
days. Corynebacteria tolerate low temperatures well. The causative agents of
diphtheria are quite resistant to drying. Disinfectants (3% phenol solution, 1%
sublimate solution, 10% hydrogen peroxide solution) kill these bacteria within
minutes.
Susceptibility of animals . Under natural conditions, animals do not get sick with
diphtheria. Of the experimental animals, guinea pigs and rabbits are the most
susceptible. With intradermal or subcutaneous infection, they develop a picture of
toxic infection with the formation of inflammation, edema, and necrosis at the
injection site. Hemorrhages are observed in the adrenal glands.
Sources of the disease . Sick people and bacteria carriers.
transmission paths . Airborne, contact-household (through dishes, toys, books,
towels, etc.).
Disease in humans : 1) diphtheria of the pharynx; 2) diphtheria of the nose.
Less common is diphtheria of the trachea, bronchi, eyes, ear, vagina and diphtheria
of damaged skin.
Pathogenesis . The entrance gates are the mucous membranes of the respiratory
tract and damaged skin. Once on the mucous membrane, diphtheria pathogens
multiply at the site of introduction and cause tissue necrosis. A film is formed that is
closely associated with the underlying tissues. Dirty gray or yellowish plaques appear
on the surface of the mucosa, consisting of destroyed epithelium, fibrin, leukocytes
and diphtheria corynebacteria. When removing the film with a cotton swab or
spatula, the mucosal surface may bleed.
In the process of reproduction of Corynebacterium diphtheria, exotoxin accumulates
in necrotic areas, which can lead to swelling of the mucous membrane and
fiber. From the mucous membrane, edema can spread to the larynx, bronchi and
cause asphyxia. The toxin circulating in the blood selectively affects the heart muscle,
adrenal glands and cells of the nervous tissue.
Diphtheria is a toxic infection. The severity of the process depends on the degree of
toxigenicity of the strain and on the body's defenses.
Immunity . Immunity is due to antitoxic and antibacterial immunity. Babies do not
get sick, as they have passive immunity, transmitted from the mother.
The presence of antitoxic immunity is judged by the Schick reaction. To set up the
reaction, 1/40 Dlm (a lethal dose of toxin for a guinea pig) contained in 0.2 ml of an
isotonic sodium chloride solution is injected intradermally in the forearm. In the
absence of antitoxin in the blood, redness and swelling (up to 2 cm in diameter)
appear at the injection site after 24-48 hours. In the presence of antitoxin, there is no
swelling and redness (the antitoxin present in the blood neutralized the injected
toxin).
The transferred disease leaves immunity. However, in 6-7% of cases, repeated
diseases are observed.
Prevention . Early diagnosis. Insulation. Disinfection. Identification of carriers of
toxigenic diphtheria bacillus.
Specific prophylaxis is carried out by the introduction of toxoid. In the USSR,
compulsory vaccination of children with the DPT vaccine is carried out - this is a
complex vaccine that includes diphtheria and tetanus toxoid and a suspension of
killed whooping cough. Vaccinate children from 5-6 months, followed by
revaccination. For revaccination, a vaccine without pertussis is administered.
specific treatment . Apply antidiphtheria antitoxic serum. The dose and frequency is
determined by the attending physician, antimicrobial drugs are also administered.

Control questions

1. What is the morphology of Corynebacterium diphtheria and what biovars are

available?
2. On what media are diphtheria bacteria grown and what is the nature of growth?
3. Relation to what carbohydrate makes it possible to distinguish gravis biovar from
other diphtheria biovars?
4. What is the route of transmission and where is the causative agent of diphtheria
most often localized in a patient?
5. What are the specific prophylaxis and specific treatment for diphtheria?

Microbiological research

The purpose of the study: the isolation of the pathogen for diagnosis. Identification
of bacteriocarriers of diphtheria according to epidemiological
indications. Identification of exotoxin in isolated culture.
Research material
1. Detachable mucous membrane of the pharynx.
2. Discharge of the nasal mucosa.
3. Detachable mucous membrane of the eye.
4. Pus from the ear.
5. Discharge of the mucous membrane of the vagina.
6. Detachable wounds.
The material for research depends on the localization of the process.
Material collection methods

Material collection methods

With any localization of the process, it is imperative to examine the mucous

membrane of the pharynx and nose. The material is collected with a cotton swab, for
which a metal wire is used, preferably aluminum, on one end of which cotton wool is
tightly wound, then the swab is mounted in a cork stopper, placed in a test tube and
sterilized in a Pasteur oven at a temperature of 160 ° C 1 swab for an hour or in an
autoclave at temperature 112°C.
Notes. 1. The material is collected on an empty stomach or not earlier than 2 hours
after eating and not earlier than 4 days after treatment with antibiotics or other
antibacterial agents. 2. If the material is taken from the pharynx and nose, then the
test tubes with both swabs are inscribed and tied together. Crops are done
separately and the study of the material from each swab is carried out as an
independent work. 3 The material collected with a dry swab should be sown no later
than 2-3 hours after collection. If it is necessary to transport the collected material,
the swab is pre-moistened with a 5% solution of glycerin in an isotonic sodium
chloride solution.
Basic research methods
1. Microbiological.
2. Bacterioscopic.
3. Biological.
Research progress
First day of research

First day of research

Second day of research

The cups are removed from the thermostat and viewed. The growth of bacteria on
Clauberg's medium may be slowed down due to the presence of inhibitors in the
medium. In this case, the cups are placed in a thermostat for another 24 hours.
Third day of research
The cups are removed from the thermostat, viewed with a magnifying glass or a
stereoscopic microscope. In the presence of suspicious colonies, some of them,
under the control of a stereoscopic microscope, are isolated on agar with 25% serum
and on a column with Piso's medium to determine the cystinase enzyme. From the
other part of the colonies, a toxigenicity test is put.
Microscopic examination of the colonies taken from Clauberg's medium, diphtheria
corynebacteria lose their specificity: there is no granularity, the size changes, the
location is preserved. When sowing them on media with serum, the morphological
specificity of diphtheria pathogens is restored.
A test for the presence of the enzyme cystinase and the determination of toxigenicity
are mandatory in the identification of diphtheria pathogens. If the result of these
experiments, carried out with a part of the colonies from Clauberg's medium, is not
clear enough or negative, then the experiment is repeated using the isolated pure
culture.
Test for cystinase . The culture under study is inoculated with a prick in the center of
the column of Pisa medium. With a positive reaction, after 18-24 hours, blackening is
observed along the injection, and a dark cloud forms around the black
rod; blackening occurs as a result of the fact that the enzyme cystinase cleaves
cystine, which is part of the Pisu medium, and the released sulfur reacts with lead
acetate - black lead sulfite is formed. Diphtheroids and pseudodiphtheria bacilli do
not contain the enzyme cystinase, therefore, when they grow on Piso's medium, the
color of the medium does not change.
Definition of exotoxin . Carried out by the method of diffuse precipitation in the
gel. The method is based on the interaction of a toxin with an antitoxin. In those
areas of the agar where these components interact, a precipitate is formed in the
form of rounded lines.
Method of determination: melted and cooled to 50 ° C Marten agar pH 7.8 is poured
into Petri dishes (exotoxin is better produced on Marten agar). The amount of agar in
the dish should be no more than 12-15 ml in order to maintain transparency - in a
thick layer, the precipitation lines are poorly visible. After the agar has solidified, a
strip of sterile filter paper moistened with anti-diphtheria antitoxic serum is applied.
The test culture is seeded with "plaques". Sowing is done with a loop. The diameter
of the plaques is 0.8-1.0 cm. The distance of the plaques from the edge of the strips
of paper is 0.5-0.7 cm; plaques of a known toxigenic strain are inoculated between
two plaques of the test culture. The test culture is considered toxigenic if the
precipitation lines are clear and merge with the precipitation lines of the control
(toxigenic) strain. If the precipitation lines intersect with the lines of the control
strain or are absent, the isolated culture is considered non-toxigenic (Fig. 50).
 Rice. 50. Detection of C. Diphtheriae exotoxin by diffuse precipitation in agar. 1 -
plaques of a non-toxigenic strain (precipitation lines cross); 2 - plaques of a toxigenic
strain (precipitation lines are connected)

Preparation of paper strips. Strips of 1.5 × 8 cm in size are cut from filter paper,
wrapped in several pieces in paper and sterilized in an autoclave at a temperature of
120 ° C for 30 minutes. Before setting up the experiment, one strip is taken out with
sterile tweezers, placed in a sterile Petri dish and moistened with anti-diphtheria
antitoxic serum. Serum is preliminarily diluted so that 1 ml contains 500 AU (antitoxic
units). The paper is moistened with 0.25 ml of serum (125 AU) and placed on the
surface of the medium. Then do the crops as described above. All crops are placed in
a thermostat. Results are recorded after 18-24 and 48 hours.
Fourth day of research
Take out the crops from the thermostat, take into account the result. Smears are
made from the culture grown on the medium with serum and stained with Loeffler's
blue.
The presence in the smears of rods characteristic in morphology, a black rod with a
cloud in the Pisu medium and precipitation lines in the agar allows us to give a
preliminary answer: "Diphtheria corynebacteria were found." The research
continues. In the absence of precipitation lines in the agar or their lack of clarity, the
study for toxigenicity must be repeated with an isolated pure culture.
For the final identification of the isolated culture and the determination of the biovar
of the pathogen, a culture is made for glucose, sucrose, starch and urea broth (to
detect the urease enzyme). Sowing on the media is done in the usual way.
Urease test . The isolated culture is inoculated into a broth with urea and an
indicator (cresol red) and placed in a thermostat. Already after 30-40 minutes, the
result can be taken into account: when sowing the true pathogens of diphtheria, the
color of the medium does not change, since they do not contain
urease. Pseudodiphtheria sticks break down urea and change the indicator - the
medium acquires a crimson-red color.
Fifth day of the study
The results are recorded (Table 50).

Table 50. Enzymatic properties of isolated pathogens

Control questions
1. What material is examined to identify the causative agent of diphtheria?
2. How is material collected for testing for diphtheria from the pharynx and nose?
3. What should be done with the swab if the collected material needs to be
transported?
4. What device is used to study colonies on Clauberg's medium?
5. What studies are carried out for the final identification of the isolated culture?
6. What methods determine the toxigenicity of corynebacterium diphtheria?
Exercise
1. Take a wire and cotton wool from the teacher and prepare 10 swabs, mount them
in a cork stopper, insert them into a test tube and sterilize.
Attention! Before sterilization, check if the swab is wrapped tightly enough.
2. Take sterile swabs from the teacher and take material from each other from the
pharynx and nose (with different swabs).
3. Study according to the table. 49 properties of causative agents of diphtheria and
corynebacteria related to them.
4. Test for toxigenicity. Make the plaques with a loop without culture.
5. Sketch the course of the study and the positive and negative results of the
toxigenicity test.
Nutrient media
Clauberg's tellurium medium : the first mixture - a mixture of 20 ml of sheep or
horse blood and 10 ml of glycerin is prepared 1.5 months in advance. On the day of
medium preparation, two other mixtures are prepared; the second mixture - 50 ml
of MPA pH 7.5 is melted and cooled to a temperature of 50 ° C, after which 2.5 ml of
the first mixture are added; third mixture - mix 17 ml of sheep's blood and 33 ml of
distilled water (the mixture is prepared sterile), heated in a water bath to a
temperature of 50 ° C. Combine the second and third mixtures, add 4 ml of 1%
potassium tellurite solution K 2 TeO 3 , quickly everything mix and pour into cups. The
medium is clear and has the color of red wine.
Wednesday Pisa . To 90 ml of molten 2% MPA (pH 7.6) add 2 ml of cystine solution
(1% solution of cystine in 0.1 N sodium hydroxide solution), mix thoroughly and add
the same volume of 0.1 N. sulfuric acid solution. The medium is sterilized for 30
minutes at a temperature of 112 ° C. To the molten and cooled to 50 ° C medium,
add 1 ml of a 10% solution of lead acetate, sterilized twice with flowing steam, mix
and add 9 ml of normal horse serum. The medium is sterilely poured into small test
tubes of 2 ml. Sowing is done by injection.
Wednesday Bunin . Dry quinosol medium is added to 100 ml of cold water (pH 7.6-
7.8), stirred and heated over low heat until the agar is melted (according to the
prescription on the label). Then the medium is boiled for 2-3 minutes until foam is
formed, after which the medium is cooled to 50°C and 5-10 ml of sterile defibrinated
blood is added. The medium is stirred and poured into Petri dishes. The prepared
medium can be stored for 3-4 days at a temperature of 4-10°C.
Tynsdale Wednesday . To 100 ml of 2% nutrient agar, melted and cooled to 50 ° C,
add: 1) 12 ml of 1% cystine solution, 0.1 N. sulfuric acid solution; 2) 12 ml of 1%
sodium hydroxide solution; 3) 1.8 ml of 2% potassium tellurite solution; 4) 1.8 ml of
2.5% sodium hyposulfite solution, 20 ml of normal horse or bovine serum. After
adding each ingredient, the medium is thoroughly mixed. Cups with the medium are
stored for 3-4 days at 10°C.
 Pathogenic mycobacteria - F. K. Cherkes
Chapter 33
Representatives of the mycobacterium Mycobacteriaceae family have the
appearance of thin, sometimes branched sticks, which resemble a mushroom. Slow
growth on nutrient media also brings them closer to fungi. These features explain
the name of the family, genus - Mycobacterium.
Mycobacteria are acid-alkaline and alcohol-resistant, which is due to the presence of
fatty substances in their cell membranes.
The genus of mycobacteria includes pathogenic and non-pathogenic
representatives. The causative agents of tuberculosis and the causative agent of
leprosy are pathogenic for humans.
Tuberculosis is widespread among animals, birds, rodents.
There are several types of tubercle bacilli:
1. Human - Mycobacterium tuberculosis
2. Bovine - Mycobacterium bovis
3. Avian - Mycobacterium avium
4. Mouse - Mycobacterium murium
5. There are mycobacteria that cause diseases in cold-blooded animals. These
include a special group of atypical mycobacteria.
Currently, atypical mycobacteria are of particular importance. They are divided
according to a number of characteristics into 4 groups: I, II, III, IV (according to
Runyon). They differ from Mycobacterium tuberculosis in their lower demands on
nutrient media. Among themselves, they differ in relation to nutrient media, growth
rate, the ability to form a pigment, as well as catalase and peroxidase
activity. Representatives of groups I and III cause diseases in humans.
Morphology . The causative agents of tuberculosis were discovered by the
river. Koch in 1882. These are thin sticks 1.5-4 × 0.3-0.5 µm in size. They are very
polymorphic: there are straight, curved, cone-shaped. As a result of the variability of
bacteria, there are acid-tolerant forms and very small, so-called Fly grains. The
variety of forms often depends on the composition of the medium, the impact of
antibiotics and chemotherapeutic agents on them. Tuberculosis bacteria are
immobile, do not have spores and capsules. Gram-positive, but they do not perceive
aniline dyes well. They are well stained red by the Ziehl-Nielsen method (see Fig. 4),
where concentrated paints and etching are used.
cultivation. The causative agents of tuberculosis are aerobes. They grow at a
temperature of 37-38 ° C and a pH of 5.8-7.0. Distinctive cultural features of tubercle
bacillus are slow growth and exactingness to nutrient media. Primarily, they grow
only on special media: the environment of Petragnani, Petrov, Levenshtein-
Jensen. They can be grown on glycerin broth, glycerin agar, glycerin
potatoes. Glycerin stimulates the growth of mycobacteria. M. bovis does not need
glycerol. The most widely used medium is Loewenstein-Jensen, which is
recommended by WHO as a standard medium for growing tubercle bacilli. Currently,
Finn II medium is also used, which differs from Lowenstein-Jensen medium in that
sodium glutamine is used instead of asparagine. On this medium, Mycobacterium
tuberculosis grows somewhat faster than on Lowenstein-Jensen medium, and the
percentage of isolation of cultures is higher. Tuberculosis bacilli can also be
cultivated on synthetic media, such as Soton's medium.
Mycobacterium tuberculosis occurs in R- and S-form. The R form is more virulent (M.
bovis is more common in the R form). On dense nutrient media, tuberculosis
pathogens form dry wrinkled cream-colored colonies with a slightly raised center and
jagged edges (see Fig. 26). In liquid nutrient media, Mycobacterium tuberculosis
grows on the 10-15th day in the form of a film, which gradually thickens, becomes
rough, wrinkled, brittle, and sometimes falls to the bottom due to gravity. The broth
under the film remains transparent.
enzymatic properties . The causative agents of tuberculosis are not biochemically
active. They found a proteolytic enzyme, which under certain conditions (acidic and
alkaline environment) breaks down the protein. They also break down some
carbohydrates, form urease. But these properties are not permanent. Therefore, the
study of enzymes has no diagnostic value.
Toxin formation . The causative agents of tuberculosis form endotoxin - this protein
substance was first isolated by R. Koch (1890) and called it tuberculin. "Old"
tuberculin is a culture liquid obtained by growing a culture in glycerin broth and
evaporated at 70 ° C to 1/10 of its original volume. The "new" tuberculin is a purified
protein derivative of tuberculin.
Tuberculin has the properties of an allergen. It does not have a toxic effect on a
healthy body. Its action is manifested only in an infected organism. Therefore, the
introduction of tuberculin is used for diagnostic purposes, in the production of
allergic tests (Pirquet or Mantoux). For this purpose, tuberculin is prepared from the
bovine type of Mycobacterium tuberculosis.
Virulent strains of tuberculosis pathogens contain a special lipid cord factor, which
promotes the adhesion of mycobacteria and their growth in the form of braids and
strands.
Antigenic structure . Mycobacterium tuberculosis contain an antigen, which includes
protein, lipoid and polysaccharide factors. This antigen causes the body to produce
antibodies (agglutinins, precipitins, complement-fixing substances, etc.). However,
these antibodies are found in low concentrations, so they are rarely used for
diagnostic purposes.
Resistance to environmental factors . Mycobacterium tuberculosis is the most stable
of the non-sporeforming forms of bacteria (resistance is due to the presence of lipids
in their shell). They endure a temperature of 100°C for 5 minutes. UV rays cause their
death only after a few hours.
In dried sputum, they live up to 10 months. At low temperatures, Mycobacterium
tuberculosis persists for a long time.
Disinfectant solutions: sublimate (1:1000), carbolic acid (5%) destroy them only after
a day. They are most sensitive to chloramine and bleach.
Susceptibility of animals . A person is very sensitive to M. tuberculosis, animals and
birds are not very sensitive. Of the experimental animals, guinea pigs are highly
sensitive to it, in which the infection proceeds in a generalized manner and usually
ends in the death of the animal.
Large and small livestock and domestic animals are susceptible to M. bovis (humans
are insensitive, but children can become infected when using the milk of sick
animals).
Of the experimental animals, rabbits are the most sensitive, in which the infection
proceeds in a generalized manner. M. avium causes disease in birds: chickens,
pigeons, pheasants, etc. However, some animals can also get sick (humans rarely
become infected).
Of the experimental animals, rabbits are sensitive. They have an acute infection.
The mouse species is pathogenic mainly for voles. In rabbits and guinea pigs, the
disease occurs in a chronic form.
Sources of infection . Human. Rarely animals.
transmission paths . The most common routes of transmission are airborne and
airborne; less food. Perhaps intrauterine infection through the placenta.
Diseases in humans and pathogenesis . Tuberculosis disease is characterized by a
variety of clinical forms. There are pulmonary (most common) and extrapulmonary
forms: tuberculosis of the stomach and intestines, kidneys, meninges, bones and
other organs.
Each of these forms can end up with a generalization of the process. With airborne
and airborne dust infection, the primary focus occurs in the lung. A tubercle is
formed in the affected organ - tubercul. The tubercle is an accumulation of
leukocytes and giant cells, inside of which are Mycobacterium tuberculosis. With
good body resistance, the connective tissue surrounds the tubercle, it calcifies and
the bacteria, remaining viable, do not go beyond the tubercle. Such is the "center of
Gon" - a calcified, small focus at the site of the primary introduction of the tubercle
bacillus (closed process).
With a closed process, tuberculosis bacilli are not excreted with sputum, urine, etc.
Thus, even with a benign course of the process, the body is not freed from
tuberculosis pathogens. It is believed that 80% of people are infected with
tuberculosis bacteria. However, they are clinically healthy. When the body gets into
unfavorable conditions, its protective functions decrease, the tubercle undergoes
necrosis, bacteria are released and involve new areas in the process, an exacerbation
occurs, caverns are formed - an open process. Sometimes there may be a
generalization of the process, which leads the body to death. More often,
tuberculosis occurs in a chronic form (a closed process). Working and living
conditions are of great importance during exacerbation.
Immunity . A person has a certain resistance, i.e., when infected, a disease does not
always occur, but an infectious (non-sterile) immunity is formed, which is
determined by a complex of protective factors: humoral, cellular, as well as the
resistance of organs and tissues.
Prevention . Early diagnosis, isolation, etc. For specific prophylaxis, the live BCG
vaccine (BCG) obtained by the French scientists Calmette and Guérin is used. This
vaccine is administered to newborns once, intradermally into the outer surface of
the shoulder. Revaccination is carried out after 7-12 years, and then every 5-6 years
up to 30 years.
Treatment . Antibacterial drugs: streptomycin, rifampicin, PAS, ftivazid, etc.

Control questions

1. By whom and when was the causative agent of tuberculosis discovered?

2. What types of tubercle bacillus are divided into? What type is pathogenic for
humans?
3. What determines the resistance of Mycobacterium tuberculosis?
4. What method is used to stain smears to detect tuberculosis mycobacteria?
5. What forms do Mycobacterium tuberculosis dissociate into and what form is
pathogenic?

Microbiological research

The purpose of the study: identification of the pathogen.

Research material
1. Sputum (tuberculosis of the lungs and bronchi).
2. Exudate from the pleural cavity (pulmonary tuberculosis, pleura).
3. Ascitic fluid and feces (intestinal form of tuberculosis).
4. Urine (kidney tuberculosis).
5. Cerebrospinal fluid (tuberculous meningitis).
6. Blood (generalization of the process).
Material collection methods
 Material collection methods

Note. Jars for collecting material should be with screw caps. Vessels for collecting
material are sterilized in an autoclave at 120°C for 20 minutes or by boiling for 1
hour.
Basic research methods
1. Bacterioscopic.
2. Luminescent.
3. Bacteriological
4. Biological.
5. Allergic.
Research progress
 Research progress

Control questions
1. On what nutrient media are mycobacterium tuberculosis grown and what is the
duration of their growth?
2. How and why is sputum treated before sowing it on nutrient media?
3. Describe the growth of tubercle bacillus on solid and liquid nutrient media.
4. What animal is most sensitive to the human type of tubercle bacillus?
Nutrient media
Levenshtein-Jensen medium: saline solution; monosubstituted potassium phosphate
- 2.4 g; magnesium sulfate - 0.24 g; magnesium citrate 10.6 g; asparagine - 3.6
g; glycerin - 12 ml; potato flour - 5 g; distilled water - 600 ml.
The reagents are dissolved in the indicated sequence at low heating and sterilized for
2 hours with flowing steam. The salt base can be prepared with a margin of 3-4
weeks.
Egg mass . 24-27 (depending on the size) fresh dietary eggs are washed with running
warm water, a brush with soap, immersed in 70% alcohol for 30 minutes, then
broken over a spirit lamp in a box with sterile tweezers into a flask with beads, stir
well and to 1 liter of egg mass add 600 ml of saline. The mixture is filtered through a
gauze filter, 20 ml of a sterile 2% solution of malachite green is added and poured
into 5 ml test tubes. Coagulation is carried out at 85°C for 45 minutes.
Wednesday Finn II . Salt base: magnesium sulfate - 0.5 g; sodium citrate - 1
g; ferroammonia alum - 0.05 g; potassium phosphate monosubstituted - 20
g; ammonium citrate monosubstituted - 20 g; sodium glutamate monosubstituted - 5
g; glycerin - 20 ml; distilled water - up to 1 liter.
The ingredients are dissolved in the specified order in warm distilled water. Set pH
6.3-6.5. Sterilize at 1 atm for 20 minutes.
Egg Wednesday . 12 eggs are washed with a brush with soap, treated with
alcohol. Break with sterile tweezers and pour into a sterile flask with beads, which
after adding each egg is shaken until a homogeneous mass is formed. Add 10 ml of a
20% aqueous solution of malachite green and 300 ml of saline. Filter through a gauze
filter and coagulate at 85°C for 30 minutes.
Soton Synthetic Medium . To 200 ml of distilled water add 4 g of asparagine, 0.5 g of
iron citrate, 2 g of citric acid, 0.5 g of magnesium sulfate, 0.5 g of basic potassium
phosphate, 60 g of glycerol, 800 ml of distilled water.
 Pathogenic anaerobes - F. K. Cherkes
Pathogenic anaerobes belong to the family Bacillaceae, genus
Clostridium. Anaerobes - an extensive group of microorganisms, among which are
pathogenic for humans: 1) tetanus clostridia; 2) clostridia gas gangrene
(polymicrobial infection); 3) Clostridia botulism.
Pathogenic anaerobes are permanent inhabitants of the intestines of animals and
humans, with the feces of which they are excreted into the external environment. In
the form of spores, they persist for a long time in soil, sea and fresh water.
Pathogenic clostridia - large sticks 4-9 × 0.6-1.2 microns in size. Young cultures are
gram-positive, old ones lose their ability to stain according to Gram. All clostridia
form oval or round spores, located terminally, subterminally, or centrally. Most
anaerobes are mobile. Flagella are located peritrichally. Clostridia produce exotoxins
of high biological activity.
Culture methods
Anaerobes are cultivated in anoxic conditions. There are several ways to remove
oxygen during their cultivation: physical and biological.
Physical Methods . Removal of oxygen mechanically. Air is removed in an anaerostat
- a hermetically sealed device with a device for pumping air. Portable (portable)
anaerostat - a small cylinder with a hermetically sealed lid and a valve for pumping
out air. Crops are placed in an anaerostat, a vacuum is created in the apparatus and
placed in a thermostat. This apparatus can be replaced by a desiccator.
Cultivation in an inert gas atmosphere. The air in the anaerostat is replaced by
nitrogen, a mixture of nitrogen with carbon dioxide or hydrogen.
Cultivation in a high column of agar with glucose. Microorganisms grow on the
bottom, protected from the air by a high layer of the medium.
Vinhal-Veyon method. Sowing is carried out in a test tube with agar melted and
cooled to 45 ° C. The contents of the tube are mixed and sucked into a Pasteur
pipette, filling it to the top. Care must be taken not to get air bubbles into the
pipette. The thin end of the pipette is sealed, lowered into a test tube with cotton
wool at the bottom and transferred to a thermostat. Isolated colonies grow in the
thickness of the agar. After sawing the capillary of the pipette, they are removed.
Biological Methods . Co-cultivation of anaerobes and aerobes. A thick layer of agar
with 5% blood is poured into a Petri dish. A groove is made in the agar along the
diameter of the dish so that the cultures do not mix. On one half, an aerobe culture
is sown, on the other - an anaerobe. The edges of the cup are filled with
paraffin. Crops are placed in a thermostat. Aerobes grow first, after they absorb all
the oxygen in the cup, anaerobes begin to grow.
Addition of reducing (oxidizing) substances to the medium. Kitt-Tarozzi medium is
used, containing 0.5% glucose solution and pieces of meat as reducing
substances. Before sowing, the medium is boiled for 20 minutes in a water bath - the
oxygen dissolved in it is removed. Quickly cool to 45 ° C and inoculate without
allowing the medium to re-saturate with oxygen. Immediately after inoculation,
sterile oil is poured into the test tube on the surface of the medium with a layer of 1-
1.5 cm to protect the inoculation from air. Crops are grown in a thermostat.
Nutrient media
Wednesday Kitt - Tarozzi . Bovine liver or meat is cut into small pieces, poured with
three times the amount of nutrient broth pH 7.4-7.6, boiled for 30 minutes. The
broth is filtered. The liver is washed on a sieve with water, distributed into test
tubes, 3-4 pieces each, pour 7-8 ml of broth. Sterilized under a pressure of 1 atm for
30 minutes.
Blood agar . 3% MTTA with 1-2% glucose pH 7.2-7.4 is mixed with 15-20% fresh
defibrinated sheep or horse blood. Pour into Petri dishes. Dry in a thermostat for 20-
30 minutes. You can use the blood of a rabbit or a guinea pig, taken from the heart
sterilely, in an amount of 5-1% of the medium.
Wilson-Blair Wednesday . 100 ml of 3% MPA with 1% glucose is melted in a water
bath, 10 ml of 20% sodium sulfite and 1 ml of 8% iron chloride solution are
added. Both solutions are prepared in sterile distilled water and boiled. The prepared
medium is poured into test tubes of 7-8 ml.
Willis-Hobbs Wednesday . Mix 400 ml Hottinger broth, 4.8 g agar, 4.8 g lactose, 1.8
ml 1% neutral red solution. Autoclave, cool to 50-55°C, add 15 ml of chicken yolk
suspension with isotonic sodium chloride solution and 60 ml of sterile skimmed milk.
There are selective media for certain species, types of bacilli and media for better
toxin formation.
Currently, casein-yeast, breech-mushroom, fish, corn and other media are widely
used.
Environmental resistance
Vegetative forms of anaerobes are not very stable. Spores are very resistant to
physical and chemical factors: they tolerate boiling from 15-20 minutes to several
hours, depending on the species, type and strain of bacilli. Also resistant to low
temperatures and drying.
Ordinary solutions of disinfectants destroy them only after a long exposure (12-14
hours). The spores of the causative agents of botulism are especially resistant.
Anaerobic toxins, like most exotoxins, are sensitive to high temperatures, direct
sunlight, and disinfectants. The exotoxin of C. botulinum is highly resistant - it is
destroyed only by 15-20 minutes of boiling.

Control questions

1. What are the conditions for cultivating anaerobes?

2. What is the main difference between the conditions for cultivating aerobes and
anaerobes?
3. What is an anaerostat?
4. What are the methods of cultivation of anaerobes?
5. What creates anaerobic conditions for cultivation on Kitt-Tarozzi medium?
 Chapter 34
The causative agent of tetanus, Clostridium tetani, was described by Nikolaier in
1884. In pure culture, Kitazato was obtained in 1889.
Morphology . C. tetani - sticks 4-8 × 0.4-1 microns in size with rounded
edges. Mobile. Flagella are located peritrichally. Capsules do not form. They form
spores of spherical shape, located terminally, which gives the bacillus the
appearance of a drumstick. Gram-positive. Old cultures sometimes lose their ability
to stain with Tram. Spores when stained with Trump or methylene blue have the
form of rings (see Fig. 4).
cultivation. The causative agent of tetanus is a strict anaerobe. Highly sensitive to
oxygen. Therefore, the rods multiply well in the depth of a tall column of
agar. Special media for their cultivation are: Weinberg's medium in the CIEM
modification, Willis and Hobbs' medium, Kitt-Tarozzi's medium, etc. Liquid media are
poured with a layer of vaseline oil to create anaerobic conditions. Before sowing,
oxygen is removed from the medium by boiling in a water bath and rapid cooling to a
temperature of 40-50°C. Tetanus pathogens grow at a temperature of 35-37°C and a
pH of 6.8-7.4. On dense nutrient media, growth appears on the 3-4th day. The grown
colonies are grayish in color, sometimes transparent with an uneven granular surface
and elongated edges - R-shape. In a tall column of agar, C. tetani form fluffy colonies,
sometimes the colonies are dark and resemble lentils. On blood media, a hemolysis
zone is noted around the colonies. When sowing causative agents of tetanus on the
Kitt-Tarozzi medium, the medium becomes cloudy. Growth on the Wilson-Blair
medium is characterized by blackening of the medium. Crops on cups are placed in
an anaerostat.
enzymatic properties . C. tetani have weak enzymatic activity. Carbohydrates do not
break down (there are strains that break down glucose). Proteolytic properties are
expressed in the reduction of nitrates to nitrites, the slow coagulation of milk and
the slow liquefaction of gelatin.
C. tetani forms fibrilysin.
Toxin formation . C. tetani produce a strong exotoxin consisting of two components:
tetanospasmin and tetanolysin. Tetanospasmin (a neurotoxin) affects the motor cells
of the nervous tissue, which leads to spasmodic muscle contraction. Tetanolizin
hemolyzes erythrocytes. A toxin obtained from a broth culture at a dose of
0.0000005 kills a white mouse weighing 20 g.
Antigenic structure . Clostridium tetanus is divided into 10 serovars. Separation into
serovars is carried out according to the H-antigen, and into serogroups - according to
the O-antigen. The causative agents of all serovars produce a toxin that is neutralized
by antitoxic serum of any type.
Resistance to environmental factors . Vegetative forms of C. tetani at a temperature
of 60-70 ° C die in 20-30 minutes. Spores are highly resistant, they can withstand
boiling for 1-1.5 hours. Spores remain in the soil and on other objects for a long
time. Direct sunlight kills them after a few hours.
Disinfectant solutions: 5% phenol solution, 1% formalin solution destroys them after
5-6 hours.
Susceptibility of animals . Under natural conditions, horses and small cattle suffer
from tetanus. Of the experimental animals, white mice, rabbits, and rats are highly
sensitive to tetanus toxin. Their disease proceeds according to the type of ascending
tetanus. It begins with contractions of the striated muscles of the hind limbs, then
the muscles of the trunk are involved, etc. Death occurs from paralysis of the heart
muscle.
Sources of infection . The causative agents of tetanus are widely distributed in
nature. Many animals are carriers of these microorganisms, so C. tetani is found in
the soil, where they enter from the intestines of animals and humans. Tetanus
infections are more common in rural areas, especially in areas with developed animal
husbandry. Spores can be carried with dust, getting on clothes and other objects.
Transmission routes and entry gates . The entrance gate is damaged skin and
mucous membranes. Tetanus is a wound infection and the incidence is associated
with trauma (especially in wartime). Wounds with deep traumatization of tissues are
dangerous, into which earth, foreign bodies, etc. are introduced. However,
penetration of a small splinter is sometimes enough to cause a disease.
Pathogenesis . Having penetrated into the depth of the tissue, spores at the site of
introduction begin to germinate into vegetative forms. Reproducing, tetanus bacillus
releases exotoxin. Tetanus toxin selectively acts on the cells of the central nervous
system and causes spasm of motor muscles. A person has descending tetanus. The
earliest signs are convulsions of the masticatory muscles (trismus), then a spasm of
the facial and occipital muscles begins. A "sardonic smile" appears. Then the muscles
of the abdomen and lower extremities contract. Death occurs from asphyxia due to
spasm of the respiratory muscles.
Immunity . There is no post-infection immunity, since the outcome of this disease is
often fatal.
Artificial immunity is achieved by the introduction of toxoid. Antitoxic immunity.
specific prophylaxis . Based on immunization with toxoid, which is a component of
DTP. Vaccinations with the DTP vaccine are carried out for all children aged 5-6
months to 12 years, followed by revaccination, as well as agricultural workers,
builders, etc. Specific treatment. Enter intramuscularly tetanus toxoid. A good result
is given by immunoglobulin obtained from the blood of donors immunized against
tetanus. In addition, tetracycline antibiotics and penicillin are administered.

Microbiological research

The purpose of the study: detection of the causative agent of tetanus and tetanus
toxin (practically rarely carried out).
Research material
1. The contents of the wound.
2. Pieces of tissue from the affected area.
3. Foreign bodies that got into the wound.
4. For prophylactic purposes, dressings, catgut, silk and preparations for
subcutaneous administration are examined for sterility (see "Sanitary
Microbiology").
5. Soil (see "Sanitary microbiology").
Material collection methods

Material collection methods

Basic research methods

1. Microscopic.
2. Biological.
3. Bacteriological (isolation of the causative agent of tetanus).
Research progress
First day of research
 First day of research

Second - third days of the study

1. If the animals have not died, continue to monitor them. Signs of tetanus in mice:
disheveled hair, stiffness of the limbs and tail ("tail pipe"), paralysis of the paw into
which the toxin is injected. Animals die in a characteristic pose: tucking their front
legs in and stretching out their hind legs. After the death of the animal, microscopic
and bacteriological examination of tissues from organs is performed.
Control mice given the toxin at the same time as the antitoxin did not develop
tetanus.
2. If the result of the biological test is negative, the crops are examined. Suspicious
colonies are subcultured to isolate a pure culture on Kitt-Tarozzi medium.
Fourth - fifth days of the study
The cultural properties of isolated microorganisms are studied. They make smears
and microscope them. The resulting culture is tested for the presence of tetanus
toxin in a biological sample (according to the same scheme, Table 51).

Table 51. Morphological, cultural and enzymatic properties of the main types of
pathogenic anaerobes

Control questions
1. Describe the morphological properties of the causative agent of tetanus.
2. What are the enzymatic properties of tetanus bacilli?
3. Toxin formation and antigenic structure of tetanus bacilli.
4. Pathogenesis of tetanus.
5. Specific prevention and treatment of tetanus.
6. On what animals and how is a biological sample placed?
 Chapter 35
Gas gangrene is a polymicrobial infection, that is, it is caused by a group of
microorganisms. They belong to the family Bacillaceae, genus Clostridium.
Main representatives: C. perfringens, C. novyi, C. septicum, C. histolyticum, C.
sordellii. Usually, the disease occurs as a result of one or more representatives of the
genus Clostridium getting into the wound and often in combination with aerobes -
staphylococci and streptococci.

Clostridium perfringens

C. perfringens was discovered in 1892 by Welch and Nettolm.

Morphology . C. perfringens are large polymorphic rods averaging 3-9 × 0.9-1.2
µm. Motionless. Cultures freshly isolated from the body have a capsule. When
exposed to unfavorable conditions, they form oval-shaped spores, located centrally
or subterminally. Gram-positive. Old cultures lose the ability to stain according to
Gram.
Cultivation . C. perfringens are anaerobes but are not very sensitive to atmospheric
oxygen. They grow well and quickly on nutrient media prepared from meat or casein
hydrolysates: 3-8 hours at a temperature of 37-42 ° C and a medium pH of 7.2-
7.4. Growth is accompanied by rapid gas formation and a decrease in pH to the acid
side. On dense nutrient media, C. perfringens form rough R, smooth S, and slimy M
colonies. Under certain conditions, colonies of the mixed O-variant appear. In the
depths of the agar column, the colonies look like lentils. In liquid media, growth is
characterized by uniform turbidity and gas formation. On blood media, C.
perfringens form a zone of hemolysis.
Enzymatic properties - C. perfringens ferments lactose, glucose, sucrose, maltose
with the formation of acid and gas. Proteolytic properties - coagulate milk, slowly (2-
7 days) liquefy gelatin. Litmus milk is coagulated with the formation of a brick-
colored clot and complete clarification of whey. They reduce nitrates to nitrites, they
do not form indole.
C. perfringens produce lecithinase, hyaluronidase, gelatinase, collagenase, and other
pathogenicity enzymes.
Toxin formation . C. perfringens secrete a complex toxic complex consisting of
several toxins, which are denoted by the Greek letters α, θ, β, etc. In addition, they
form an enterotoxin. The main toxic complex is α-toxin, which has a large and
comprehensive biological activity.
Antigenic structure . C. perfringens is divided into five serovars, which are
designated by capital Latin letters A, B, C, D and E. These serovars differ from each
other in antigenic and biochemical properties of their toxins.
Serovar A is a natural intestinal inhabitant, but can cause foodborne infections in
humans. Serovar B causes intestinal symptoms in lambs. Serovar C causes necrotizing
enteritis in humans and disease in cattle. Serovar D causes enterotoxemia in animals.
Resistance to environmental factors . Vegetative forms of C. perfringens are not
very stable: they are detrimental to disinfectants in the usual concentrations used in
laboratories.
Spores of some strains can withstand boiling for several minutes. The most resistant
microbes are serovar A.
Susceptibility of animals . Under natural conditions, C. perfringens causes disease in
domestic animals. Of the experimental animals, guinea pigs, rabbits, pigeons, and
mice are sensitive to them. In infected animals, tissue necrosis occurs at the site of
toxin injection. There may be clostridia in the blood.

Clostridium novyi

C. novyi discovered by Novy in 1884

Morphology . C. novyi - large, straight or slightly curved rods 4-22 × 1.4-1.6
µm. Often arranged in chains. Mobile - peritrichous. In the external environment,
they form oval spores located subterminally (the width of the spores may be
somewhat wider than the cell). They do not have capsules. Gram-positive. Old
cultures may be gram negative.
Cultivation . C. novyi are strict anaerobes. Highly sensitive to atmospheric
oxygen. They grow on casein, carbohydrate and meat-peptone media at a
temperature of 37-43 ° C and a pH of 7.4-7.6. On dense nutrient media, round,
translucent colonies with a granular surface and fringed edges grow after 48
hours. In an agar column, they form flaky, clump-shaped colonies with a compact
center. On liquid nutrient media, they grow with the accumulation of gas and the
precipitation of films. On blood agar around the colonies, a zone of hemolysis is
observed.
enzymatic properties . C. novyi are less active than C. perfringens. They only ferment
glucose and maltose, producing acid and gas. Proteolytic properties: milk is slowly
curdled, gelatin is slowly liquefied. Indole and hydrogen sulfide do not form.
Of the pathogenicity enzymes, phospholipase was found.
Antigenic structure . C. novyi is divided into four serovars: A, B, C, D, differing in
antigenic properties and the toxins they synthesize.
Toxin formation . C. novyi synthesize several toxins, denoted by the Greek letters α,
β, γ, etc.
Exotoxins have necrotic, hemolytic and lethal effects. In addition, they disrupt the
permeability of the walls of blood vessels, which leads to the formation of a gel-like
edema.
Resistance to environmental factors . Vegetative forms of C. novyi are
unstable. Spores persist in the external environment for many (25-30) years. Boiling
kills them in 40-60 minutes, direct sunlight - in a day. The usual concentrations of
disinfectant solutions destroy them in 15-20 minutes.
Susceptibility of animals . Mammals and birds (pigeons) are sensitive to C.
novyi. From experimental animals: guinea pigs, rabbits, mice. When C. novyi culture
is injected subcutaneously, they develop a gelatinous jelly-like edema, sometimes
with gas formation. The animal dies after 24 hours.

Clostridium septicum

Clostridium septicum was discovered by L. Pasteur in 1877.

Morphology . C. septicum is a polymorphic rod 3-4 × 1.1-1.6 µm in size (filamentous
forms up to 50 µm in length are found). The sticks are mobile - peritrichous. Spores
are located subterminally, sometimes centrally. The capsule does not form. Gram-
positive. Old cultures may stain Gram negative.
Cultivation . C. septicum are strict anaerobes. They grow well on meat and casein
media with the addition of 0.5% glucose at a temperature of 37-43 ° C and a medium
pH of 7.4-7.6. On blood glucose agar, they form colonies in the form of intertwining
threads, around which there is a small zone of hemolysis. In the depth of the sugar
agar column there are colonies with a compacted center and filaments extending
from the edges. In the MPB form a uniform turbidity, followed by loose sediment
and gas formation.
enzymatic properties . C. septicum have saccharolytic properties: they break down
glucose, lactose, maltose with the formation of acid and gas. Mannitol and glycerin
do not ferment. Proteolytic properties: liquefy gelatin, milk coagulates slowly. They
convert nitrates to nitrites, break down proteins with the release of hydrogen sulfide
and ammonia. They do not form indole (see Table 51).
Antigenic structure . C. septicum have O- and H-antigens. According to H-antigens, 6
serovars were established in them using the agglutination reaction.
Toxin formation . C. septicum exotoxin consists of several substances: β, θ, γ, etc.
The main substance, α-toxin, has lethal, necrotic and hemolytic properties. In
addition, fibrinolysin and collagenase were found in the filtrates of C. septicum
cultures. All these factors play an important role in pathogenesis.
Resistance to environmental factors . Vegetative forms quickly die in the presence
of atmospheric oxygen. The spores are less resistant than the spores of other
clostridia.
Susceptibility of animals . Under natural conditions, domestic animals are sick: large
and small cattle.
Of the experimental animals, guinea pigs are sick. After intramuscular injection of C.
septicum culture into the paw, intense edema develops at the injection site, which
spreads over the entire anterior abdominal wall and the animal dies in 24-48
hours. From the affected tissue, when pressed, a bloody-foamy fluid is released.

Clostridium histolyticum

C. histolyticum was isolated by Weinberg in 1916.

Morphology . Small sticks 1.6-3.1 × 0.6-1 µm. Mobile - peritrichous. They form
spores located subterminally. Gram-positive.
Cultivation . C. histolyticum are facultative anaerobes. Grow on meat and casein
media. On blood agar, they form small shiny colonies with smooth edges. There is a
small area of hemolysis around the colonies.
enzymatic properties . C. histolyticum do not have saccharolytic
properties. Proteolytic properties are pronounced: they liquefy gelatin, lyse pieces of
meat placed in a liquid nutrient medium, and hydrogen sulfide is formed (see Table
51).
Toxin formation . In the filtrates of C. histolyticum, α-toxin is found, which has lethal
and necrotic properties. In addition, β-factor, which destroys collagen (collagenase),
was found in the filtrates. This toxin selectively acts on pancreatic cells. The
significance of C. histolyticum in human pathology has not been fully elucidated.

Clostridium sordellii

C. sordellii was first isolated and studied by Sordelli in 1922.

Morphology . C. sordellii - sticks 3-4 × 1.1-1.5 µm. Mobile - peritrichous. Spores are
oval, located subterminally or ovally. Gram-positive.
Cultivation . C. sordellii are facultative anaerobes. On the surface of dense nutrient
media, they form grayish-white, somewhat convex colonies. On blood agar, a narrow
zone of hemolysis is given. In the depth of the agar, the colonies are lentil-shaped. In
liquid meat and casein nutrient media, they grow rapidly, forming mucus.
enzymatic properties . C. sordellii have saccharolytic properties: they break down
glucose, maltose, fructose, do not break down lactose and sucrose. Proteolytic
properties are expressed in the slow liquefaction of gelatin and curdled whey, they
form indole, hydrogen sulfide and urease (see table. 51).
C. sordellii produce lecithinase, hyaluronidase, hemolysin, fibrinolysin.
Toxin formation . C. sordellii secrete a highly active toxin with lethal properties
similar to C. novyi α-toxin.
Resistance to environmental factors . Vegetative forms are not stable. Spores are
stable and persist in the soil for a long time.
Susceptibility of animals . In experimental animals, C. sordellii causes a disease
similar to gas gangrene.
Sources of infection . * Clostridium gas gangrene enters the environment from the
intestines of animals, more often herbivores (under poor sanitary and hygienic
conditions, they can be found on human skin).
*
 ( The following material applies to all considered representatives of the genus Clostridium.  )
Transmission routes and entry gates . If tissues are damaged, especially with
extensive lacerations, and lumps of earth, scraps of clothing, shell fragments get into
the wound, a disease can develop. In peacetime, gas gangrene can occur after
surgery, drug injections, community-acquired abortions, etc.
Pathogenesis . Spores or vegetative forms of cells that have fallen into the wound
multiply and secrete exotoxin. In the process of reproduction, Clostridium necrotizes
healthy tissue. The process develops especially intensively in muscle tissue, since
there is a large amount of glycogen, which is a good environment for the
development of anaerobes. Most often, the infection occurs with deep wounds,
when "blind pockets" are formed, which are poorly supplied with oxygen and
favorable conditions are created for the development of clostridium. The released
exotoxins cause intoxication phenomena.
Clostridia often occur in association: the combined action of C. perfringens and C.
novyi toxins causes a more severe reaction than the action of separate toxins. In the
pathogenesis of anaerobic infection, the accompanying flora (staphylococci,
streptococci, etc.), as well as the reactivity of the macroorganism, are of great
importance.
Immunity . Antitoxic and antibacterial, but the leading role belongs to the
antitoxin. A person has natural immunity, which occurs as a result of the presence of
clostridium in the intestines. After the disease, fragile immunity remains. More
stable immunity is created by immunization with toxoid.
Prevention is carried out by surgical treatment of the wound (excision, incisions). For
specific prophylaxis, adsorbed polyanatoxin is used, containing toxoids of all
representatives of gas gangrene. For seroprophylaxis in case of wounds (more often
in wartime), anti-gangrenous serum is administered: 10,000 IU each of C.
perfringens, C. novyi, C. septicum, i.e., a total of 30,000 IU. A mixture of anaerobic
phages is also used.
Treatment . For specific treatment, antitoxic serum is used at 50,000 IU of each
Clostridium, that is, a total of 150,000 IU. Serum is administered
intravenously. Antibiotics are also used: penicillin and sulfa drugs and oxygen
therapy.

Microbiological research

The purpose of the study: the identification of anaerobic pathogens, their toxin.
Research material
1. Exudate from the wound.
2. Pieces of altered tissue from the wound.
3. Foreign bodies that got into the wound.
4. Blood (generalization of the process).
Material collection methods

Material collection methods

Basic research methods

1. Microscopic.
2. Bacteriological.
3. Biological.
Research progress
First day of research
 First day of research

Second - fourth days of the study

Take out the crops from the thermostat. In the presence of growth - blackening on
the Wilson-Blair medium, hemolysis zones around the colonies on blood agar - a
pure culture is isolated. Morphology, mobility and enzymatic activity are studied (see
Table 51).
When pathogens of gas gangrene are detected, a biological sample is placed on mice
to determine the type of pathogen. To do this, the test material is poured into 0.9 ml
in 5 test tubes and 0.6 ml of the corresponding antitoxic sera are added to each: C.
perfringens, C. novyi, C. septicum, C. histolyticum, C. sordellii. In the last, 6th test
tube, isotonic sodium chloride solution is added - control.
The mixture of toxin with antitoxic serum is left at room temperature for 40 minutes
to neutralize the toxin. 0.5 ml from each tube is administered intravenously to two
mice. The animals are being monitored.
The death of animals can occur within 5-6 hours to 3-4 days. Mice treated with
homologous antiserum toxin remain alive. If the result of the biological test is
negative, the experiment is repeated with the isolated pure culture according to the
same scheme.
Given the rapid development of the clinical symptoms of gas gangrene, it is
necessary to quickly give an approximate conclusion about the type of pathogen (in
order to apply urgent serotherapy). To do this, a smear-imprint is made from the test
material, treated with immunofluorescent species-specific serum and studied by the
immunofluorescent method (see Chapter 2).

Control questions

1. What are the morphological and cultural features and properties of gas gangrene
pathogens?
2. Which of the causative agents of gas gangrene has a capsule and is immobile?
3. Which of the causative agents of gas gangrene has the most pronounced
biochemical properties?
4. What methods are used for laboratory diagnosis of gas gangrene?
5. What method determines the presence of a toxin?
 Chapter 36
Clostridium botulinum (from Latin botulus - sausage) were discovered by Van
Ermengen in 1896. They were isolated from ham, which caused mass poisoning.
Morphology . The causative agents of botulism are rods 4-9 × 0.6-1 µm in size with
rounded ends. The sticks are polymorphic: there are short forms and long
filaments. The causative agents of botulism form spores located subterminally. The
spores are wider than the sticks and therefore the stick with the spore has the
appearance of a tennis racket. C. botulinum does not have capsules. Mobile -
peritrichous. Young cultures stain Gram-positive.
Cultivation . C. botulinum are strict anaerobes. Grow at a temperature of 25-37 ° C
and pH 7.3-7.6. They are cultivated on casein, meat and other media. On blood
glucose agar, microbes produce irregularly shaped colonies with filamentous
processes. In agar, the colonies resemble cotton balls in a column, sometimes the
colonies look like lentil grains. On blood agar in Petri dishes, colonies grow in the
form of dewdrops with a shiny surface and smooth or jagged edges (R-shape). On the
liver broth, clostridia grow with the formation of turbidity and subsequent
precipitation, while the broth is clarified.
Enzymatic properties (see table. 51). Saccharolytic properties: break down lactose,
glucose, maltose and glycerol with the formation of acid and gas. Proteolytic
properties: melt pieces of the liver, break down egg white, liquefy gelatin, peptonize
milk, form hydrogen sulfide and ammonia.
Toxin formation . C. Botulinum produce poison, the most powerful of all biological
toxins (1 microgram of botulinum toxin contains 100,000,000 lethal doses for a white
mouse). The toxin consists of two components: neurotoxin and hemagglutinin.
Antigenic structure . According to the antigenic properties of the neurotoxin, all
strains are divided into seven serovars: A, B, C, D, E, F, and G. Each serovar is
characterized by specific immunogenicity. Serovar A, B, and E toxins are the most
common cause of botulism, while serovar C, D, and F are less common. Serovar G
toxins are poorly understood.
Resistance to environmental factors . Vegetative forms of C. botulinum die at 80°C
after 30 minutes. Spores are persistent. They withstand boiling for several hours (up
to 5 hours). In large pieces of meat, large-capacity cans, spores persist even after
autoclaving. In a 5% phenol solution, spores persist for a day. Botulinum exotoxin
withstands boiling for 10 minutes. It is resistant to sunlight, low temperatures and
disinfectants.
Susceptibility of animals . Small and large cattle, horses, rodents and birds are
sensitive to the causative agents of botulism. Of the experimental animals, white
mice, guinea pigs, rabbits, and cats are sensitive.
Sources of infection . The causative agents of botulism are widespread in nature:
soil, water, where they enter with the feces of animals and fish. C. botulinum live and
reproduce in soil. A person becomes infected through the use of products containing
pathogens and exotoxin.
transmission paths . Food (when eating contaminated meat, vegetables and canned
fish, mushrooms, sturgeons, etc.). Canned foods prepared at home are especially
dangerous.
Pathogenesis . The entrance gate is the mucous membrane of the intestinal
tract. Neurotoxin, which is formed during the reproduction of vegetative forms of
causative agents of botulism, is not sensitive to proteolytic enzymes of the
gastrointestinal tract. The pathological process is caused by a neurotoxin, which is
absorbed into the blood through the intestines, spreads throughout the body,
affecting the central nervous system. Mainly affected: cells (nuclei) of the medulla
oblongata, cardiovascular system. In patients, changes in the organs of vision, a
disorder of respiratory and swallowing functions are noted.
Immunity . There is no natural resistance. Humans are highly sensitive to C.
botulinum toxin. The transferred disease does not leave immunity.
Prevention . Prevention of the possibility of contamination of food products, the
correct production technology for the manufacture of canned food and other
products. Prevention of botulism in everyday life: home canning products should be
boiled in a water bath (or saucepan) for 15-20 minutes before use.
Specific prevention and treatment . People who have consumed products that may
contain the causative agent of botulism or botulinum toxin are injected with anti-
botulinum polyvalent antitoxic serum types A, B, E. After establishing the type of
toxin, anti-botulinum serum of the type that corresponds to the type of isolated
strain is administered.

Microbiological research
The purpose of the study: detection of C. botulinum, botulinum toxin, determination
of the serovar.
Research material
1. Vomit.
2. Gastric lavage.
3. Cal.
4. Blood.
5. Food leftovers.
Material collection methods

Material collection methods

Basic research methods

1. Biological.
2. Bacteriological.
3. The bacterioscopic method is practically not used, because it is impossible to
distinguish clostridine by morphology.
Research progress
First day of research
First day of research
 Rice. 51. Scheme of the neutralization reaction of botulinum toxin in mice. 1 - a
mixture of anti-botulinum sera; 2 - antibotulinum sera A, B, C, E (white mice are
alive; black mice are dead)

Second - fourth days of the study

1. Examine the animals. Disease and death of animals can occur within 1-4 days. The
disease is characterized by the appearance of rapid breathing, relaxation and
retraction of the muscles of the abdominal wall (wasp waist), convulsions, paralysis,
after which the death of the animal occurs. Mice injected with anti-botulinum serum
centrifugate remain alive.
If botulinum toxin is found in the sample, a neutralization reaction is performed with
type-specific diagnostic sera A, B, C, E, F, G (see Fig. 51) (serum D is not produced in
the USSR). A separate syringe is taken for each serum. Mice receiving serum
homologous to the toxin (type) remain alive.
2. Remove the crops from the thermostat. In the presence of suspicious colonies,
they are isolated on the Kitt-Tarozzi medium to obtain a pure culture of the
pathogen and again put the neutralization reaction, as described above.
Fifth - sixth days of the study
The biological properties of the isolated culture are studied: morphology, mobility,
enzymatic properties. In case of a negative result of a biological sample with native
material, it is repeated with an isolated culture according to the same scheme - to
determine the presence and type of botulinum toxin.

Control questions

1. What are the morphology and cultural properties of botulism pathogens?

2. What are their enzymatic properties?
3. What material should be examined if botulism is suspected?
4. What are the main laboratory tests for botulism?
5. How to put a biological sample and neutralization reaction with anti-botulinum
sera?
 Pathogenic spirochetes - F. K. Cherkes
Spirochetes belong to the Spirochaetaceae family. They are thin, convoluted
microorganisms, the body of which consists of an axial thread and a spiral cytoplasm
surrounding it, which gives them a corkscrew shape.
Ultrasections revealed a three-layer outer membrane. In some spirochetes,
filamentous formations are visible at the ends in an electron microscope. They do
not form flagella, capsules, spores. Very mobile. They have four types of movement:
translational, flexion, wave-like and rotational (see chapter 5). Spirochete sizes range
from 5 to 500 microns in length and 0.3-0.75 microns in width.
Spirochetes pathogenic for humans include representatives of the following genera:
1. Treponema - causative agents of syphilis and yaws
2. Borrelia - causative agents of relapsing fever epidemic, endemic and Vincent's
tonsillitis
3. Leptospira - causative agents of leptospirosis.
Morphologically, spirochetes differ from each other in the number and depth of
helical whorls and in relation to coloration. The main staining method according to
Romanovsky is Giemsa. In this way, borrelia are best stained - in a blue-violet color,
worse - treponema - in a pale pink color. Spirochetes are studied in a living state (see
Fig. 4).
Diseases caused by spirochetes are called spirochetosis. Characteristic for the clinic
of spirochetosis is the cyclical course.
 Chapter 37
Treponema pallidum - the causative agent of syphilis is included in the genus
Treponema (from Latin trepo - turn, nemo - thread).
T. pallidum was discovered by F. Shaudin in 1905. I. I. Mechnikov, P. Erlich, D. K.
Zabolotny and others made a great contribution to the study of syphilis.
Morphology . T. pallidum is a spiral thread measuring 8-18 × 0.08-0.2 microns with
small, uniform curls. Number of curls 12-14. The ends of the treponema are pointed
or rounded. Treponemas are mobile. They have four types of movement. According
to Romanovsky - Giemsa are painted in a pale pink color, so they are called T.
pallidum - pale treponema. Poor staining is due to the low content of
nucleoproteins. Spirochetes can be detected in Burri-stained preparations with silver
plating. In addition, they are studied in a living state - in a dark field.
The causative agents of syphilis do not have spores and capsules (see Fig. 4).
Cultivation . Pale treponemas are very demanding on nutrient media. On artificial
nutrient media, they grow only in the presence of pieces of rabbit brain or kidneys
and ascitic fluid. Grow slowly, 5-12 days at a temperature of 35-36 ° C under
anaerobic conditions. Pale treponemas reproduce well in the chick embryo (by
transverse fission). When grown on artificial nutrient media, treponemas lose their
virulence. Such cultures are called cultural. Cultures grown in chick embryos are
called tissue cultures. They usually retain virulence.
Treponema does not have enzymatic properties . However, the culture strains differ
in their ability to form indole and hydrogen sulfide.
Toxin formation . Not installed.
Antigenic structure . Pale treponema contains several antigenic complexes:
polysaccharide, lipid and protein. Serogroups and serovars have not been
established.
Resistance to environmental factors . Pale treponemas are unstable. A temperature
of 45-55 ° C destroys them after 15 minutes. They are resistant to low
temperatures. When frozen, they keep up to a year. Spirochetes are sensitive to salts
of heavy metals (mercury, bismuth, arsenic, etc.). Ordinary concentrations of
disinfectants kill them within minutes. They are sensitive to benzylpenicillin, bicillin,
etc. Under the influence of certain environmental factors and antibacterial drugs,
treponemas can form cysts. In this form, they are in the body for a long time in a
latent state.
Susceptibility of animals . Under natural conditions, animals do not get sick with
syphilis. However, on monkeys, as I. I. Mechnikov and E. Roux showed, it is possible
to reproduce the clinical picture of syphilis: a hard chancre is formed at the injection
site. It has now been shown that when rabbits, guinea pigs are infected, ulcers form
on the skin at the injection site or elsewhere. On rabbits, by passages, it is possible to
preserve the isolated strain of treponema for a long time.
Sources of infection . A sick man.
transmission paths . Household contact (direct contact), predominantly sexual
contact. Sometimes syphilis can be transmitted through objects (dishes,
linens). From a mother with syphilis, the disease is transmitted through the placenta
to the child (congenital syphilis).
Pathogenesis . The entrance gates are the mucous membranes of the genital tract
and oral cavity.
The primary period - spirochetes enter the mucous membrane, and after an
incubation period (an average of 3 weeks), an ulcer is formed at the site of
introduction, which is characterized by dense edges and a bottom - a hard
chancre. The formation of a hard chancre is accompanied by an increase in lymph
nodes. The primary period lasts 6-7 weeks.
The secondary period - the causative agents of syphilis spread throughout the body
through the lymphatic and circulatory pathways. At the same time, roseola, papules,
and vesicles form on the skin and mucous membranes. The duration of this period is
3-4 years.
The third period - develops with untreated syphilis. During this period, granulation
growths are formed in organs, tissues, bones, and vessels - gummas or gummous
infiltrates, prone to decay. This period can last several years (in a latent form). The
patient during this period is not contagious. With untreated syphilis (in some cases),
after many years, damage to the central nervous system can occur: with brain
damage - progressive paralysis, with damage to the spinal cord - dorsal tabes. These
diseases occur when treponema is localized in the brain tissue, which leads to severe
organic and functional changes in the body.
Immunity . There is no natural immunity. With syphilis, "non-sterile" infectious
immunity develops. It is called a chancre, since when re-infected, a hard chancre
does not form, but all subsequent periods develop. In syphilis, IgC and IgM are
detected, as well as IgE reagins, which bind complement in the presence of a
cardiolipid antigen.
Prevention . Sanitary and educational work, early detection of patients with
syphilis. specific prophylaxis. Not developed.
Treatment . Penicillin, bicillin, bioquinol, etc.

Control questions

1. Describe the spirochete morphology and staining methods.

2. What is a hard chancre?
3. What material for research will you take in different periods of syphilis?
4. What is the immunity for syphilis?

Microbiological research

The purpose of the study: detection of pale treponema and serodiagnosis.

Research material
1. The contents of the hard chancre (primary period).
2. Contents of roseola, papules, vesicles (secondary period).
3. Blood (secondary, third and fourth periods).
Material collection methods
 Material collection methods

Basic research methods

1. Microscopic.
2. Immunofluorescence reaction (RIF).
3. Serological: 1) Wasserman reaction (RSK);
2) sedimentary reactions.
4. Treponema immobilization reaction (RIT).
Research progress
 Research progress

Serological diagnostics
Wasserman reaction. The reaction is set according to the principle of the
complement fixation reaction (Table 52). It differs in that a nonspecific antigen can
be used in the Wasserman reaction. For example, a lipoid extract from a bovine
heart is a cardioantigen. Due to the non-specificity of antibodies that react with this
antigen, they are called reagins. The reaction with a nonspecific antigen is explained
by the fact that the content of globulins in the patient's blood serum increases and
the degree of their dispersion changes. Globulins, entering into combination with
lipid extracts, form a complex that binds complement, and therefore hemolysis does
not occur (in the hemolytic system). The absence of hemolysis - a positive reaction -
serologically confirms the diagnosis of syphilis.
 Table 52

Note. 1) ++++ complete hemolysis delay; - hemolysis; 2) non-specific antigen No. 1

(lipoid fraction of a bovine heart); 3) specific antigens No. 2 and 3 prepared from
treponema cultures.
sedimentary reactions . 1. Kahn reaction. The patient's serum is inactivated at 56°C
for 30 minutes. 0.6% cholesterol is added to the antigen (bovine heart lipid extract)
to increase the sensitivity of the reaction (Table 53).
 Table 53

Accounting for the result: the appearance of precipitation is noted as a positive

reaction .
2. The Sachs-Vitebsky reaction (cytocholic sedimentary reaction) is a modification of
the Kahn reaction. The authors used a more concentrated antigen, to which
cholesterol is added, which contributes to a more rapid formation of the precipitate.
Treponema immobilization reaction (RIT) . This is the most specific reaction in the
diagnosis of syphilis.
At present, a method for this reaction has been developed: a suspension of
treponemas is obtained from a crushed testicle of a rabbit infected with T. pallidum
and stored in a special medium that does not inhibit the mobility of treponemas. 1.7
ml of a suspension of tissue treponemas is added to the test tube, 0.2 ml of the test
serum, 0.1 ml of fresh complement are added.
Controls: in the 1st test tube, instead of the tested serum, the serum of a healthy
person is added; in the 2nd - inactivated guinea pig serum is poured. All test tubes
are placed in a desiccator or anaerostat, filled with a mixture of gases (1 volume of
carbon dioxide and 19 volumes of nitrogen) and placed in a thermostat at 35 ° C.
Then the test material is applied to glass and the mobility of treponemas is studied in
a dark field. The principle of the reaction is that the serum of a patient with syphilis
in the presence of complement inhibits the movement of pale
treponema. Determine the percentage of immobilized treponema.
The result is considered positive if immobilized treponems are above 50%; weakly
positive - from 30-50%; negative - below 20%.
Control questions
1. What material is used for laboratory diagnosis of syphilis in different periods of the
disease?
2. What are the methods of laboratory research in the diagnosis of syphilis?
3. What antigens should be used in the Wasserman reaction?
4. What ingredients are needed to perform the treponem immobilization test
(RIT)? What material is taken from the subject, what is determined in it?
 Chapter 38
The causative agents of relapsing fever belong to the family Spirochaetaceae, genus
Borrelia.
According to the nature of the carrier, relapsing fever is divided into epidemic,
transmitted by lice, and endemic, the carriers of which are ticks.
Borrelia of relapsing fever differ from other spirochetes in that they have large,
uneven curls and are well stained with aniline dyes, including Romanovsky-Giemsa
purple (see Fig. 4).

epidemic relapsing fever

The causative agents of epidemic (lousy) relapsing fever were described by O.

Obermayer in 1868.
Morphology . Borrelia Obermeier are a spiral thread with 5-8 unequal curls and
pointed ends. Their dimensions are 10-18 × 0.3-0.5 µm. They have all four types of
movement inherent in spirochetes.
Cultivation . On artificial nutrient media, they grow only in the presence of serum or
ascitic fluid, tissues or organs of animals. They grow slowly, 7-12 days, under
anaerobic conditions at a temperature of 30-35 ° C and a pH of 7.2-7.4. In liquid
media, even with abundant reproduction, there is no visible growth. In recent years,
Borrelia have been cultivated in the chick embryo.
Borrelia do not have enzymatic properties .
Toxin formation . Form endotoxin (little studied).
Antigenic structure . Little studied.
Resistance to environmental factors . Borrelia are very sensitive to high
temperatures: 45-50°C inactivate them after 30 minutes, at 0°C they remain for
several days. They are more resistant to low temperatures. When frozen, they keep
for several months. When dried, they die quickly. Normal concentrations of
disinfectants kill them in a few minutes.
Susceptibility of animals . Under natural conditions, animals do not suffer from
relapsing fever. Under experimental conditions, it is difficult to infect monkeys, white
mice, and hamsters with Obermayer's Borrelia.
Sources of infection . A sick man.
Carriers . Lice that remain infectious throughout life (30-40 days); Borrelia are not
transmitted transovarially to offspring.
mechanism of infection . The louse, having sucked the patient's blood, becomes
contagious in 5-7 days. During this time, spirochetes from the intestines of lice
penetrate into the hemolymph, where they accumulate. When combing, a person
damages the body of the louse and rubs the hemolymph into the place of scratching.
Pathogenesis . In 1874, G. Minkh, and in 1881, I. I. Mechnikov, in experiments on
themselves, proved that the pathogens of relapsing fever circulate in the blood (they
infected themselves with the blood of the patient).
Once in the human body with infected lymph, Borrelia multiply in the cells of the
macrophage system, then enter the bloodstream. The first febrile attack of the
disease occurs. Antibodies accumulate in the patient's blood - spirochetolizins, which
lyse Borrelia. The resulting endotoxin causes intoxication phenomena - temperature
and other functional disorders. But part of the borrelia, located in the depths of the
tissues, are preserved, multiply and give rise to a new generation of borrelia that are
not sensitive to the existing lysines. When they enter the bloodstream, a second
attack of the disease begins. Lysines are formed in the body, dissolving the second
generation of Borrelia. There are several such attacks (3-5). Usually, each subsequent
attack is shorter than the previous one, and the intervals between them (apyrexia)
are longer.
In patients with relapsing fever, the phenomenon of thrombocytobaria is still
observed - in the capillaries of internal organs, platelets are adsorbed on the surface
of spirochetes, an aggregate of "spirochetes and platelets" is formed. This aggregate
disrupts local blood circulation, and Borrelia lose their mobility.
Immunity . It is characterized by the presence of antibodies: spirochetolizins,
agglutinins, thrombocytobarins. However, he is unstable.
Prevention . Fight against pediculosis, improvement of sanitary and hygienic
conditions. Specific prophylaxis has not been developed.
Treatment . Tetracycline, penicillin, levomycetin and arsenic preparations.
Control questions
1. Describe the morphology of relapsing fever borrelia and cultivation methods.
2. Who first showed that the pathogens of relapsing fever circulate in the patient's
blood?
3. What is the mechanism of infection and pathogenesis of relapsing fever?
4. What is immunity caused by and what is the "thrombocytobaria phenomenon"?

Endemic relapsing fever

The causative agents of endemic relapsing fever are several types of Borrelia: B.
persica, B. duttonii and others. B. duttonii were found in the blood of a patient by F.
Ross in 1904.
Morphology . Borrelia of tick-borne relapsing fever are similar to the causative
agents of epidemic relapsing fever.
Cultivation . B. duttonii can be grown on special media. They are grown at a
temperature of 30-35 ° C and a pH of 7.2-7.4 under anaerobic conditions.
enzymatic properties . Not detected.
Antigenic structure . There are several variants of Borrelia that can be differentiated
using the biological method.
Resistance to environmental factors . The same as in pathogens of epidemic
relapsing fever.
Susceptibility of animals . Rodents get sick: mice, hamsters, gerbils. Of the
experimental animals, guinea pigs, rats and white mice are sensitive.
Sources of infection . The source and natural focus are rodents: mice, hamsters,
gerbils and ticks of the genus Ornithodoros. In the body of ticks, Borrelia persist for
life and are transmitted transovarially.
transmission paths . Transmissible. A person becomes infected by the bite of a tick,
in the saliva of which there are Borrelia. A papule forms at the site of the bite.
Pathogenesis . Similar to the pathogenesis of epidemic relapsing fever, but the
number of attacks is greater. Clinically, the disease is milder.
Immunity . In endemic foci, immunity is acquired in early childhood and is
determined by the presence of spirochetolizins and other antibodies. Mostly visitors
are sick. After the illness, immunity is unstable. There is no cross-immunity with
epidemic relapsing fever.
Prevention . Destruction of rodents and insects. Each natural focus is characterized
by its own type of pathogen. Specific prophylaxis has not been developed.
Treatment . Antibiotics: tetracycline, penicillin, chloramphenicol, etc.

Microbiological research

The purpose of the study: identification of the pathogen and differentiation of

pathogens of epidemic relapsing fever from endemic.
Research material
The blood of the sick
Material collection methods

Material collection methods

Basic research methods

1. Microscopic.
2. Immobilization of Borrelia.
3. "Loading" reaction of Borrelia with platelets (rarely used).
4. Fluorescence microscopy (see chapter 3).
Research progress
 Research progress

Control questions

1. What material is used to diagnose epidemic and endemic relapsing fever?

2. List the research methods and which one is the main one.
3. How to differentiate epidemic relapsing fever from endemic?

Exercise

Draw the causative agents of relapsing fever, stained according to the Romanovsky-
Giemsa method.
 Chapter 39
Borrelia vincentii (Vincent's Borrelia) cause ulcerative tonsillitis, ulcerative stomatitis
and other ulcerative necrotic processes. In smears from ulcers stained by Gram or
diluted Pfeiffer fuchsin, a spirochete is found in combination with a gram-negative
fusiform bacillus B. fusiformis (therefore, ulcerative angina is called
fusospirochetosis).
Morphologically, Vincent's spirochetes are indistinguishable from oral cavity
saprophytes.
B. fusiformis is characterized by uneven staining of the cytoplasm. In the center, the
color is paler, therefore, under microscopy, the impression of a double stick is
created.
It is believed that infection with ulcerative angina occurs through dirty dishes or
other objects that fall into the patient's saliva.
Diagnostics . Microscopy of stained preparations made from ulcer discharge. The
presence of Borrelia in combination with the bacterium Fusiformis is a diagnostic
sign.
 Chapter 40
Pathogenic leptospira are included in the family Spirochaetaceae, genus
Leptospira. Discovered in 1915 by Japanese explorers Inado and Ido.
The genus Leptospira is a group of microorganisms that cause diseases in animals
and humans.
Morphology . Leptospira - spiral threads, 6-20 × 0.1-0.25 microns long. They have
numerous (12-18) small curls closely adjacent to each other. The ends of the spirals
are bent in the form of hooks. There are also hookless strains. Leptospira are very
mobile. They have all forms of movement.
They are poorly stained with aniline dyes, therefore, in order to detect them in
pathological material and in cultures, the method of microscopy of living leptospira
in a dark field or the silvering method is used - with this method they are stained
brown.
Cultivation . Leptospira are strict aerobes. They reproduce from liquid and semi-
liquid nutrient media containing rabbit serum at a temperature of 28-30 ° C and a
medium pH of 7.2-7.4. Grow slowly (7-10 days). Environments during reproduction of
leptospira remain transparent. Growth is determined by opalescence or by
microscopy of preparations in a crushed drop (in a dark field). Leptospira can also be
grown on dense nutrient media containing rabbit serum. Growth appears on the 5-
7th day. When growing leptospira on artificial nutrient media, they lose their
virulence.
enzymatic properties . Enzymes were found in leptospira: catalase, oxidase, lipase,
etc.
Toxin formation . The presence of endotoxin in leptospira has not been proven.
Antigenic structure . The antigenic structure of Leptospira is studied in a
microagglutination reaction with monoreceptor sera. Based on this reaction,
leptospira are divided into 19 serogroups and 169 serovars. Each serogroup and
serovar has a name.
Resistance to environmental factors . Leptospira are sensitive to high temperatures:
56 ° C kills them after 30 minutes. At low temperatures (- 70-80 ° C) they are stored
for a long time. Leptospira are very sensitive to desiccation. They are sensitive to the
action of acids, bile. Normal concentrations of disinfectants kill them in a few
minutes.
Leptospira survive in water for up to two months, in dairy products and bread for
several hours.
Susceptibility of animals . Rodents are most susceptible to leptospira, but
artiodactyls, large and small cattle, pigs, dogs and even predatory animals are sick. In
animals, the disease usually occurs in a chronic form.
Of the experimental animals, guinea pigs and golden hamsters are the most sensitive
to leptospira. However, only some Leptospira serovars can cause disease in them. In
rodents, the disease also occurs in a chronic form and leptospira are excreted in the
urine.
Sources of infection . Sources of infection are sick animals - agricultural and rodents
(mainly rats). They infect the surrounding soil, open water bodies (rivers, ponds,
wells) with urine. In addition, they contaminate food.
transmission paths . Contact-household (when bathing and performing various
works in water contaminated with leptospira of sick animals; when caring for sick
animals), food (through water and food products).
Pathogenesis . The entry gates are damaged skin and intact mucous membranes of
the mouth, eyes and gastrointestinal tract. The primary affect on the skin and
mucous membranes is not formed. Leptospirosis occurs in icteric and anicteric
form. Having entered the body, leptospira through the lymphatic pathways
penetrate into the blood, where they circulate. With the blood flow, they penetrate
into the parenchymal organs and are localized in the liver and kidneys. By this time,
antibodies are formed that destroy leptospira.
In the process of disintegration of leptospira, a toxic substance is released, which
leads to intoxication and hemorrhages in parenchymal organs. In severe cases,
jaundice and acute renal failure (nephritis) develop in humans and in animals
susceptible to leptospira.
In mild forms of the course, lesions of parenchymal organs are detected only with
special research methods.
Immunity . Associated with the formation of agglutinins and spirochetolizins, the
maximum concentration of which is reached in 3-4 weeks (antibodies are in high
titers of 1:1000 and above). Immunity is maintained for a long time.
Prevention . Rodent control, swamp drainage and other land reclamation activities,
as well as food protection from rats and mice. Farm animals are vaccinated.
specific prophylaxis . Vaccination of people working in the outbreak. Leptospirosis
vaccine is a suspension of heat-killed leptospira several serovars.
Treatment . Penicillin, tetracycline, antileptospiral immunoglobulin (prepared from
the most common Leptospira serogroups).

Control questions

1. Tell us about the morphological and cultural properties of leptospira.

2. What is the stability of leptospira in the external environment?
3. Pathogenicity for animals, sources of infection and routes of transmission.
4. Name the entrance gate. What is the pathogenesis of leptospirosis?
5. Immunity. Due to what antibodies does it form?

Microbiological research

The purpose of the study: the identification of leptospira in patients and their
detection in the external environment. Serovar definition.
Research material
1. Blood.
2. Urine.
3. Cerebrospinal fluid.
4. Water sources and foodstuffs.
5. Sectional material.
Material collection methods
 Material collection methods

Basic research methods

1. Microscopic.
2. Bacteriological (with microscopy).
3. Serological.
4. Biological.
Research progress
 Research progress

Control questions
1. What is the material for research in case of suspected leptospirosis?
2. List the main research methods.
3. Tell us how a biological sample is placed.
Nutrient media
Wednesday Tersky . A phosphate mixture is prepared: 1) disubstituted sodium
phosphate - 11876 g; 2) distilled water - 1 l; 3) monosubstituted potassium
phosphate - 9078 g (prepared solutions are stored in a dark place). The mixture is
sterilized and 10% heated rabbit serum is added. After sowing, the medium is
covered with a layer of liquid paraffin or vaseline oil.
Fairworth-Wolff medium . For 1 liter of medium: 1) peptone - 1 g; 2) sodium chloride
- 0.5 g; Zermsen buffer mixture - 100 ml; 4) distilled water - 900 ml; 5) 10%
inactivated rabbit serum.
 Rickettsia - F.K. Cherkes
Rickettsiae are a special group of polymorphic bacteria that are intracellular
parasites. They are included in the Rickettsiaceae family.
The first representative of this group was identified in 1909 by the American scientist
Ricketts while studying the etiology of Rocky Mountain fever. He died from this
disease.
In 1913, the Czech scientist Provacek also found similar microorganisms in the blood
of patients with typhus. Infected with typhus, the scientist also died.
And in 1916, the Portuguese scientist Rocha-Lima, on the basis of long-term
observations, established that the causative agents of Mexican typhus, European
typhus and other similar diseases are varieties of microorganisms discovered by
Ricketts, and named them rickettsia in honor of the discoverer. And in memory of
Provacek, the causative agent of typhus was named Provacek's rickettsiae.
A. A. Krontovsky, M. A. Krontovskaya, P. F. Zdrodovsky, E. S. Galinevich and others
made a great contribution to the study of typhus.
Among the rickettsia there are varieties that are pathogenic for humans, animals and
arthropods.
In humans, rickettsia causes various febrile diseases, which are called rickettsiosis.
According to P.F. Zdrodovsky, human rickettsiosis is divided into 5 groups: a group of
typhus, a group of tick-borne spotted fevers, a group of tsutsugamushi, a group of Q
fever, a group of paroxysmal rickettsiosis.
The classification of rickettsiosis is based on a number of features: the properties of
the pathogen, epidemiology and clinic of the disease.
On the territory of the USSR, some of the rickettsiosis are found: epidemic typhus
(European), Brill's disease, endemic typhus, Q fever.
Morphology . Rickettsiae are small (0.2-1 microns), polymorphic microorganisms,
among which there are rod-shaped, cocci-shaped and filamentous (10-30 microns
long) representatives.
Rickettsia do not have spores, capsules, are immobile. Gram-negative. According to
Romanovsky - Giemsa and according to the Zdrodovsky method, they are painted
red. The structure of the cell wall is similar to the structure of the wall of gram-
negative bacteria.
Cultivation . Rickettsia reproduce inside the host cell. Aerobes. They have their own
metabolism, behave independently in the cell. However, they are energetically
dependent parasites on the cell.
In the host cell, each type of rickettsia multiplies only in certain places: in the
cytoplasm, nucleus or vacuoles of cells. They multiply well in the tissues of sensitive
animals and arthropods. In laboratory practice, the method of infection of chicken
embryos and tissue culture is most often used.
enzymatic properties . Not expressed.
Toxin formation . Rickettsia produce heat-labile endotoxin.
Antigenic structure . Rickettsia have two antigens: group thermostable and specific
thermolabile.
Resistance to environmental factors . Rickettsia are not very resistant to high
temperatures. Exceptions are the causative agents of Q fever. All rickettsia are
resistant to low temperatures and drying. They are sensitive to
antibiotics. Sulfamides do not inhibit their growth.
 Chapter 41

Typhus

Typhus is caused by Rickettsia provazekii.

Long before the discovery of the causative agent of typhus, the Russian doctor O. O.
Mochutkovsky, in experiments on himself, showed that the causative agent of this
disease circulates in the patient's blood.
Morphology . The causative agents of epidemic typhus - Rickettsia Provacek - are
polymorphic. More often they have the form of cocci or dumbbells, there are
filamentous forms. Average sizes from 0.8-2.0 × 0.3-0.6 µm. When stained according
to the Zdrodovsky method, they become red.
Cultivation . They multiply in the cytoplasm of host cells, louse intestinal epithelium,
and vascular endothelium. More often they are cultivated in the yolk sac of a chicken
embryo. A cloudy plaque forms at the breeding site on the 8-13th day. The optimum
temperature for their development is 35°C.
Toxin formation . Rickettsia Provacec produce endotoxin. It is not received in its pure
form. However, its sensitivity to temperature (when heated, it quickly collapses)
gives the right to assume that it is of protein origin. Toxin, when rickettsia penetrates
into the body, affects the cells of the vascular endothelium, which leads to an
increase in capillary permeability.
Antigenic structure . Rickettsia Provacec contain two antigens. One of them is
superficial, thermolabile. According to its chemical composition, it is a lipid
polysaccharido-protein complex. This antigen is non-species-specific and is common
with the antigens of the causative agents of endemic typhus, as well as with the
antigens of Proteus OX 19 , OX 2 . The second - protein-polysaccharide complex is
species-specific and is located deep in the cell.
E. Weil and A. Felix discovered the ability of Proteus OX 19 to give a positive
agglutination test with the serum of patients with typhus. This reaction, named after
scientists, was widely used for diagnostic purposes. The authors believed that
Proteus OX 19 was also the causative agent of typhus. However, with the
accumulation of material and evidence of the etiological role of rickettsia in typhus, it
was established: 1) Proteus OX 19 is not the causative agent of typhus; 2) Proteus OX
19 gives positive agglutination tests, with sera of patients with typhus, because it has
an antigen common with Provacec's rickettsiae; 3) the Weil-Felix reaction is not
always specific, and it was no longer used in the diagnosis of typhus, replacing the
antigen in the agglutination reaction with a diagnosticum from Rickettsia Provacek.
Resistance to environmental factors . At high temperatures, especially in a humid
environment, Prowaceca rickettsia die quickly.
In the dried feces of lice, rickettsia persist for a long time. Ordinary disinfectant
solutions destroy them quickly.
Susceptibility of animals . Under experimental conditions, white mice, guinea pigs
and monkeys can be infected. The clinical picture of typhus is reproduced on
monkeys. Infected white mice develop specific pneumonia.
Sources of infection . A sick man.
transmission paths . Transmissible. In 1909, the French scientist Nicole, in
experiments on monkeys, established that Prowacek rickettsiae are transmitted by
body lice. It was further shown that head lice can also be vectors.
mechanism of infection . Having sucked the patient's blood, the louse becomes
infectious on the 4-5th day. During this time, rickettsia multiply in the intestinal
epithelial cells of the louse. Having accumulated there, they destroy epithelial cells,
enter the intestinal lumen and are excreted in large quantities with the feces of the
louse. Once on the skin of a healthy person, the louse bites him and immediately
excretes rickettsia with feces. A person combs the bite site and rubs rickettsia into
the wound. So the pathogens are in the internal environment of the human body.
Pathogenesis . Once in the human body, rickettsiae are introduced into the cells of
the vascular endothelium. They multiply, destroy cells, enter the blood in large
quantities - rickettsiaemia occurs. The process in the vessels is characterized by
inflammation and the formation of blood clots, which leads to blockage of small
blood vessels. Around the thrombosed vessels of the brain, the formation of
granulomas occurs - inflammation of the type of meningoencephalitis.
Typhoid fever begins acutely, there is a high temperature, general symptoms of
intoxication, severe headache, and a roseolous-petechial rash appears.
Immunity . Antimicrobial and antitoxic. After the transferred disease -
lifelong. Antibodies that neutralize the toxin, agglutinins, complement-fixing
antibodies, etc. are found in the blood.
Diseases of typhus were often associated with national disasters (war, famine, etc.)
and lice.

Brill disease

In recent years, a number of data have been accumulated on the long-term

preservation of Provacec's rickettsiae in the body of a person who has had
typhus. With a decrease in the body's resistance, they can cause a recurrence of the
disease many years later (10-30 years), i.e., this is an endogenous relapse of
epidemic typhus. For the first time this disease was described by N. Brill, and N.
Zinsser proved that its causative agent is Rickettsia Provacek. Brill's disease is
characterized by a mild, benign course. A diagnostic feature in this disease is a
negative agglutination reaction with Proteus OH 19 (Weil-Felix) and positive
agglutination with Provacek's rickettsiae. There is another opinion that Brill's disease
is a reinfection, i.e., re-infection, and a mild course is due to the presence of
immunity acquired as a result of a previous illness.
Prevention . Isolation of patients and extermination of lice. specific
prophylaxis . Currently, a chemical vaccine has been developed, prepared from a
concentrated surface antigen of Rickettsia Provacec.

Control questions

1. Give a description of rickettsiae and methods of their cultivation.

2. List the causative agents of the main rickettsiosis.
3. Tell us about the source of the disease, the ways of transmission and the
mechanism of infection of epidemic typhus.
4. What is Brill's disease?

endemic flea typhus

The causative agents of endemic typhus were discovered by H. Muser in 1928 and
were named Muser's rickettsiae in his honor. Now they are called R. typhi
Morphology . Small coccoid (within 1 µm in diameter) or rod-shaped (0.3-0.6 × 1.5
µm) microorganisms. They are less polymorphic than Rickettsia
Prowaceca. According to the Zdrodovsky method, they are painted red. Gram-
negative.
Cultivation . Rickettsia Musera reproduce well in the yolk sac of a chicken embryo at
a temperature of 35 ° C. Growth is characterized by the formation of plaques. In
arthropods, they multiply in the nucleus and cytoplasm of intestinal epithelial cells.
Toxin formation . Rickettsia Musera produce an endotoxin that differs from
Rickettsia Provacec toxin, which can be detected by a neutralization reaction.
Antigenic structure . Rickettsia Musera have two antigenic complexes. One -
thermostable - common with the Provacec rickettsia antigen and antigens against
OX 19 and OX 2 . The second one is thermolabile, species-specific, which makes it
possible to differentiate Muser's rickettsiae from Prowacek's rickettsiae.
Resistance to environmental factors . Rickettsia Musera is not very stable in the
external environment, but in the dried state and at low temperatures they persist for
a long time. Ordinary concentrations of disinfectant solutions destroy them quickly.
Susceptibility of animals . Endemic typhus affects rodents, mainly mice and rats. Of
the experimental animals, guinea pigs are sensitive, with intraperitoneal infection
they develop periorchitis (scrotal phenomenon).
Sources of infection . Endemic typhus is a zoonotic infection. The main sources in
nature are rats and mice.
transmission paths . Transmissible, food, contact-household, Carriers, can be rat
fleas and ticks (ticks transmit rickettsia transovarially).
Pathogenesis . Endemic typhus is a blood infection. The pathogenesis is similar to
that of typhus. Clinically it is easier. The disease is characterized by fever and
rash. The disease is endemic.
Immunity . After the disease, persistent immunity develops due to antimicrobial and
antitoxic protective factors.
Prevention . Destruction of insects, rodents and improvement of sanitary and
hygienic conditions. Specific prophylaxis is carried out by immunization with a
vaccine containing killed Muser's rickettsiae. Vaccinate people living in endemic foci
and at risk of infection.
Treatment . Tetracycline antibiotics.
Control questions
1. What sources of infection and ways of transmission of endemic typhus do you
know?

Microbiological research

The purpose of the study: detection of antibodies to the pathogen and

differentiation of typhus from endemic (and other rickettsiosis).
Research material
Blood
Material collection methods

Material collection methods

Basic research methods

1. Serological methods:
a) complement fixation reaction (RCC);
b) agglutination reaction;
c) reaction of indirect hemagglutination (RIHA);
d) toxin neutralization reaction;
e) immunoluminescent method.
2. Biological sample.
Research progress
 Research progress

Control questions
1. What serological methods are used to differentiate epidemic typhus from
endemic?
2. What serological reactions are used to differentiate epidemic typhus from Brill's
disease?
 Chapter 42
Q fever is an acute infectious disease, first described in the 30s of the XIX century in
Australia (from the English query - unclear).
In 1939, the causative agent of this fever was isolated from the patient's blood,
identified by F. Burnet, and named Burnet's rickettsiae in his honor. In the USSR, Q
fever has been registered since 1948.
The causative agent of Q fever belongs to the genus Coxiella.
Morphology . C. burnetti are small, polymorphic microorganisms, lanceolate, rod-
shaped, 0.3-0.8 µm long, spherical, 0.3-0.5 µm in diameter. Gram-
negative. According to the Zdrodovsky method, they are painted red.
Cultivation . Burnet's rickettsiae reproduce well in the yolk sac of the chick
embryo. The optimum temperature for their reproduction is 35 ° C. Inside the host
cells, rickettsia multiply mainly in vacuoles. Growth is detected by the appearance of
plaques.
enzymatic properties . Not expressed.
Toxin formation . The toxin has not been identified, but rickettsia contain an allergen
that can sensitize the body with the formation of granulomas.
Antigenic structure . Burnet's rickettsia have two antigens: phases I and II. The phase
I antigen is surface and is a polysaccharide. The phase II antigen is located inside the
cell. Its chemical nature has not been elucidated. With prolonged cultivation in a
chicken embryo, Coxiella burnetti loses the ability to form I antigen. This ability is
restored after passages through the body of the guinea pig.
Resistance to environmental factors . Burnet's rickettsiae are quite resistant. They
withstand temperatures of 80-90°C for 30 minutes. Pasteurizing milk does not
destroy them. They are stored for a long time in dairy products: cottage cheese,
butter, kefir and withstand the action of UV rays for 1.5 hours. At low temperatures,
especially in ice conditions, they are stored for several months. In sterile water for 3-
4 months. Rickettsia Burnet resistant to the action of gastric juice, 5% formalin
solution and 1% phenol solution.
Susceptibility of animals . Under natural conditions, Burnet's rickettsiae are found in
cows, sheep, mules, dogs, horses, rodents, birds, and ticks. In animals, the disease is
characterized by fever. The disease occurs in them more often in a chronic
form. Rickettsiae are excreted in milk, urine, feces.
Of the experimental animals are sensitive: white mice, guinea pigs, rabbits.
Sources of infection . There are often pets.
Ways of transmission of Coxiella burnetii are diverse: 1. Air-dust (treatment of wool
of infected animals causes specific pneumonia).
2. Food (use of food products contaminated with secretions of sick animals or feces
of infected insects).
3. Transmissible (bite of ticks infected with Burnet's rickettsiae. Ticks transmit
rickettsiae to offspring transovarially).
Pathogenesis . Once in the body, rickettsiae penetrate into the blood and lymph -
rickettsiaemia occurs, then - into the cells of organs and tissues. In the human body,
rickettsia undergo phagocytosis, but they are not lysed in the phagocyte (incomplete
phagocytosis). The clinical picture depends on the mechanism of infection. There are
the following forms: pneumonic, influenzal and meningoencephalitic. Each of them is
manifested by a number of features.
Immunity . Strong and long-lasting due to the presence of complement-fixing
antibodies, agglutinins, etc.
Prevention . Destruction of rodents, insects. Pet supervision. The milk is
boiled. Animal secretions are rendered harmless. Patients are hospitalized. specific
prophylaxis. In places with high incidence, people are immunized with a vaccine
prepared from live Burnet's recketsia strain M-44 (the vaccine is effective, but
reactive).
Treatment . Tetracycline antibiotics.

Control questions

1. What is the morphology and cultural properties of Burnet's rickettsia?

2. What is the stability of Burnet's rickettsia in the external environment?
3. What are the sources and routes of transmission of Burnet's rickettsia?

Microbiological research
The purpose of the study: detection of antibodies to the pathogen, isolation and
identification of the pathogen.
Research material
Blood
Material collection methods

Material collection methods

Basic research methods

1. Serological.
2. Biological.
Research progress
 Research progress

Control questions
1. List the diagnostic methods for suspected Q fever.
2. What is the purpose of re-setting RSK?
 Viruses
Viral diseases arose in ancient times, but virology as a science began to develop
at the end of the 19th century.

In 1892, the Russian botanist D. I. Ivanovsky, studying the mosaic disease of

tobacco leaves, found that this disease is caused by the smallest microorganisms
that pass through finely porous bacterial filters. These microorganisms are called
filterable viruses (from Latin virus - poison). Later it was shown that there are other
microorganisms that pass through bacterial filters, so the filterable viruses began to
be called simply viruses.

The question of the origin of viruses is the subject of much research and
discussion. Some scientists suggest that viruses are descendants of non-cellular
forms of living parasitic microorganisms. Others believe that viruses arose as a
result of the regressive evolution of single-celled microorganisms. Still others think
that viruses originated from cellular elements that became autonomous systems.

A great contribution to the study of viruses was made by Soviet virologists: M.

A. Morozov, N. F. Gamaleya, L. A. Zilber, M. P. Chumakov, A. A. Smorodintsev,
V. M. Zhdanov and others.

Viruses are a non-cellular form of the existence of living matter. They are very
small. According to the figurative expression of V. M. Zhdanov, "their size in
relation to the size of medium bacteria can be compared with the size of a mouse in
relation to an elephant." It became possible to see viruses only after the invention
of the electron microscope.

Currently, many methods are used to study viruses: chemical, physical,

molecular biological, immunobiological, and genetic.

All viruses are divided into those affecting humans, animals, insects, bacteria
and plants.

Viruses have a wide variety of forms and biological properties, but they all have
common structural features. Mature particles of viruses are called virions.

Unlike other microorganisms that contain both DNA and RNA, the virion
contains only one of the nucleic acids - either DNA or RNA.

The nucleic acid of viruses can be single-stranded or double-stranded. Almost all

viruses containing RNA have single-stranded RNA in their genome, and those
containing DNA have double-stranded DNA. In accordance with the two types of
genetic substance, viruses are divided into RNA- and DNA-containing. DNA-
containing ones include 5 families, RNA-containing ones - 10 families.

Virus classification *
*
 ( These data are for only some of the human pathogenic viruses. )

Virus classification

The structure of the virion . In the center of the virion is a nucleic acid, which
is surrounded by a capsid (from the Greek kanca - a box). The capsid is made up of
protein subunits called capsomeres. The mature virus is chemically a
nucleocapsid. The number of capsomeres and the way they are stacked (Fig. 52)
are strictly constant for each type of virus. For example, the polio virus has 32
capsomeres, while the adenovirus has 252 capsomeres. Capsomeres can be stacked
in the form of a polyhedron with uniform symmetrical faces - a cuboidal shape (for
example, adenovirus). Spiral (spherical) fold is characteristic of influenza
viruses. There may be a type of symmetry in which the nucleic acid has the
appearance of a spring around which capsomeres are stacked, in which case the
virus is rod-shaped - the virus that causes tobacco leaf disease.
 Rice. 52. Schematic representation of the location of capsomeres in the capsid of
viruses. a - influenza virus; b - adenovirus; c - herpes virus; d - polio virus

The phage has a complex type of symmetry: the head is cuboidal, and the
process is rod-shaped (spermoid shape) (see Fig. 21, 22).

Thus, depending on the way of laying, viruses are divided into cuboidal,
spherical, rod-shaped and spermatozoal forms.

Some viruses, which have a more complex structure, have a shell called
peplos. It is formed when the virus leaves the host cell. In this case, the viral capsid
is enveloped by the inner surface of the cytoplasmic membrane of the host cell and
one or more layers of the supercapsid membrane are formed. Only some viruses
have such a shell, for example, rabies, herpes, and encephalitis viruses. This shell
contains phospholipids that are destroyed under the influence of ether. Thus, by
exposure to ether, it is possible to distinguish a virus having a peplos from a virus
with a "naked capsid".

In some viruses, capsomeres in the form of spikes protrude from the outer lipid
layer of the envelope (these spikes are blunt). Such viruses are called peplomers
(for example, influenza virus, see Fig. 52).

The nucleic acid of the virus is the carrier of hereditary properties, and the
capsid and the outer shell have protective functions, as if protecting the nucleic
acid. In addition, they contribute to the penetration of the virus into the cell.

Virus sizes . Viruses are measured in nanometers. Their value fluctuates in a

wide range from 15-20 to 350-400 nm.

Methods for measuring viruses : 1) filtration through bacterial filters with a

known pore size; 2) ultracentrifugation - large viruses settle faster; 3)
photographing viruses in an electron microscope.

The chemical composition of viruses . The amount and content of DNA and

RNA viruses are not the same. For DNA, the molecular weight ranges from 1
10 6 to 1.6 10 8 , and for RNA it ranges from 2 10 6 to 9.0 10 6 .

Proteins in virions are found in a small number, they consist of 16-20 amino
acids. In addition to the capsid proteins, there are also internal proteins associated
with the nucleic acid. Proteins determine the antigenic properties of viruses, and
also, due to the dense packing of polypeptide chains, protect the virus from the
action of host cell enzymes.

Lipids and carbohydrates are found in the outer shell of complex virions. The
source of lipids and carbohydrates is the host cell envelope. Polysaccharides, which
are part of some viruses, determine their ability to cause erythrocyte agglutination.

Virus enzymes . Viruses do not have their own metabolism, so they do not need
metabolic enzymes. However, some viruses revealed the presence of enzymes that
facilitate their penetration into the host cell. For example, neuraminidase was found
in the influenza A virus, which cleaves off neuraminic acid contained in the
membranes of animal cells (erythrocytes, etc.). In phages - lysozyme, which
destroys the cell membrane, phosphatase, etc.

Detection of viral antigens . Viral antigens in infected host cells can be

detected using the immunofluorescence method. Preparations containing cells
infected with viruses are treated with specific immune luminescent sera. When
viewed under a fluorescent microscope, a characteristic glow is observed in the
places of accumulation of viral particles. The type of virus is determined by the
correspondence of the specific luminescent serum that caused the glow.

The introduction of the virus into the cell, its interaction with the host cell
and reproduction (reproduction) are composed of a number of successive stages.

Stage 1. Begins with the adsorption process at the expense of virion and cell
receptors. In complex virions, receptors are located on the surface of the envelope
in the form of styloid outgrowths (influenza virus), in simple virions, on the surface
of the capsid.

Stage 2. Penetration of the virus into the host cell proceeds differently for
different viruses. For example, some phages pierce the shell with their offshoot and
inject the nucleic acid into the host cell (see Chapter 8). Other viruses enter the cell
by drawing in the viral particle with the help of a vacuole, i.e., at the site of
introduction, a recess is formed in the cell membrane, then its edges close and the
virus enters the cell. This retraction is called viropexis.

Stage 3. "Undressing the virus" (disintegration). For its reproduction, the viral

nucleic acid is released from the protein coats protecting it (envelope and
capsid). The undressing process may begin during adsorption, or it may occur when
the virus is already inside the cell.

Stage 4. At this stage, the replication (reproduction) of nucleic acids and the
synthesis of viral proteins occur. This stage occurs with the participation of DNA
or RNA of the host cell.

Stage 5. Assembly of the virion. This process is provided by the self-assembly

of protein particles around the viral nucleic acid. Protein synthesis may start
immediately after viral nucleic acid synthesis, or after an interval of several
minutes or several hours. Some viruses self-assemble in the cytoplasm. Others have
host cells in the nucleus. The formation of the outer shell (peplos) always occurs in
the cytoplasm.

Stage 6. The release of the virion from the host cell occurs by leakage of the
virus through the cell membrane or through the hole formed in the host cell (in this
case, the host cell dies).

Types of interaction between virus and cell . The first type - a productive

infection - is characterized by the formation of new virions in the host cell.
 The second type - abortive infection - is that the replication of the nucleic acid is
interrupted.

The third type is characterized by the incorporation of a viral nucleic acid into
the DNA of the host cell; there is a form of coexistence of the virus and the host
cell (virogeny). In this case, synchronous replication of viral and cellular DNA is
ensured. In phages, this is called lysogeny.

microscopic examination. In certain viral infections, specific intracellular

bodies are observed in the cytoplasm or nuclei of the cells of the host organism -
inclusions that are of diagnostic value (Babes-Negri bodies in rabies, Guarnieri
bodies in smallpox, etc.). The sizes of viral particles and inclusion bodies can be
artificially increased by special methods of processing preparations with mordant
and impregnation (for example, the Morozov silvering method) and observed with
immersion microscopy. Smaller virions, lying beyond the visibility of an optical
microscope, are detected only with electron microscopy. There are different points
of view regarding intracellular inclusions. Some authors believe that they are an
accumulation of viruses. Others believe that they arise as a result of the cell's
reaction to the introduction of viruses.

Virus Genetics . Modification (non-inherited changes) in viruses is determined

by the characteristics of the host cell in which the virus reproduces. Modified
viruses acquire the ability to infect cells similar to those in which they were
modified. Different viruses show modification in different ways. For example, the
shape of "negative spots" (phage colonies) changes in phages.

Mutation - in viruses occurs under the influence of the same mutagens that cause
mutation in bacteria (physical and chemical factors). A mutation occurs during the
replication of nucleic acids. Mutations affect various properties of viruses, such as
sensitivity to temperature, etc.

Genetic recombination in viruses can occur as a result of the simultaneous

infection of a host cell with two viruses, while the exchange of individual genes
between the two viruses can occur and recombinants are formed containing the
genes of two parents.

Genetic reactivation of genes sometimes occurs when an inactivated virus is

crossed with a full-fledged virus, which leads to the rescue of the inactivated virus.

Spontaneous and directed genetics of viruses is of great importance in the

development of the infectious process.
 Resistance to environmental factors . Most viruses are inactivated by high
temperatures. However, there are exceptions, for example, the hepatitis virus is
heat-resistant.

Viruses are not sensitive to low temperatures; ultraviolet rays of the sun have an
inactivating effect on viruses. Scattered sunlight acts on them less actively. Viruses
are resistant to glycerin, which makes it possible to keep them in glycerol for a long
time. They are resistant to antibiotics (when cultivating viruses, the test material is
treated with antibiotics to suppress the bacterial flora).

Acids, alkalis, disinfectants inactivate viruses. However, some formalin-

inactivated viruses retain their immunogenic properties, which makes it possible to
use formalin to produce vaccines (rabies vaccine).

Susceptibility of animals . The range of susceptible animals for some viruses is

very wide, for example, many animals are susceptible to rabies viruses. Some
viruses infect only one type of animal, for example, the canine distemper virus only
infects dogs. There are viruses to which animals are not sensitive - for example, the
measles virus, etc.

Organotropism of viruses . Viruses have the ability to infect certain organs,

tissues and systems. For example, the rabies virus infects the nervous system. The
smallpox virus is dermotropic, etc.

Isolation of viruses in the environment . From a sick body, viruses can be

excreted with feces, for example, the polio virus and other enteroviruses. The
rabies virus is excreted with saliva, the influenza virus - with the discharge of the
nasopharyngeal mucosa, etc.

The main ways of transmission of viruses . Airborne (flu, smallpox), food

(poliomyelitis, hepatitis A), contact household (rabies), transmissible
(encephalitis).

Antiviral immunity . The human body has an innate resistance to certain

viruses. For example, a person is not susceptible to the canine distemper
virus. Animals are not susceptible to the measles virus. In these cases, antiviral
immunity is based on the absence of cells capable of supporting the reproduction of
viruses.

Antiviral immunity is determined by both cellular and humoral protective

factors, nonspecific and specific. nonspecific factors. A powerful inhibitor of the
reproduction of viruses is a protein substance - interferon. In a healthy body, it is
contained in a small amount, and viruses contribute to the production of interferon
and its amount increases significantly. It is nonspecific, as it blocks the
reproduction of various viruses. However, it has tissue specificity, i.e., cells of
different tissues form unequal interferon. It is believed that its mechanism of action
is that it prevents protein synthesis in the host cell and thereby stops the
reproduction of the virus.

Specific factors of antiviral immunity include virus-neutralizing antibodies,

hemagglutinating and precipitating.

Methods for culturing viruses . Viruses reproduce only in viable cells. They

are cultivated: in chicken embryos (Fig. 53), tissue cultures of humans and various
animals, in the body of sensitive animals, susceptible arthropods.

Rice. 53, Chicken embryo. 1 - chorion-allantois: 2 - allantoic cavity; 3 - amniotic

cavity; 4 - yolk sac; 5 - air bag; 6 - shell membrane

In the first period of the development of virology, the main method for studying
viruses was the artificial infection of animals, but this method is complex, and
besides this, animals turned out to be immune to many viruses.

Of great importance in the development of virology was the introduction of

methods for cultivating viruses in chicken embryos and in cell culture of human
and animal tissues.

Infection of chicken embryos . For the reproduction of viruses, chicken

embryos of 7-12 days of age are used, incubated in a thermostat at 37 ° C. A
necessary condition for the proper development of the embryo is the observance of
a certain air humidity, which can be created by placing a vessel with water in a
thermostat.

The suitability of a chicken embryo for infection is determined by the presence

of embryonic movements and a developed network of blood vessels on the chorion-
allantoic membrane when translucent with an ovoscope.

The cultivation of viruses in chicken embryos is carried out in different places of

the embryo, which is infected (see Fig. 53):

1) on the chorion-allantoic membrane,

2) into the allantoic cavity;

3) into the amniotic cavity;

4) into the yolk sac.

Infection of chicken embryos is carried out in a box using sterile

instruments. Before infection, chicken embryos are wiped twice with a cotton swab
moistened with alcohol.

Infection on the chorion-allantoic membrane. After disinfection, the eggs are

carefully cut off a piece of the shell from the blunt end, the shell membrane is
removed - this reveals the chorion-allantoic membrane. Infectious material in the
amount of 0.1-0.2 ml is applied to the chorion-allantoic membrane with a syringe
or a Pasteur pipette. After infection, the hole is closed with a cap and the gap
between it and the chicken embryo is filled with paraffin.

On the other side of the egg, the name of the infectious material and the date of
infection are written with a simple pencil.

Infection in the amniotic cavity. The egg is ovoscoped and on the lateral side a
site is selected where the chorion-allantois is devoid of large blood vessels. This
area is marked with a pencil. The eggs are placed on a stand in a horizontal
position, disinfected, and a hole in the shell is pierced with a special sterile spear to
a depth of 213 mm, through which a needle with infectious material is inserted
directly into the amniotic cavity at the same distance. In order to prevent the
injected liquid from flowing back, a puncture is first made above the air bag, after
which both holes are filled with paraffin.
 Infection in the allantoic cavity. Infection is carried out in a dark box. The
airspace is marked, the shell above the airspace is disinfected, and the syringe
needle with the material is inserted through the hole in the shell towards the
embryo. If the needle has entered the allantoic cavity, then a shift in the shadow of
the embryo is observed. After infection, the hole is filled with paraffin.

Infection in the yolk sac. The shell is disinfected. The egg is placed on the stand
with the blunt end to the right so that the yolk sac is facing up. A hole is pierced
above the air chamber in the center. Through the hole in the shell in a horizontal
direction to a depth of 2-3 mm, a syringe needle is inserted, which enters the yolk
sac. The material is injected in a volume of 0.2-0.3 ml. After the introduction of the
material, the hole is waxed.

The temperature regime and duration of incubation depend on the biological

properties of the introduced virus.

Infected eggs are checked daily by candling to check the viability of the
embryo. If the embryos die on the first day, then the cause of this is usually trauma
during infection. Such eggs are derived from experience.

If necessary, separately examine each component of the embryo, the material is

collected in a certain order: the allantoic fluid is sucked off, then the amniotic fluid,
the chorion-allantoic membrane is cut, the amniotic membrane, the embryo, the
yolk sac are separated, and only after that the chorion-allantoic membrane is
removed, separating it from the inner shell surface. The presence of the virus in the
infected embryo is determined by the characteristic changes in the chorion-allantoic
membrane of the infected chicken embryo.

Viruses that do not have hemagglutinating activity are detected using RSK.

To detect the virus in the allantoic or amniotic fluids of infected embryos, RHA
is placed (hemagglutination is caused by allantoic or amniotic fluids or a
suspension prepared from the chorion-allantoic membrane).

Cultivation of viruses in cell culture . For the accumulation of viruses in

sensitive cell cultures, tissues of humans and various animals are used. Single-layer
cultures of primary trypsinized and transplanted cell lines received the greatest
practical application.

Single-layer cell cultures are grown in flat glass vessels-mattresses. Cell

suspension in a liquid nutrient medium at a temperature of 37°C allows you to get
"in vitro" a layer of cells with a specific histological structure. The presence of
viruses in tissue cultures is detected by the change (degeneration) of cells. The type
of viruses is determined by neutralizing the action of viruses when the
corresponding type-specific sera are added to the virus-containing material.

These methods allow to take into account the results of the study faster and are
more economical. In cases where viruses do not cause a cytopathic effect
(degeneration) and do not develop in chicken embryos, animal infection methods
are used (see Chapter 11).

For the cultivation of viruses, transplantable cells are used, which are more often
obtained from malignant tumor cells.

Monolayer cultures are obtained from human, chicken, and animal embryos.

The advantage of single-layer cell cultures is the simplicity of the technique and
ease of accounting.

The ability of cells to reproduce outside the body is related to the degree of
tissue differentiation. Less differentiated tissues have a greater ability to proliferate
(connective, epithelial tissue).

The essence of the methods in the preparation of primary tissue cultures is the
destruction of the intercellular tissue and separation of cells for the subsequent
production of a monolayer.

Dissociation of cells is carried out by exposing the tissue to proteolytic enzymes,

most often trypsin. Trypsin solution contributes to the separation of cells while
maintaining their ability to reproduce. Nutrient medium is necessary for growing
cell culture. The composition of the medium is complex, it includes a number of
ingredients: amino acids, glucose, vitamins, mineral salts, coenzymes, etc. The
tissue culture is obtained under strictly aseptic conditions. Antibiotics (500 units of
penicillin and 250 units of streptomycin per 1 ml) are added to the medium to
suppress the growth of the bacterial flora.

The prepared tissue is poured with a 0.25% solution of warmed trypsin and
incubated in a thermostat at 37 ° C. During the incubation, the tissue is periodically
stirred by rotating the flask. Trypsinized cells are centrifuged at 800-1000 rpm for 5
minutes.

Trypsinization and centrifugation are carried out very carefully so as not to

injure the cells. After centrifugation, the supernatant is removed, and the cell
sediment is placed in a small volume of nutrient medium. To obtain a
homogeneous mass, the cell suspension is filtered through one layer of gauze in a
funnel (sterile). Cell suspension is checked for sterility by inoculation of 0.1 ml in 2
test tubes with sugar broth.

The success of cell cultivation depends on the inoculum dose, therefore, after
trypsinization, cells are counted in the Goryaev chamber. After counting, the cell
suspension is diluted with a nutrient medium in such a way that 1 ml contains
500,000-1,000,000 cells and poured into test tubes and mattresses. The tissue
culture tubes are incubated in an incubator in an inclined position.

Seeded cultures are examined daily under a low magnification microscope to

determine the nature of their growth. Normal proliferating cells are light and grow
in a single layer. If the cells are dark, granular and do not proliferate, which may be
the result of contamination (poor handling of dishes or contamination of
ingredients), then such cultures are removed from the experiment.

Changing the nutrient medium 2-3 days after sowing improves the intensity of
proliferation.

Normal, well proliferating cells are infected with the test material.

Inoculated cultures are predominantly obtained from malignant tumors. Strain

Hela - cell culture of cervical cancer of a woman named Helena (obtained in
1950); strain Hep-2 isolated from a patient with cancer of the larynx. The growth of
these cells is maintained in the laboratory by passages. Their peculiarity lies in the
fact that they multiply for a long time. At present, these cells have gone through
thousands of generations. In the process of passages, they lose some morphological
and biochemical properties - they undergo mutations. However, they remain quite
suitable for the cultivation of viruses in them. The culture of these cells is used by
laboratories around the world.

Reproduction of the virus in cell culture occurs at different times depending on

the properties of the virus and the type of cells.

The presence of the virus is judged by cytopathic action. Under the microscope,

cell degeneration is observed. The time of cytopathic action and its nature depend
on the dose and properties of the virus.

In some viruses, a cytopathic effect is detected after a few days (pox virus), in
others - after 1-2 weeks (hepatitis virus, etc.).
 Currently, hundreds of viruses are known to infect humans. The fight against
viral infections is carried out by different methods. Most effective immunization. In
this way, smallpox was eliminated, and the incidence of poliomyelitis was
reduced. Public prevention is important in the fight against viral infections - the
destruction of stray dogs (rabies control), personal prevention, etc.

However, these measures cannot ensure the elimination of all viral

diseases. Scientists are persistently looking for ways in which it would be possible
to infect the virus without damaging the cell in which it is located.

Therefore, it is natural that in the program of the CPSU, virology is named one
of the leading branches of natural science, which should receive priority
development in the coming years.

Basic Methods for Investigating Viruses . 1. Hemagglutination reaction,

hemagglutination delay reaction, indirect hemagglutination reaction. Complement
binding reaction.

2. Neutralization reaction of viruses in tissue culture.

3. Method of immunofluorescence.

4. Histological method - detection of inclusions (Babesh-Negri bodies - with

rabies; Pashen bodies - with smallpox, etc.).

5. Biological method.

RNA viruses
Chapter 43
Influenza is a common acute infectious disease of the respiratory tract, which
has an epidemic character and affects large masses of people. The influenza
pandemic (1918-1920), dubbed the "Spanish flu", swept almost 1.5 billion people
and claimed 20 million human lives.

As early as the beginning of the 20th century (1904), M. I. Afanasyev and P. B.

Vaks suggested the viral nature of the infection. However, this was established only
in 1933, when C. Smits, C. Andrews and P. Laidlaw isolated from patients and
studied varieties of influenza viruses, called influenza A viruses, in 1940 T. Francis
and T. Medzhill studied viruses influenza B, and in 1947 R. Teyler discovered the
influenza C virus.
 Influenza pathogens belong to the Orthomyxoviridae family (from Latin
myxomatosis - slimy). They have a pronounced tropism for the mucous membranes
of the upper respiratory tract.

This family includes viruses that cause disease in humans, animals, and birds.

Morphological structure . The structure of influenza virions is complex. It

contains a single-stranded RNA, a helical nucleocapsid, and a protective lipid-
carbohydrate-protein shell. The number of capsomeres has not been precisely
determined. Virions are mostly spherical in shape, but filamentous virions are also
found. The shell has protrusions (thorns) - ash meters (see Fig. 53). RNA is
synthesized in the nucleus of the host cell. Viral proteins are synthesized in the
cytoplasm. The formation of virions occurs near the cell membrane.

Cultivation . The influenza virus multiplies well in the chicken embryo (in the
cells of the amniotic or allantoic cavities). It can also be cultivated in the cell
culture of the kidneys of monkeys, human embryos, etc.

Antigenic structure . Influenza viruses have an S antigen, according to which,

in the complement fixation reaction, they are divided into types A, B, C.

Virus A has two more antigens: hemagglutinin and neuraminidase (see Fig. 53).

Hemagglutinin has four subtypes (H 0 , H 1 , H 2 , H 3 ), and neuraminidase has
two (N 1 and N 2 ). The combination of these antigens determines the subtype of the
virus, which changes with different epidemics. Thus, the influenza epidemic in
1933 was caused by the type A influenza virus, which had the formula
AH 0 N 1 . AH 1 N 1 was isolated during the epidemic of 1947, AH 2 N 2 - in 1957
(Singapore strain), AH 3 N 2 - in 1968 (Hong Kong strain), in 1977 the AH 1 N type
virus returned again 1 .

The variability of the combination of these antigens determines the variability of

the A virus. The B virus is more stable. The most resistant type C. Types and
subtypes of viruses do not give cross immunity.

Influenza viruses share a common antigen with human erythrocyte isoantigens.

Resistance to environmental factors . A temperature of 65 ° C kills influenza

viruses in 5-10 minutes. At 50 ° C, it loses its infectious properties after a few
minutes. At room temperature, the virus is inactivated after a few hours. In an
acidic, alkaline environment, under the influence of ether and disinfectant
solutions, it dies very quickly. The virus is very sensitive to UV rays and
ultrasound. It is resistant to the action of glycerin, in which it can be stored for
several months.

Susceptibility of animals . During epidemic outbreaks, pets can adapt to the

influenza virus. This was revealed by an increase in their antibody titer. But the
epidemic role of animals is unknown. Maybe they are the guardians of the virus in
nature and play a role in its circulation.

From experimental animals, it is possible to infect ferrets through the respiratory

tract, intranasally - white mice. The disease in them is characterized by damage to
the lungs, often leading to their death.

Sources of infection . A sick man. transmission paths . Airborne (when

talking, coughing, sneezing).

Pathogenesis . The virus enters the mucous membrane of the upper respiratory

tract. Having penetrated into the human body, it is introduced into the epithelial
cells of the upper respiratory tract, from where it passes into the bloodstream and
causes intoxication. In the mucosa, the virus causes cell death. This creates
conditions for the penetration of other microorganisms that cause a secondary
infection - pneumonia, bronchitis, etc. Bacteria localized in the upper respiratory
tract are activated - autoinfection occurs. In addition, the influenza virus activates
chronic diseases such as tuberculosis, etc.

Immunity . It is formed mainly due to antihemagglutinins, which have virus-

neutralizing properties - they prevent the adsorption of viruses on a sensitive
cell. Antineuraminidase antibodies play an important role. Protection of the
organism is also determined by interferon and other inhibitors found in human
blood serum.

Usually, immunity is type- and strain-specific. The change of subtypes of the A

virus excludes reliable protection of the organism against this infection, since there
is no cross-immunity, and it has not yet been possible to predict the direction of
variability.

Prevention . Isolation of the patient, ventilation of the room, wet cleaning, etc.

It is also important to prevent cooling, which reduces the production of such a
protective factor as interferon.

specific prophylaxis . In the USSR, a live vaccine containing attenuated viruses

of type A and B is used. The vaccine is administered intranasally. Received a live
vaccine for children, which is administered orally. Research is underway to create a
vaccine from hemagglutinins, neuraminidase.

For individual protection, interferon and oxalin ointment are used (lubricate the
nasal mucosa).

However, anti-influenza measures cannot be considered sufficiently effective. In

the USSR, 40-50 million people a year get sick with influenza, which damages
people's health and the country's economy. Therefore, a comprehensive influenza
program is currently being developed, in which many (more than 20) research
institutes are participating.

Treatment . With influenza caused by virus A, rimantadine has been used. To

prevent secondary infections, various antibacterial drugs are used.

Virological diagnostics
The purpose of the study: to identify the virus and determine its
type; determination of antibodies in blood serum.

Research material

1. Imprints of the mucous membrane of the nasal cavity (in the acute stage of the
disease).

2. Nasopharyngeal discharge.

3. Blood.

Material collection methods

 Material collection methods

Basic research methods

1. Cultivation of swabs from the nasopharynx in chicken embryos.

2. Rhinocytoscopic examination.

3. Immunofluorescence (see chapter 12).

4. Serological study of paired sera in RSK and RTGA.

Research progress
 Research progress

Control questions
1. Describe the morphological structure of the influenza virus.

2. What methods of cultivating influenza viruses do you know?

3. What is the antigenic structure of the influenza virus and what types do you
know of it?

4. What type of influenza virus is most susceptible to variability and what are
the names of the isolated strains (designations)?

5. What is the stability of the influenza virus in the external environment?

6. Why do secondary infections often occur with influenza?

7. How and what material is collected to detect the influenza virus?

8. What determines the instability of immunity in influenza?

9. What are the main methods of research in the laboratory diagnosis of

influenza.

Chapter 44
The causative agent of rabies belongs to the family Rhabdoviridae
(rhabdoviruses).

This family includes rabies, vesicular stomatitis and other viruses that cause
disease in animals and insects.

For thousands of years, all mankind has suffered from this terrible disease -
rabies. The mention of this disease is found in the Iliad by Homer, the works of
Aristotle and Avicenna. In the 1st century BC e. The Roman scientist Celsius
proposed to burn the bitten places with a red-hot iron. This painful event saved
only if the wound was small and cauterization was performed immediately after the
bite. You can talk about many more means, but they all turned out to be ineffective.

Rabies was first studied by L. Pasteur in 1880.

 In 1886, a group of Odessa doctors, at their own expense, sent N. F. Gamalei to
Pasteur in Paris to get acquainted with the method of preparing a rabies
vaccine. After his return, a laboratory was opened in Odessa, where an anti-rabies
vaccine was produced.

Morphological structure . The causative agent of rabies has a rod-shaped

(bullet-shaped) shape, one end of which is flat, the other is elongated. Size 80-180
nm. The virion contains a single-stranded RNA surrounded by a capsid. Outside,
the capsid is covered with a membrane, which includes glycopreteids and
glycolipids. In the shell there are awl-shaped formations (peplomers).

In the cytoplasm of virus-affected cells, specific inclusions are formed,

described by Babesh (1892) and Negri (1903). Therefore, they are called Babesh-
Negri bodies. The size of these bodies is from 3-4 to 20 microns. They are of
different shapes, most often spherical, but there are oval and polygonal. Acidic
dyes turn them ruby red.

Babesh-Negri bodies are located in the cytoplasm of the nerve cells of the
brain. The detection of Babesh-Negri bodies is of diagnostic value.

Cultivation . The rabies virus is cultivated in the brain tissue of mice, chickens,

rabbits, in chicken embryos, embryos of calves, sheep and cell cultures of various
animal species.

Antigenic structure. Rabies viruses do not have antigenic varieties. There are

two rabies viruses: wild, circulating in animals, virulent, and in humans, called a
street virus. Another rabies virus was obtained by L. Pasteur in the laboratory by
successive, long passages (133 times) of the street virus through the brain of a
rabbit. At the same time, the duration of the incubation period for infecting a rabbit
was reduced from 21 to 7 days. Further passages no longer changed the incubation
time, it was fixed at 7 days, and the virus was called fixed (virus fixe). During
passage, the virus adapted to the rabbit brain and lost its ability to cause disease in
humans, dogs, and other animals. However, it has completely retained its antigenic
properties, so it is used for the preparation of an anti-rabies (against rabies)
vaccine.

Susceptibility of animals . Rabies affects many animals, domestic and wild,

even birds. However, dogs, wolves, foxes, and bats get sick more often. Bats get
sick without obvious signs of disease and may be the guardians of the rabies virus
in nature.

Sources of infection . Sick animals.

 transmission paths . The rabies virus is transmitted by direct contact from sick
animals (bites) or when the saliva of a sick animal gets on a damaged surface of the
skin or mucous membranes.

Pathogenesis . From the moment of a bite or licking to human disease, it takes

from 15-45 days to 3-6 months (cases of incubation for more than a year are
described).

The duration of incubation depends on the gate of infection, the nature of tissue
damage.

The shortest incubation period is for bites to the face and head.

From the site of introduction, the viruses spread along the nerve trunks and enter
the cells of the central nervous system. The largest amount of the virus is
concentrated in the hippocampus, medulla oblongata, nuclei of cranial nerves and
in the lumbar part of the spinal cord. In nerve cells, the virus reproduces
(multiplies). As a result of damage to the nervous system, increased reflex
excitability appears: convulsions, especially of the respiratory and swallowing
muscles. There is shortness of breath and hydrophobia (hydrophobia). One idea of
drinking causes severe painful convulsions in patients. Death occurs in 4-5
days. Lethality 100%.

The clinical picture of rabies in dogs: the animal becomes sullen, salivation
appears. The dog begins to devour inedible things - stones, chips, etc. Then a
period of excitement begins. The dog runs in a straight line with its head bowed
low. Attacks meeting people, animals without barking and bites them. The period
of excitation is replaced by paralysis and death of the animal.

Immunity . Postinfectious immunity is not well understood. The mechanism of

immunity that occurs after vaccination is associated with virus-neutralizing
antibodies that appear 2 weeks after vaccination, as well as with the interference of
vaccine and street viruses. The phenomenon of interference is that a fixed virus
reaches the cells of the nervous system much faster, multiplies in them and
prevents the introduction of a street virus. Immunity is maintained for 6 months.

Prevention . Destruction of rabid animals, stray dogs. Registration of dogs and

their mandatory vaccination. In case of a bite, treat wounds immediately.

specific prophylaxis . The introduction of the anti-rabies vaccine proposed by

Pasteur. The vaccine is a suspension of the brain tissue of an animal (rabbit, mouse,
etc.) infected with a fixed rabies virus. There are 2 types of vaccines: Fermi and
Phillips. They differ from each other in the quantity and quality of the
preservative. The Fermi vaccine contains 1% phenol, Phillips - glycerin.

Recently they have been using the Flory vaccine. It is a live rabies vaccine made
from viruses cultured in bird embryos. The principle of operation is the same
(interference of viruses).

Vaccination is subject to all bitten or licked sick or suspicious of rabies

animals. There are no contraindications, however, people are not vaccinated
without sufficient indications, since even a fixed virus can lead to
complications. Vaccinations are done as early as possible, they are carried out
repeatedly. The vaccine is administered subcutaneously into the abdomen. The
number of vaccinations is prescribed by the doctor.

For bites of dangerous localization and to increase the effectiveness of vaccines,

anti-rabies immunoglobulin is also used, which is obtained from the serum of
horses hyperimmunized with a fixed virus. Immunoglobulin has the ability to
neutralize the effect of the virus. In addition, it prevents post-vaccination
complications (allergic encephalomyelitis, etc.).

In the USSR and other countries of the world, research is being carried out
aimed at obtaining an anti-rabies vaccine that does not cause complications.

Treatment . Not developed.

Virological diagnostics

Post-mortem diagnosis is based on: 1) detection of Babesh-Negri bodies in brain

cells; 2) detection of a specific antigen by the method of fluorescent antibodies; 3)
isolation of the virus by bioassay on laboratory animals.

1. Babesh-Negri bodies can be detected in stained smears and prints from non-

fixed brain tissue and in histological sections (even if the brain is rotting). To detect
them, several preparations are made (since there may be few bodies). When stained
according to Muromtsev, the cytoplasm of nerve cells is blue, and the bodies of
Babesh-Negri are purple with a pink tint and pronounced dark purple granularity.

In the brain cells of animals with rabies, Babesh-Negri bodies are found more
often than in sections made from the salivary glands.

2. The method of fluorescent sera has not yet received wide practical

application.
 3. The bioassay method on laboratory animals is carried out only in special
laboratories.

The work, storage and transfer of non-fixed material is carried out according to
the rules for working with especially dangerous infectious material (see "Especially
dangerous infections").

Control questions
1. What is the shape and size of the rabies virus?

2. What are Babesh-Negri bodies?

3. What is a fixe virus?

4. What is the susceptibility of animals to the rabies virus?

5. How is the rabies virus transmitted?

6. What rabies virus is used to prepare the vaccine (street or fixe virus?)

7. What is the basis for virological diagnosis in case of suspected rabies?

Chapter 45
The disease of poliomyelitis has been known for a very long time. In an
Egyptian temple, a bas-relief depicting an Egyptian priest was discovered with one
leg thinner than the other (dry leg) and the other foot in the "horse foot" position -
now known to be the result of polio.

Polio viruses
The causative agents of poliomyelitis are part of the Picornaviridae family (from
Latin pico - small, rna - RNA-containing).

This family includes three genera, of which enteroviruses are of the greatest
importance in human pathology: causative agents of poliomyelitis, Coxsackie and
ECHO (Enteric cytopathogenic human orphan viruses) (orphan - orphan).

The viral etiology of polio was established by K. Landsteiner and E. Paperer in

1909 in experiments on monkeys.
 Morphological structure (see Fig. 52) Poliovirus belongs to small viruses (15-
30 nm). The virus consists of a single-stranded RNA and a protein capsid
consisting of 32 capsomeres. The shape of the virus is cuboidal. There is no outer
shell. Carbohydrates and lipids were not detected. Infectious properties are
associated with RNA.

Cultivation . The polio virus multiplies well in the cell culture of monkey

kidneys, fibroblasts, human embryos and transplanted cell cultures. Reproduction
of the virus is accompanied by a cytopathic effect (cell degeneration).

Antigenic structure . Three serological types of the poliovirus are known,

which are designated by Roman numerals (I, II, III). Type I virus has the greatest
epidemiological significance (it causes diseases in 65-90% of cases). Type II virus
is found in 10-12%, type III virus causes some sporadic diseases.

Poliovirus serotypes differ in the neutralization reaction. They do not induce

cross immunity.

Resistance to environmental factors . Boiling kills the virus quickly. A

temperature of 50 ° C destroys it after 30 minutes. The virus can survive up to 3
months at room temperature. It tolerates low temperatures well. In cold stools,
poliovirus persists for up to 6 months. In milk - up to 3 months. Polioviruses persist
for a long time in the soil, open water bodies, which is of epidemiological
significance. Viruses are resistant to the effects of gastric juice and 1%
phenol. Sensitive to formalin, chloramine, hydrogen peroxide, potassium
permanganate, etc.

Susceptibility of animals . Monkeys (chimpanzees, macaques) are sensitive to

the poliovirus type I and III. Rodents (cotton rats, hamsters, mice) are sensitive to
the type II virus. Experimental infection causes them a disease accompanied by
paralysis.

Sources of infection . Sick people and virus carriers.

transmission paths . Food (the virus is excreted with faeces up to 40 days). The

disease can be transmitted by flies (flies carry the virus on their paws, abdomen)
and through environmental objects; airborne (in the first days of illness, the virus is
released from the nasopharynx).

Pathogenesis . The entrance gate is the mucous membrane of the upper

respiratory tract and the digestive tract. Once in the body on the nasopharyngeal
mucosa, the virus enters the lymph nodes of the pharyngeal ring and the small
intestine, where it reproduces (reproduction). Virus-neutralizing antibodies
accumulate in the blood, blocking the penetration of the virus into the central
nervous system. In the event that the virus nevertheless penetrates the central
nervous system, it is localized in the motor cells of the anterior horns of the spinal
cord and in the gray matter of the subcortex, where it causes an inflammatory-
degenerative process. The result is flaccid paralysis, often of the lower extremities.

Clinically, poliomyelitis occurs in three forms: abortive, non-paralytic

(meningeal) and paralytic (in 1% of cases).

Poliomyelitis is more common in children aged 5 months to 5-6 years. The polio

virus is shed in feces and nasopharyngeal mucus.

Immunity . After the illness, lifelong immunity remains, due to the resulting

virus-neutralizing antibodies to homologous types of the virus.

Prevention . Early diagnosis and isolation of patients.

Specific prophylaxis is carried out by active immunization. The first vaccine

was proposed by Salk in the 1940s. This vaccine consisted of formalin-inactivated
polioviruses I, II and III. However, it did not cause the formation of stable
immunity and was very painful when administered intramuscularly.

The second vaccine was proposed by Sabin. It consisted of live attenuated

(weakened) mutant strains of three types of poliomyelitis. These strains were
devoid of infectious properties, but retained immunogenicity.

In the 60s, MP Chumakov and A. A. Smorodintsev, using a weakened strain

obtained by Sabin, developed a method for preparing a vaccine in the form of
dragee candies, which greatly facilitated its use. Mass vaccination of children led to
the almost complete eradication of polio in the USSR, for which the authors
received the Lenin Prize.

The mechanism of action of the vaccine is the interference of viruses, i.e., the
vaccine strain of the virus, populating the intestinal cells, blocks the reproduction
of the wild strain, as well as the formation of virus-neutralizing antibodies.

Treatment . Symptomatic. Immunoglobulin is used.
 Virological diagnostics

The purpose of the study: to identify the virus and determine its
type; serodiagnosis.

Research material

1. Feces of patients (taken on the 1st and 2nd week of the disease).

2. Nasopharyngeal discharge (taken in the first days of the disease).

3. Pieces of the brain and spinal cord, the contents of the small and large
intestines, lymph nodes (sectional material).

Material collection methods

Material collection methods

Basic research methods

1. Isolation of viruses by infecting various cell cultures.

2. Serological study (neutralization reaction) and RSK.

Research progress

Isolation of viruses is carried out by infecting the studied material with two
types of cultures - primary and transplanted cells.
 Research progress

The presence of the virus is judged by the cytopathic effect on the cells.

In the absence of cell degeneration within 7-10 days after infection, the next
passage is carried out. For the second inoculation, the culture fluid taken from the
first passage is used and a new cell culture is infected.

In the absence of cell degeneration in the second passage, the result is

considered negative.

If the result is positive, the isolated virus is typed with commercial sera against
three types of poliomyelitis virus by neutralization in cell culture and CSC.

Serological diagnostics

Retrospective diagnostic methods. To confirm the diagnosis of poliomyelitis, a

neutralization reaction is performed with paired sera of the patient. One serum is
taken at the beginning of the disease, the second - a month after the onset of the
disease. Both sera are tested simultaneously in a neutralization reaction. To do this,
the sera are heated for 30 minutes at 56°C and diluted with Hank's
solution. Dilutions are made from 1:4 to 1:1024. Each dilution is mixed with a
standard dose of type I, II and III virus. After an hour of contact (at room
temperature), each mixture is infected with 2 test tubes with a suspension of cell
culture and placed in a thermostat for 4-9 days. The results of the reaction are
judged by the change in the color of the medium (from crimson to yellow). A color
change indicates the presence of antibodies. The color change occurs because the
cells remain viable, form metabolic products, changing the pH reaction and,
accordingly, the color of the medium. The viability of the cells is ensured by the
neutralization of the virus with the appropriate serum.

A positive reaction is considered only with a fourfold increase in the titer of

virus-neutralizing antibodies, that is, if, for example, the titer of the first serum was
1:8, and in the second, taken after 1-2 months, the titer was not less than 1:32.

Coxsackie viruses
The Coxsackie virus was first isolated in 1948 by G. Doldorf and I. Sickles in
the city of Coxsackie in the USA from the feces of children with poliomyelitis-like
diseases.

Morphological structure . Coxsackieviruses are small viruses (20-30

nm). They include RNA and protein, the shape is cuboidal.

Cultivation . They are cultivated in the same way as poliovirus.

Antigenic structure . According to the antigenic structure and pathogenic

properties, Coxsackie viruses are divided into 2 groups: A and B.

Group A includes 24 serological types, which differ in the neutralization

reaction.

Group B includes 6 serotypes, which also differ in the neutralization reaction.

Resistance to environmental factors . Boiling destroys Coxsackie viruses

quickly, 50-55 ° C - after 30 minutes. Viruses tolerate low temperatures well. At a
temperature of -70 ° C, they remain for about 3 months. Viruses are resistant to
various concentrations of hydrogen ions, the action of ether, 5% lysol, but are
sensitive to hydrochloric acid, formaldehyde, etc.
 Susceptibility of animals . Type A virus causes paralysis in neonatal mice, and
serotype 7 causes polio-like illness in monkeys and cotton rats. Type B virus causes
spastic paralysis in newborn mice.

Sources of infection . Coxsackieviruses are widely distributed in nature. They

are excreted with feces and from the nasopharynx from patients and virus carriers.

transmission paths . Food (when using contaminated water, food and in contact

with environmental objects infected with a virus; viruses can be carried by flies),
airborne (the patient releases viruses when sneezing, coughing in the first days of
illness).

Pathogenesis . The entrance gate is the mucous membrane of the nasopharynx

and the digestive cycle. Coxsackie viruses are characterized by neuro- and
myotropy. Coxsackie A viruses can cause paralytic diseases similar to polio. Group
B coxsackieviruses cause aseptic serous meningitis, aseptic myocarditis and other
enteroviral diseases.

Immunity . After the illness, persistent type-specific immunity remains.

Prevention . Sanitary measures: isolation of patients, quarantine. Specific

prophylaxis has not been developed.

Treatment . Symptomatic.

Virological diagnostics

The purpose of the study is to isolate the virus and determine its type.

The material for research, the methods of its collection and the main research
methods are the same as in poliomyelitis.

Diagnostic methods . Virus isolation is carried out in the same way as in

poliomyelitis: inoculation of the test material on primary and continuous cell
cultures. Some Coxsackie A serotypes are difficult to adapt in cell culture.

The isolated viruses are identified in RSK and neutralization reactions.

To differentiate the Coxsackie virus type A from type B, a biological test is put:
they infect newborn white mice (suckers).
 Type A virus causes them flaccid paralysis without encephalitis, and type B
virus causes convulsions and paralysis, in addition, damage to internal organs is
observed - the liver, pancreas, etc. In case of a disease caused by Coxsackie
viruses, a retrospective serodiagnostic method is also used, reactions are put
neutralization with paired sera (see chapter 45).

ECHO viruses
In 1941, D. Enders, while studying poliomyelitis, isolated a virus from the
patient's intestines, which differed in serological properties from the poliomyelitis
virus and other intestinal viruses, and called it orphan - an orphan. Further work
showed that there are a large number of viruses similar to it, and the whole group
was called ECHO - Enteric cytopathogenic human orphan viruses.

Morphological structure . The size of the ECHO virus is 10-15 nm. In its

structure and reproductive ability, it differs little from polioviruses and
Coxsackieviruses.

Cultivation . ECHO viruses, as well as polio and Coxsackie viruses, are

cultivated in primary and continuous cell lines.

antigenic properties . ECHO viruses include 32 serotypes that do not confer

cross-immunity.

Resistance to environmental factors . The same as that of Coxsackie viruses.

Susceptibility of animals . Animals are not susceptible to ECHO viruses.

Sources of infection, transmission routes and entry gates . Same as polio and

coxsackie viruses.

Pathogenesis . ECHO viruses cause a variety of diseases - they cause aseptic

serous meningitis, enterovirus fever, epidemic exanthems, myocarditis and other
febrile diseases.

Immunity . ECHO viruses create strong immunity due to virus-neutralizing

antibodies.

Prevention . General sanitary measures as in other intestinal infections, but it

should be remembered that hypothermia and overheating contribute to the spread
of enterovirus diseases. Specific prophylaxis has not been developed.
 Treatment . Symptomatic.

Virological diagnostics

The material for the study, the methods of its collection, the main methods of
research and diagnosis are the same as for other enteroviral diseases. However, as a
tissue cell culture, it is better to use primary trypsinized ones.

Control questions
1. What viruses are included in the Picornaviridae family?

2. What is the size and morphological structure of poliomyelitis, Coxsackie and

ECHO viruses?

3. What methods of cultivation of polioviruses do you know? What causes the

reproduction of viruses in cell cultures?

4. What types of polioviruses do you know? Which of them has the greatest

epidemiological significance?

5. Sources of the disease, gates of infection and pathogenesis in poliomyelitis.

6. Tell us about the specific prevention of poliomyelitis.

7. What material should be taken for research and what are the main methods
used for diagnosis in cases of suspected poliomyelitis?

8. Tell us about Coxsackie viruses.

9. Tell us about ECHO viruses.

DNA containing viruses

Chapter 46
The family Poxviridae contains several genera with diverse hosts. Variola virus
is pathogenic for humans.

The disease of smallpox has been known since time immemorial (about 3000
BC) and it was common in all countries of the world.
 One of the ancient historians wrote: "No people, no race, no rank, no age, no sex
spared smallpox. Everything trembled before it." Smallpox is terrible for its
contagiousness. In Germany in the 18th century, 80,000 people died of
smallpox. The Russian Tsar Peter II, the Austrian Emperor Joseph, the French King
Louis XIV, the English Queen Anna, the famous Russian actress
Komissarzhevskaya and others died of smallpox.

It is difficult for us now to imagine the crushing force with which the smallpox
virus wielded. But this scourge of mankind has been broken by science. Smallpox
epidemics have stopped.

And in the last few years, not a single case of smallpox has been reported
worldwide.

The etiology of smallpox was established by the end of the 19th century. In
1892, Guarnieri, in histological sections made from the cornea of the eyes of a
rabbit infected with smallpox material, found spherical and crescent-shaped
inclusions ranging in size from 3-4 to 10 microns, stained red according to
Romanovsky-Giemsa. These inclusions were called Guarnieri bodies. And in 1906,
in the contents of smallpox pustules, Pashen discovered smallpox corpuscles, in
preparations processed by the silvering method according to Morozov. These
corpuscles were called Pashen-Morozov bodies.

Morphological structure . Smallpox virus is large, 300-350 nm in size,

cuboidal in shape. On ultrasections of smallpox virions, a lipoprotein membrane
was found, under it a viroplasm, which contains a nucleocapsid. The DNA of the
smallpox virus is double-stranded. Several enzymes have been isolated from the
nucleocapsid of the virion.

Cultivation . The variola virus develops well in chicken embryos on the

chorion-allantoic membrane. Its reproduction is characterized by the formation on
the shell of white, dense punctate plaques with a shiny surface, about 1 mm in size.

The virus can also be cultured in primary and continuous human and animal cell
cultures. Here, growth is characterized by a cytopathic effect (degeneration of cells
after 48-72 hours).

Antigenic structure . The smallpox virus has several antigens: soluble (L-

thermolabile and S-thermostable), nucleoprotein NP antigen. Smallpox viruses
share antigens with the smallpox vaccine virus and human erythrocytes of group A
and AB.
 Resistance to environmental factors . At a temperature of 100 ° C, viruses die
instantly. A temperature of 60 ° C destroys them in an hour. Smallpox viruses
tolerate low temperatures and drying well - they remain in smallpox crusts for a
long time. Disinfectant solutions (30% chloramine, lysol) inactivate smallpox
viruses after 30 minutes. They are more resistant to phenol and ether, and smallpox
viruses persist for months in 50% glycerin.

Susceptibility of animals . Small and large cattle are susceptible to the

smallpox virus. Under experimental conditions, monkeys, guinea pigs, rabbits, etc.
are easily infected. However, it is only possible to reproduce a disease that is
clinically similar to a human disease only in monkeys.

In newborn white mice, the virus causes smallpox encephalitis.

Sources of infection . Sick people.

transmission paths . Airborne and airborne (the virus is transmitted by

coughing, talking, through dishes, as well as through dust particles on clothing).

Pathogenesis. The smallpox virus penetrates through the mucous membrane of

the respiratory tract and through the skin. Having entered the body, viruses are
localized in the regional lymph nodes. Having multiplied there, they enter the
bloodstream, causing viremia. Secondary reproduction (reproduction) occurs in the
lymphoid tissue and is accompanied by clinical manifestations of the disease: high
fever, headache, loss of consciousness, etc. Possessing dermotropic properties,
viruses enter the epidermis. Papules, vesicles and pustules form on the skin and
mucous membranes. Smallpox papules are characterized by transparent contents
and look like pearls with a mother-of-pearl luster. At the site of the appearance of
pustules, necrosis is formed, after healing of which scars remain. Scarring of the
mucous membrane of the eyes leads to blindness (in 25% of cases). The mortality
rate for smallpox is high, with hemorrhagic form - 100%. In this form, the pustules
fill with blood - black pox.

There are mild forms of smallpox when the disease occurs without fever and
rash.

Immunity . People who have been ill have lifelong immunity. It is caused by

virus-neutralizing, hemagglutinating and complement-fixing antibodies. Artificial
immunization followed by revaccination gives stable immunity. It is believed that
interferon is also a protective factor.
 Prevention . Early diagnosis, isolation, disinfection, prevention of smallpox
importation from other countries, quarantine, etc.

specific prophylaxis . In the fight against smallpox, specific prophylaxis is of

great importance. For many years before our era in the east, there were different
methods of dealing with smallpox. In India, Iran - crushed crusts from the pustules
of patients with a mild form were rubbed into the skin of healthy people, and in
China they were applied to the mucous membranes of the nose.

In 1796, the English physician E. Jenner, after long-term observations, used the
contents of cowpox pustules to vaccinate people. Hence the name - vaccine (from
Latin vacca - cow).

The vaccine prepared in this way was used for a long time. Then a method was
developed for obtaining ovovaccines (the virus was accumulated in a chicken
embryo). This method is more convenient to manufacture and more economical.

Currently, the vaccine is prepared from the virus grown in cell culture.

In March 1919, V. I. Lenin signed a decree on mandatory smallpox

vaccination. After mass immunization, smallpox in the USSR was eliminated.

In 1958, at the initiative of the USSR, at the XI Assembly of the WHO, a

decision was made to eliminate smallpox throughout the world through mass
vaccination. As a result, in recent years, not a single case of smallpox has been
recorded in the world, and in 1981, on the recommendation of WHO, the
mandatory vaccination against smallpox was canceled.

Virological diagnostics

The purpose of the study: to identify the causative agent of smallpox. Work with
the smallpox virus is carried out under strictly controlled conditions (see
"Particularly Dangerous Infections").

Research material

1. Contents of papules, vesicles, pustules.

2. Detachable mucous membrane of the nasopharynx.

3. Blood (from the 5th day of illness) is taken to detect specific antibodies.
 Material collection methods

Material collection methods

Basic research methods

1. Method of immunofluorescence (express diagnostics) (see chapter 12).

2. Reaction of RSK, RTGA and RNGA (see chapter 12).

3. Isolation of the virus in chicken embryos and cell culture Hela, Hep-2.

4. Detection of Guarnieri bodies in infected cells.

5. Detection of Paschen bodies in the contents of vesicles (morozov stain).

Control questions
1. What is the size and structure of the pox virion?

2. What are the main methods for cultivating the variola virus?

3. The pathogenesis of natural smallpox.

4. Immunity and specific prophylaxis? By whom and when was the first decree
on compulsory vaccination against smallpox signed?

5. What are the main methods for diagnosing smallpox?

Unclassifiable viruses
Chapter 47
Infectious hepatitis has been known for a long time. Hippocrates also described
the contagious form of jaundice. But only in 1883, the Russian doctor S.P. Botkin,
after lengthy observations and research, came to the conclusion that this is a disease
of an infectious nature. In honor of Botkin, infectious hepatitis was named after
him "Botkin's disease".

The assumption of a viral etiology of hepatitis was made in 1937 when studying
an outbreak of jaundice after mass immunization of soldiers.

Currently, it is known that viral hepatitis combines two independent diseases:

hepatitis A - infectious, hepatitis B - serum. The causative agents of these diseases
belong to different qualification groups.

Hepatitis A virus
Identified by Feiston and others in 1973 in the feces of a patient using electron
microscopy.

Morphological structure . It is a small virus (20-25 nm). The type of nucleic

acid has not been definitively established. It is believed to contain RNA. The shape
of the virus is cuboidal. It consists of a nucleoid and an outer shell.

Type A virus is called HAV (hepatitis A virus).

Cultivation . Viruses do not reproduce in cell cultures, but they can be

cultivated in the body of South American monkeys.

Antigenic structure . No serotypes have been found in the hepatitis A virus. It

interacts only with HAV antibodies.

Resistance to environmental factors . Type A viruses are quite stable. Their

complete inactivation occurs only when boiling for 30-40 minutes. They tolerate
low temperatures, drying, exposure to acids, ether, and are not destroyed by UV
rays. The usual concentrations of disinfectant solutions destroy them in 40-60
minutes.

Susceptibility of animals . Monkeys (chimpanzees) are susceptible to hepatitis

A virus.

Sources of infection . Sick people with a clinically pronounced icteric and

anicteric form of the disease.
 transmission paths . HAV is transmitted predominantly through food. Flies can
be mechanical carriers. The virus is more often transmitted in the first days of
illness.

Pathogenesis . The main route of penetration of the virus is through the mucous

membrane of the gastrointestinal tract. Once in the gastrointestinal tract, HAV
penetrates into the epithelial cells of the intestinal mucosa, and from there into the
blood. Viremia occurs. With blood, viruses spread throughout the body and affect
parenchymal organs. HAV has the highest tropism for hepatocytes (liver cells), in
the cytoplasm of which the virus reproduces. At the same time, protein and
carbohydrate metabolism is disturbed. Bile acids appear in the blood, the feces
become discolored, and the urine darkens. Violation of carbohydrate metabolism
leads to an increase in the blood serum of enzymes: aldolase and
transferase. Hepatitis A virus most often infects children aged 1 to 15 years.

Immunity . Postinfectious immunity is stable. Antibodies belong to IgM and

IgG (see chapter 12).

Prevention . Isolation of patients, disinfection of feces, sputum of patients,

general sanitary measures.

specific prophylaxis . Immunoglobulin is administered to children aged 3 to 15

years who have been in contact with a patient with hepatitis, which reduces the
incidence, and in case of illness, it alleviates the severity of the course.

Treatment . Symptomatic.

Hepatitis B virus
Parenteral serum hepatitis is caused by type B virus. It is called HBV (hepatitis
B virus).

In 1961, Bloomberg and co-authors discovered an antigen in the blood serum of

patients, which was named Australian. And in 1970, Dane also found larger
particles in the blood serum of patients with hepatitis, which they called Dane
particles.

Morphological structure . The HBV virus occurs in three morphological forms.

1. Small spherical particles - 22 nm in diameter.

 2. Tubular particles of different lengths. It is believed that these two forms are
free protein particles - capsids.

3. Virus-like particles - 40 nm (described by Dane and called Dane

particles). They have double stranded DNA. They found lipids, polypeptides,
carbohydrates and a double shell. Currently, they are considered a full-fledged
virion - HBV itself.

Cultivation . Hepatitis B virus is reproduced in the culture of human embryonic

hepatocytes, in diploid human liver cells, and also in the organs and tissues of
chimpanzee monkeys. The virus is difficult to culture, making diagnosis difficult.

Antigenic structure . Dane's particles were found to have HBV antigen. This is

a complex antigen, which includes: polypeptides, carbohydrates, lipids. Antibodies
formed against it are called anti-HB S . In the core of the Dane particle there is a
cor-antigen - antibodies to it are called anti-HBc.

Resistance to environmental factors . At a temperature of 60 ° C, the virus

persists for 3-4 hours. Low temperatures do not affect it. In frozen blood products,
it remains viable for up to 20 years. HBV are resistant to ether. 5% formalin
solution inactivates them after 12 hours, 3% chloramine solution - after 2 hours.

Sources of infection . Blood of patients and antigen carriers.

transmission paths . Type B hepatitis is transmitted parenterally by injection of

vaccines, serums, drugs, non-sterile hepatitis virus-infected needles, syringes,
scarifiers.

Pathogenesis . The incubation period for type B hepatitis is 2-6 months (for

hepatitis A - 3-6 weeks). Having penetrated the hematogenous, lymphogenous
route, HBV enters the liver, and then the disease develops in the same way as with
hepatitis caused by HAV. Differential diagnosis of hepatitis A and B according to
clinical data is difficult.

Prevention . Of greatest importance for the prevention of type B hepatitis is the

correct sterilization of instruments and the selection of donors when using their
blood. Donated blood is tested for the presence of the HB S antigen.

Preventive measures for medical personnel working with HBV-infected material

are based on special care when working with blood (treatment of hands
contaminated with blood, 2% hydrogen peroxide solution, etc.).
 specific prophylaxis . Immunoglobulin is administered to persons at risk of
infection.

A vaccine prepared from the HB S  antigen is currently being developed .

Treatment . Symptomatic.

Virological diagnostics

For the diagnosis of hepatitis A and B, biochemical parameters of the blood

serum of patients are widely used, which reflect the functional state of the
liver. The violation of the pigment metabolism of liver function is judged by the
amount of free and bound bilirubin in the serum. To detect a violation of the
protein-synthetic function, a thymol test is performed. All these studies are carried
out in clinical laboratories.

Virological tests for diagnosing HAV . Virological diagnosis is carried out by

determining antigens and antibodies in the blood. For this purpose, the most
sensitive serological methods are used: RPG (gel precipitation reaction), counter
immunoelectrophoresis, etc. Serological methods have been developed for the early
and retrospective diagnosis of viral hepatitis: in the acute form, the diagnosis is
reduced to the detection of the virus in the feces of patients by immunoelectron
microscopy with the serum of convalescents .

Retrospective diagnosis of hepatitis A is carried out by the method of studying

paired blood sera of a patient in RSK and RPG (see Chapter 12).

Virological tests for diagnosing HBV . 1. In the acute stage, diagnosis is

carried out by detecting the HBS antigen in the patient's blood serum.

2. Detection of anti-HB S in CSCs. Anti-HBS S appear only 2-4 weeks after the

onset of the disease and circulate in the blood for a long time.

3. Retrospective diagnosis of hepatitis B is carried out by determining anti-

HB S in paired blood sera of the patient in the gel precipitation reaction (see
Chapter 12).

Control questions
1. What diseases are caused by hepatitis viruses and what are the modes of
transmission?
 2. What is the resistance of hepatitis viruses?

3. What immunoglobulins cause immunity after infectious hepatitis A?

4. What are Dane particles?

5. What indicators are used for laboratory diagnosis of hepatitis A and B?

6. What studies are carried out for retrospective diagnosis in hepatitis A and B?

Oncogenic viruses
The idea that tumors are of infectious origin and are caused by viruses has been
expressed for a long time.

At the beginning of the 20th century, a number of scientists (V. Ellerman, O.

Bang, P. Raus) experimentally proved that some viruses are capable of causing
tumor diseases (chicken leukemias and sarcomas, etc.). Over the past decades, the
viral etiology of a number of tumors has been experimentally proven: breast cancer
in mice, rabbit fibromas, human skin papilloma, etc.

More than 150 oncogenic viruses are now known. Some of them are well
studied.

The main difference between oncogenic viruses and other viruses is that they do
not have a cytopathic effect on cells, but change (transform) them in the direction
of unhindered reproduction, leading to the formation of a tumor.

Oncogenic viruses, like all other viruses, are divided into DNA- and RNA-
containing. DNA-containing oncoviruses include: papovaviruses, adenoviruses,
herpes viruses, etc. RNA-containing viruses have several types: A, B, C, D (all
these types are similar in their physicochemical properties). RNA-containing
viruses include the pathogens of Rous sarcoma, avian leukemia, etc.

There are different theories explaining the mechanism of action of DNA-

containing oncogenic viruses. The virogenetic theory of L. A. Zilber (1961)
received wide recognition. The essence of this theory lies in the fact that viral DNA
is integrated into the genome of the host cell and can remain there in a latent state,
as was experimentally shown in experiments with temperate phages (see Chapter
8). Under certain conditions, for example, when exposed to a number of physical
and chemical factors, the viral genome changes, upsets cellular regulation, which
leads the cell to uncontrolled division and tumor formation. However, the virus
cannot be detected in a mature tumor. It is believed that the virus in this case plays
the role of a trigger.

The ability to transform a cell varies among different viruses. For example, some
viruses transform only one in 1000 cells, others only one in 100,000 cells. And the
Rous sarcoma virus transforms every cell it infects.

Despite the fact that some oncogenic viruses have been isolated, studied, and
even the tumors caused by them have been experimentally reproduced in animals,
there is still no reliable evidence of the viral nature of all tumors.
 Part III. Sanitary microbiology
One of the basic principles of Soviet health care is prevention, since it is better to
prevent the onset of a disease than to treat already developed ones.
That is why the Soviet Union has organized a wide network of sanitary and
prophylactic institutions, institutes dealing with questions of sanitation and hygiene,
and about 5,000 sanitary and epidemiological stations.
Sanitary microbiology deals with the study of microorganisms and the processes they
cause in the environment (water, soil, air, food, etc.).
The main task of sanitary microbiology is to prevent the occurrence of infectious
diseases, which is achieved by studying the ecology of microorganisms, developing
practical measures to combat infectious diseases.
The direct detection of pathogenic microorganisms in the external environment
presents significant difficulties, since they are found in it inconsistently and in small
quantities. Therefore, they use indirect indicators of environmental contamination -
the identification of sanitary-indicative microorganisms.
Sanitary-indicative microorganisms are permanent inhabitants of the surfaces and
cavities of the human and animal body, excreted from the body in the same ways as
pathogenic microorganisms. Therefore, the more sanitary-indicative microorganisms
are revealed, the greater the likelihood of pathogenic microorganisms entering the
environmental objects.
For each object of the external environment, there are certain sanitary-indicative
microorganisms - evaluation criteria for bacteriological indicators. For example, in
relation to intestinal infections, the role of such indicators belongs to Escherichia coli
- permanent inhabitants of the intestines of humans and animals.
For conducting sanitary and microbiological studies, there are special state all-Union
standards - GOSTs or guidelines that allow you to assess the compliance of the
microflora identified in the environment with hygienic requirements.
GOSTs or guidelines provide for:
1. Sampling rules.
2. The amount of material taken.
3. Conditions of transportation.
4. Research methods.
5. The purpose of the study.
6. Criteria for evaluating the results obtained.
An accompanying document is attached to each sample taken, indicating:
1. Sample name (water, soil, food, etc.).
2. Place of sampling and number.
3. Date (year, month, day, hour).
4. The purpose of the study.
5. Where the sample is sent for research.
6. Signature of the person who took the sample.
Note. In some cases, for example, when studying water sources, soil, the
meteorological conditions at the time of sampling are noted.
Transportation of selected samples is always carried out at a temperature not higher
than 6-8 ° C, so that there is no reproduction and death of microorganisms. This
temperature is maintained with the help of rubber bags filled with warm water in
winter and ice in summer. For transportation and storage of the taken samples, it is
better to use cool bags or containers with ice.
In the laboratory, the received samples are recorded in journals. Bacteriological
examination should be carried out no later than 3-6 hours from the moment of
taking the material.
 Sanitary-bacteriological study of water - F.K. Cherkes
Water to be tested:
1) centralized water supply;
2) from wells of various types;
3) open reservoirs (rivers, lakes, seas);
4) swimming pools;
5) waste water.
Note. Samples of chlorinated water are taken into bottles with a dechlorinator
(hyposulfite).
Water sampling . Water is taken from open reservoirs using special bottles or bottles
equipped with weights. It is recommended to take a water sample at a depth of 10-
15 cm from the surface (since the surface is exposed to atmospheric factors) and at a
distance of 1.5 m from the shore (water near the shore may be contaminated with
soil microflora).
For tap water sampling, sterile vials with a capacity of 500 ml are used, closed with
cotton-gauze stoppers and covered with paper caps.
The tap is pre-fired with a swab moistened with alcohol, after which the water is
drained for 10-15 minutes and collected in vials. The filled vials are closed with
sterile stoppers.
Note. 333 ml of water are examined (Table 54).
 Table 54. GOST 16963-73 empirical table

In the distribution network of the water supply system, water sampling is carried out
depending on the number of people living in the service area.
Standard research methods are regulated for central water supply water (GOST
18963-73) and provide for:
1. Determination of the total number of microorganisms (in 1 ml of the test water
there should be no more than 100).
2. Determination of the if-index and if-titer (if-index is 3, if-titer is 333 and higher; for
Moscow and Leningrad, if-index is not more than 2, and if-titer is more than 500).
3. Research according to epidemiological indications for pathogenic microflora
(pathogenic microorganisms should not be detected).

Determination of the total number of bacteria

According to GOST 18963-73, the total number of bacteria is the number of

microorganisms that are contained in 1 ml of the test water, capable of forming
colonies visible to the naked eye (or when magnified with a magnifying glass) during
the day at a temperature of 37 ° C.
First day of research
In the study of tap water, 2 cups are inoculated. 1 ml of undiluted water is added to
one of them, and 1 ml of water diluted 10 times (that is, 0.1 ml of the original
sample) is added to the other.
When examining more polluted water, 1 ml of water diluted 100 times is
inoculated. This corresponds to 0.01 and 1 ml of water diluted 1000 times (0.001 ml),
etc. To obtain such volumes, tenfold dilutions are prepared sequentially, 1 ml of each
dilution is added to the cup and poured into a thin layer (12-15 ml ) melted and
cooled to 45 ° C nutrient agar. For a uniform distribution of the test water, the dishes
filled with agar are mixed by rotating them. After the agar has solidified, the
inoculations are placed in a thermostat and incubated at 37°C for 24 hours.
Second day of research
Cups with crops are removed from the thermostat and the number of grown
colonies is counted. Take into account only those dishes where the number of
colonies is in the range of 30-300. If there are few colonies, they are counted with
the naked eye or with a magnifying glass.
If there are many colonies, then the count can be carried out using a special device
for counting microbial colonies (Fig. 54).
Rice. 54. Device for counting colonies of microorganisms. 1 - table for Petri dishes; 2
- a needle with a spring device; 3 - indicator of the counter; 4 - toggle switch to turn
on the pulse counter; 5 - toggle switch to turn on the meter lighting lamp

The calculated number of colonies is multiplied by the dilution and the number of
microbes in 1 ml of the test water is found out.

Definition of BGKP

The presence of BGKP (bacteria of the Escherichia coli group) is an indicator of fecal
contamination, the intensity of which is characterized by:
Coli index - the number of E. coli found in 1 liter of water.
Coli-titer - the smallest amount of water in which the presence of Escherichia coli * is
detected .
*
 ( If-titer and if-index is one indicator, differently expressed. )
To detect BGKP in water, two methods can be used: titration (fermentation) and the
method of membrane filters.
 Titration method
For the study of water, the accumulation medium is glucose-peptone (GPS) Eikman
medium with an indicator and fermentation tubes. The medium is prepared
concentrated (10 times) and normal concentration - for sowing 1 ml of water.
First day of research
The test water is inoculated in 100 ml in 3 flasks, 10 ml in 3 test tubes (with a
concentrated medium) and 1 ml in 3 test tubes (with a medium of normal
concentration) - a total of 333 ml. The cultures are incubated in a thermostat at 37°C
for 24 hours.
Second day of research
Take out the crops from the thermostat and view them.
In the presence of turbidity in the flasks or test tubes, they are inoculated with a loop
on the sectors of the Endo medium in Petri dishes. The cultures are incubated in a
thermostat at 37°C.
Third day of research
Take the cups out of the thermostat. Swabs are made from suspicious colonies. In
the presence of gram-negative rods, a test is made for oxidase activity. A positive
test for oxidase gives the right to give a negative answer.
Oxidase test . 1st method: 2-3 colonies of each type are removed from the Endo
medium and applied to the surface of filter paper moistened with dimethyl
paraphenylenediamine. A positive reaction is characterized by blue strokes made
from the colonies.
2nd method: the reagent can be applied to an isolated colony on Endo's medium
(red colony turns blue) (Fig. 55).
Rice. 55. Determination of coli-index of water by titration method
A negative test for oxidase indicates the presence of BGKP in the water. In this case,
calculate if-index and if-titer using standard (empirical) tables of GOST 16963-73 (see
table. 54).
These tables provide for every possible combination of culture volumes from which
E. coli is isolated.
Membrane filter method
To filter water, you can use a Goldman funnel with a capacity of 700-800 ml.
First day of research
A measured volume of the test water is poured into the funnel of the mounted and
sterilized Seitz filter device. Using a pump, a vacuum is created in the receiving vessel
(usually water is filtered through filters No. 2 and 3). At the end of filtration, remove
the filter with sterile or fire-burnt tweezers and place it on the Endo medium in a
Petri dish so that the surface with microbes settled on it faces upwards (3-4
membrane filters can be placed on one dish).
Crops are incubated in a thermostat at a temperature of 37 ° C for 18-24 hours.
Second day of research
Cups with crops (filters) are removed from the thermostat. The absence of suspicious
colonies gives the right to give a negative answer.
All red and pink colonies with or without a metallic sheen are subject to
registration. Smears are made from the grown colonies, stained according to Gram
(Fig. 56).
 Rice. 56. Determination of coli-index of water by the method of membrane filters

In the presence of gram-negative rods, a test for oxidase is put. A positive oxidase
test gives the right to give a negative answer. With a negative oxidase test,
inoculation is carried out on a semi-liquid medium with glucose and an indicator or
on a HPS medium with fermentation tubes - to detect the fermentation of
carbohydrate to acid and gas. In the presence of acid and gas, the coli index is
calculated. For example, 3 colonies grew on all filters on Endo medium, 300 ml of
water was passed through the filter.
Calculation. 300 ml - 3 colonies
1000 ml - x
x = 10; if-index 10
Note. The titration method is more accurate and can be used in the presence of
impurities in the water. The membrane filter method is more economical and makes
it possible to give an answer on the 2nd day.

Detection of fresh fecal contamination

To determine the presence of fresh fecal Escherichia coli in the water, water is sown
(3 volumes) on a lactose-peptone medium with boric acid. Incubate at 43°C for 24
hours. The presence of acid and gas indicates fresh faecal contamination.
According to epidemiological indications, salmonella, shigella, enteroviruses are
determined in water.
Note. Enterococci are a commonly accepted additional indicator of faecal
contamination of drinking water. When conducting a bacteriological study, all groups
of enterococci are determined, although fecal streptococci are predominantly of
sanitary importance, the detection of which is an indicator of fresh fecal
contamination.
Control questions
1. What is the main task of sanitary microbiology?
2. What are sanitary indicative microorganisms?
3. What is coli-index and coli-titer?
4. What methods of determining BGKP do you know?
Exercise
Determine the total number of microbes in the water sample under
study. Environment GPS (Eikman).
Nutrient media
GPS (Eikman) concentrated . In 1 liter of water dissolve 100 g of peptone, 50 g of
sodium chloride. The mixture is heated to a boil, filtered, 100 g of glucose is added,
pH is set to 7.4-7.6 and poured into 10 ml flasks with a capacity of 250 ml, 1 ml into 3
test tubes (concentrated medium) and 1 ml into 3 test tubes with medium of normal
concentration (in all containers, the medium is adjusted to the desired concentration
with sterile water).
Note. In the study of especially polluted waters, large dilutions are made (for
example, 10 -6 , 10 -7 , etc.).
 Sanitary and bacteriological study of the soil - N. A.
Belskaya
Soil is the main habitat for many microorganisms (see Chapter 6). From the soil,
microbes enter the water and seed the air.
The microbiological study of the soil is essential. It is carried out when choosing a site
for the construction of children's institutions, sports grounds, hospitals, hospitals,
military camps, waterworks and other facilities.
Sanitary and microbiological analysis of the soil includes the definition of:
1) the total number of bacteria in 1 g of soil;
2) the titer of sanitary indicative microorganisms BGKP and C. perfringens;
3) thermophilic bacteria in 1 g of soil;
4) according to epidemiological indications, a study is carried out for the presence of
pathogenic microorganisms (salmonella, shigella, tetanus clostridium, botulism,
some viruses, etc.).
Soil sampling . The choice of a place for soil sampling is determined by the sanitary
doctor and bacteriologist, depending on the purpose and objectives of the study. On
the surveyed area up to 1000 m 2, two plots with an area of 25 m 2 are
allocated . One should be located near sources of pollution (landfills, dustbins,
cesspools, etc.), the other - at a distance from them (control). On each plot of 25
m 2 , five sampling points are planned: four at the corners and one in the center, or
five points along the diagonal of the plot.
To study the surface layer of the soil, samples are taken with a sterile spatula or
scoop at a depth of up to 20 cm. A whole piece of soil is dug from individual points of
the site with a spatula. The upper layer 1.5-2.0 cm thick is removed with a sterile
knife and 200-300 g of soil is collected from the middle of the piece with a sterile
spoon. A mixed sample composed of five individual soil samples must weigh at least
1 kg.
When examining samples from deep soil layers (from 0.75 to 2 m), a special drill with
a cavity is used. At a given depth, the drill cavity opens, fills with soil, then closes
mechanically, and the drill is removed to the surface.
Soil samples taken for analysis are transferred to sterile jars with cotton-gauze
stoppers and covered with sterile parchment paper. Each jar is labeled with the date
and sample number. The accompanying document notes the nature of the soil, the
location of pollution sources, the area of the surveyed territory, data characterizing
the climate of the area, etc.
All samples are placed in a wooden box with nests and immediately transported to
the laboratory. If it is not possible to start the study of the soil on the same day, then
it is allowed to store the samples in a refrigerator at 1-2 ° C during the day.
Preparation of soil samples for research. Soil samples taken in one area from several
points are mixed well, freed from large inclusions (rubble, stones, roots, glass). 200-
300 g is separated from the average sample and introduced into a sterile dish. Then
the soil is crushed in a sterile mortar, sieved through a sterile sieve onto sterile paper
and a 30 g sample is taken for research. A soil sample is poured into a sterile flask
with a capacity of 500 ml and 270 ml of sterile tap water is added, a soil dilution of
1:10 is obtained. The soil suspension is shaken for 10-15 minutes, and from the
prepared dilution 1:10 without settling, a series of consecutive tenfold dilutions is
prepared according to the generally accepted method. In the analysis of clean soils,
they are limited to 3-4 dilutions (up to 1:1000, 1:10000), in the study of
contaminated soils, dilutions are used - up to 1:100000, 1:1000000.
The determination of the total number of bacteria in the soil is carried out similarly
to the study of water. Indicators of the total number of bacteria for different types of
soils are presented in Table. 55.
Definition of BGKP
The definition of BGKP as an indicator of fecal contamination is carried out by two
methods: titration and the method of membrane filters.
Titration method
First day of research
From the initial dilution of soil suspension 1:10, 10 ml is taken with a sterile pipette,
which corresponds to 1 g of soil, and inoculated into vials with 50 ml of Kessler
medium. Then, 1 ml of each soil dilution is inoculated into test tubes with floats
containing 9 ml of the same medium. The crops are grown in a thermostat for 24
hours at 37°C.
 Second day of research
Crops are viewed (if growth is retarded, crops are left on the third day). The absence
of gas formation and turbidity in the fermentation vessels with the Kessler medium
after 48 hours makes it possible to give a negative answer.
If there is gas formation and turbidity in the media, or only turbidity from these
vessels, a loop is sown on the sectors of the Endo medium in Petri dishes. Plates with
inoculations are incubated in a thermostat at 37°C for 24 hours.
Third day of research
Seeing crops. Lack of growth on Endo's medium gives the right to a negative answer.
If colonies typical for Escherichia coli grow on Endo's medium, then smears are made
from them, stained according to Gram and microscoped. When gram-negative rods
are detected in smears, a test for oxidase is put. If the oxidase test is negative, then
the enzymatic properties of the isolated culture are checked by inoculation on a
semi-liquid medium with glucose. The cultures are placed in a thermostat for 24
hours at 37°C.
Fourth day of research
Seeing crops. The appearance of acid and gas in the medium confirms the presence
of Escherichia coli in the studied soil dilution.
The coli-titer of the soil is determined by the smallest volume in which BGKP is found
(the coli-titer indicators for various types of soils are presented in Table 55).

Table 55

Membrane filter method

The method of membrane filters is used in the study of lightly polluted soils. 10 ml of
soil suspension from dilutions 1:10, 1:100, 1:1000 are passed through sterile
membrane filters No. 3. The further course of the study is similar to the definition of
Escherichia coli in water. The method of membrane filters allows to reduce the study
period to two days. The results of the analysis are expressed by the coli index. The
soil coli index is the number of Escherichia coli in 1 g of soil.
Note. Kessler's medium contains lactose, which is fermented by CGBs, and gentian
violet, which inhibits the growth of gram-positive microflora.
Determination of C. perfrinrens titer
Of all the prepared soil dilutions (from 1:10 to 1:1,000,000), 1 ml is introduced into
two parallel rows of sterile test tubes. One row of test tubes is heated at 80°C for 15
minutes to remove non-spore-bearing microflora. Then, 9 ml of molten and cooled
to 45 ° C Wilson-Blair medium, prepared ex tempore, are poured into all test
tubes. The test tubes are rotated between the palms so that the inoculum is evenly
distributed in the nutrient medium, and they are quickly lowered into cold water to
remove oxygen and cool the medium. The crops are grown at 43°C for 24 hours.
C. perfringens grows deep in the medium as black colonies. Gas formation is
recorded by the rupture of the nutrient medium. In smears prepared from colonies,
gram-positive large rods with oval-shaped spores located centrally or subterminally
are found.
The limiting dilution of soil suspension, which gives the growth of C. perfringens on
the Wilson-Blair medium, means the titer of this microbe in the soil (see Table
55). The presence of C. perfringens in the soil is an indirect indicator of the presence
in it of other clostridia - the causative agent of tetanus (C. tetani), the causative
agent of botulism (C. botulinum).
In the soil, the number of thermophilic bacteria in 1 g is also determined. Soil, in
which there are many Escherichia coli and few thermophiles, can be considered as
contaminated with faeces.
Nutrient media
Wednesday Kessler . To 1 liter of distilled water add 10 g of peptone, 50 ml of ox
bile. The mixture is boiled for 20-30 minutes, filtered through cotton wool, 10 g of
lactose are added and the volume is adjusted to 1 liter. The pH is set to 7.4-7.6. Add
4 ml of a 1% aqueous solution of gentian violet. The medium is poured into flasks
and test tubes with floats. Sterilize for 15 minutes at a pressure of 0.5 atm (112 °
C). The medium is purple.

Control questions

1. In what cases is a sanitary and bacteriological study of the soil carried out?
2. What definitions include the sanitary-bacteriological analysis of the soil?
3. How is soil sampling carried out?
4. What methods determine the presence of BGKP in the soil?

Tasks

1. Prepare a series of successive dilutions of 1:100, 1:1000, 1:10000 from soil

suspension at a dilution of 1:10 and determine the microbial number in this soil
sample.
2. Take from the teacher ready-made crops of soil dilutions on the Wilson-Blair
medium, determine the titer of C. perfringens. Make colony smears, Gram
stain. Look for C. perfringens in the smears under the microscope and show the
teacher. Sketch the results of microscopy in a notebook.
 Sanitary and bacteriological examination of air - F. K.
Cherkes
Among the environmental factors that affect human life, air occupies a leading
place. The science that studies the microflora of the air is called aeromicrobiology.
The air is not a favorable environment for the development of microorganisms, as it
does not contain nutrients and is in constant motion. Therefore, most
microorganisms quickly disappear from the air. However, some of them are more
resistant, such as tubercle bacillus, spores of clostridium, fungi and others, can
remain in the air for a long time.
There are more microorganisms in the air of cities than in the air of forests and
fields.
The number of microorganisms in the air decreases with height. For example, at an
altitude of 500 m above Moscow , 2-3 bacteria are found in 1 m 3 of air, and at an altitude of 1000 m
- half as much.

The number of microorganisms in the premises is usually greater than in the air of
open places.
GOST does not standardize methods for conducting air research. Previously, much
attention was paid to the definition of hemolytic streptococci as indicators of indoor
air pollution by microflora located in the human nasopharynx. Currently, more
attention is paid to the direct detection of pathogenic and opportunistic
microorganisms in the air.
Sanitary and bacteriological examination of air is carried out in a planned manner: in
hospitals, operating rooms, children's institutions, etc.
In a sanitary-bacteriological study, determine:
1. The total number of bacteria in 1 m 3 of air.
2. The presence of pathogenic and conditionally pathogenic microorganisms in 1
m 3 of air.
Detection of microorganisms in the air is carried out with the help of special devices
and special environments (diagnostic and differential diagnostic).

Air sampling methods

There are two main methods of air sampling for research: 1) sedimentation - based
on the mechanical settling of microorganisms; 2) aspiration - based on the active
suction of air (this method makes it possible to determine not only the qualitative,
but also the quantitative content of bacteria).
sedimentation method
Petri dishes with a nutrient medium (MPA) are installed in an open form horizontally,
at different levels from the floor. The method is based on the mechanical
sedimentation of bacteria on the surface of agar in Petri dishes. The medium plates
are exposed for 10 to 20 minutes, depending on the expected air pollution. Elective
media are used to identify pathogenic flora. The exposure in these cases is extended
to 2-3 hours. After exposure, the cups are closed, delivered to the laboratory and
placed in a thermostat for 24 hours at a temperature of 37 ° C. The next day, the
grown colonies are studied. This method is mainly used indoors.
( Aspiration method )
Bacteriotrap Rechmensky. Before use, the device is filled with sterile soda. The
operation of the device is based on pulling air through it with the help of an
aspirator. In this case, the liquid in the device is sprayed. After the end of suction, the
liquid through which air was passed is inoculated at 0.1-0.2 ml per MPA in Petri
dishes. If necessary, use elective media increase the inoculum dose (0.3-0.5 ml). The
liquid obtained in the receiver can be used to infect animals (for example, in studies
conducted to detect viruses, rickettsia, etc.).
Dyakonov's device is also based on trapping bacteria in a liquid through which air is
passed.
The device PAB-1 is designed for bacteriological examination of large volumes of air
within a short period of time. Air samples are obtained at a rate of 125-150
l/min. The principle of operation of the device is based on capturing microorganisms
on an electrode of opposite charge. The high speed of air sampling in this device and
the possibility of seeding it on various nutrient media is important for the detection
of pathogenic and opportunistic bacteria (for example, Pseudomonas aeruginosa in
surgical departments, etc.).
Krotov's apparatus. The action is based on the principle of air jet impact on the
medium in Petri dishes. The device consists of three parts: an air sampling unit, a
rotameter, and an electrical part of the feeding mechanism.
The investigated air is sucked into the slot of the device with the help of a centrifugal
fan rotating at a speed of 4000-5000 rpm and hits the surface of the open Petri dish
with the medium. Microorganisms in the air are deposited on nutrient agar. To
evenly distribute microorganisms over the entire surface, the table with the cup on it
rotates. Air is removed from the device through an air tube, which is connected to a
rotameter showing the speed of air being drawn through the device.
The disadvantage of Krotov's device is that it needs electricity, so it can not be used
in all conditions.
First day of research
The selected samples are placed in a thermostat at 37°C for 18-24 hours.
Second day of research
The cup is removed from the thermostat and the colonies are counted. Bacterial air
pollution is expressed by the total number of microbes in 1 m 3 of it.
Calculation. For example, 125 liters of air were passed in 10 minutes, 100 colonies
grew on the surface.
100×1000

The number of microbes in 1 m of air = = 800

125

To determine Staphylococcus aureus, sampling is carried out on yolk-salt agar. Cups

with crops are incubated in a thermostat at 37°C for 24 hours and kept at room
temperature for 24 hours to detect the pigment. Colonies suspected of S. aureus are
subject to further identification (see Chapter 14).
In children's institutions, the air is checked for the presence of salmonella. To do this,
air is inoculated into a cup with bismuth-sulfite agar medium.
Detection of pathogenic bacteria and viruses in indoor air is carried out according to
epidemiological indications. To identify tuberculosis pathogens, a POV device is used;
Shkolnikova's medium is used as a trapping medium.

Control questions

1. Is the air a favorable environment for the development of microorganisms?

2. In what institutions is a planned study of air microflora carried out?
3. Tell the device of Krotov's apparatus.

Task

In 10 minutes, 250 liters of air were passed through. 150 colonies grew. Calculate the
number of colonies in 1 m of air.

Exercise

Take 4 Petri dishes with MPA medium, open them and place them at different levels
from the floor. After 20 minutes, close the cups and place in the thermostat. The
next day, count the number of grown colonies, determine the degree of air pollution.
 Sanitary and bacteriological examination of milk and
dairy products F. K. Cherkes, N. A. Belskaya
Milk and dairy products are a favorable environment for the reproduction of
microorganisms.
In the manufacture of some dairy products: cottage cheese, kefir, curdled milk,
fermented baked milk and others, special microflora is used, for example, lactic
streptococci, lactic acidophilus bacilli, etc. The microflora used to prepare these
products is specific to them and is not taken into account.
Nonspecific microflora found in milk and dairy products are aerobic bacteria: CGB,
staphylococci, etc.
The pathogens of tuberculosis, brucellosis, salmonellosis, anthrax, polio virus,
anaerobic bacilli, etc. can be transmitted with milk.
The contamination of milk and dairy products with non-specific microflora can occur
at the time of milking, transportation, storage, etc.
The study of milk and dairy products is carried out in accordance with GOST 9225-68.
Sampling . Samples of liquid and semi-liquid products after their thorough mixing are
taken in an amount of 50-100 ml into sterile flasks. Samples of butter, cheese,
cottage cheese are taken with a sterile probe from the depth of the product. Before
taking a sample of butter, cottage cheese, the top layer of the product is carefully
cleaned, and the surface of the cheese at the sampling point is cauterized with a hot
knife. From packaged products, take 2 samples in the original packaging. The
samples taken are accompanied by a document stating:
1. Sample number.
2. Name and grade of the product.
3. Date of manufacture.
4. Date and hour of sampling.
5. Amount of necessary research.
6. Position and signature of the person who took the sample.
Microbiological examination of the product should be carried out no later than 4
hours from the moment of sampling. During transportation, the temperature should
not exceed 6°C.
GOST for milk and dairy products provides for the determination of the total number
of bacteria in 1 g (ml) and the determination of the titer of citrate-negative (citrate-
negative) varieties of BGKP (coli-titer).

Determination of the total number of bacteria

Preparation of samples for research . Tenfold dilutions are prepared from milk and
other dairy products (according to the generally accepted method). The number of
dilutions for each type of product is prepared taking into account the most likely
microbial contamination (Table 56).

Table 56

Note. To determine the total number of bacteria, one should choose those dilutions
that, when sown on plates, grow at least 50 and no more than 300 colonies.
Sowing . 1 ml of each dilution is added to 2-3 sterile Petri dishes and 12-15 ml of
nutrient agar, melted and cooled to 45°C, are poured. Cups are pre-
labeled. Immediately after pouring, the contents of the cup are stirred (by slight
rotational rocking) to evenly distribute the inoculated material. Crops are placed in a
thermostat at 37 ° C for 48 hours.
At the end of the incubation period, the dishes are removed and the number of
colonies is counted using a counter. The number of colonies grown on each plate is
multiplied by the appropriate dilution. The results obtained for individual dishes are
added, divided by the number of dishes and the arithmetic mean is obtained, which
is an indicator of the total number of bacteria in 1 g (ml).
The relevant GOSTs regulate the quality of products, which is established according
to acceptable indicators: the total number of microbes and coli-titer. An example for
two types of products is presented in Table. 57.

Table 57. Indicators of the total number of bacteria and coli-titer in milk

Note. For other dairy products, there is also a GOST stipulating the permissible
number of microbes in 1 ml (g) of the product. The letters A and B indicate the
category of the product.
In fermented milk products (kefir, curdled milk, cottage cheese, sour cream, etc.)
containing abundant specific microflora, the total number of bacteria is not
determined, but the composition of the microflora is controlled. To do this,
preparations are prepared from fermented milk products and stained with
methylene blue. In the field of view of the preparation should be only
microorganisms specific to this product. For example, for curdled milk - lactic acid
streptococci and sticks; for kefir - lactic acid streptococci and sticks, single
yeast. Microscopy reveals spoilage microorganisms (molds and large amounts of
yeast).

Definition of BGKP

The contamination of milk and dairy products with bacteria of the Escherichia coli
group is determined by the fermentation method. Fermentation titer is the smallest
amount of products, expressed in grams or milliliters, in which E. coli is
present. According to GOST 9225-68, only citrate-negative varieties of Escherichia
coli are taken into account (Fig. 57).
Rice. 57. Determination of coli-titer of milk
 fermentation method
First day of research
Sowing of milk and lactic acid products is carried out in 6 test tubes with 5 ml of
Kessler medium. In 3 test tubes, 1 ml of the whole product is inoculated, in the other
3 test tubes, 1 ml from a dilution of 1:10 (0.1 ml). The cultures are incubated in a
thermostat at 43°C for 18-24 hours.
Second day of research
From each fermented tube, inoculation is made on the sector of the Endo medium
and incubated at 37 ° C for 18-24 hours.
Third day of research
In the absence of typical CGB colonies, the product is considered uncontaminated
with Escherichia coli.
If colonies typical of CGB are present, smears are made, Gram-stained, and
microscopically examined. When gram-negative rods are detected, a sample is
placed on oxidase and inoculated on a medium with glucose and Coser's medium.
Fourth day of research
Record the results. The presence of acid and gas on the medium with glucose and
the absence of growth on the medium of Coser indicates the presence of citrate-
negative varieties of Escherichia coli. If the titer is calculated according to the
table. 58.

Table 58

Note. The calculation of the coli-titer for butter, cheese, curd products, ice cream
and canned milk is carried out according to other tables specified in GOST.
The presence of pathogenic microorganisms in milk and dairy products is
unacceptable.
Control questions
1. How is the total number of microbes determined in milk and dairy products?
2. How is the coli-titer determined?
3. What microorganisms can be found in milk and dairy products?
Nutrient media
Wednesday Kessler . See p. 484.
Coser Wednesday . To 1 liter of distilled water add 1.0 g of monopotassium
phosphate, 0.2 g of magnesium sulfate, 2.5-3.0 g of sodium citrate. The solution is
sterilized in an autoclave at 1 atm for 15 minutes, 10 ml of a 0.5% alcohol solution of
bromthymol blue is added and poured into sterile test tubes.
medium with glucose . See chapter 7.
Yolk-salt environment . See chapter 14.
 Sanitary and bacteriological examination of cream and
cream products - F. K. Cherkes
Sanitary and bacteriological examination of the cream is carried out in accordance
with guidelines No. 1351-75, approved by the USSR Ministry of Health, which provide
for:
1. Determination of the titer of Escherichia coli.
2. Determination of plasma coagulating staphylococci in 1 g of products.
Sampling . Cream samples are taken from the surface and from the layer. The
sample is removed with a sterile spoon in the amount of 50 g and placed in a sterile
glass dish.

Determination of the titer of Escherichia coli

First day of research

The collected material is placed in a thermostat or water bath at a temperature not
exceeding 43-45 ° C. For research, the lower part of the melted cream is used.
3 tenfold dilutions are made from the test material: 1:10, 1:100, 1:1000.
10 ml of a 1:10 dilution are inoculated into a flask with 50 ml of Kessler medium. The
remaining dilutions (1:100 and 1:1000) are inoculated 1 ml in 5 ml of Kessler medium
in test tubes. Crops are placed in a thermostat at a temperature of 43 ° C for 24
hours.
Second day of research
Of all the inoculations with the Kessler medium, they are sown on sectors of the Petri
dish with the Endo medium and placed in a thermostat at a temperature of 37 ° C for
24 hours.
Third day of research
In the absence of suspicious colonies give a negative answer.
Colonies suspicious of Escherichia coli are smeared and Gram-stained. In the
presence of gram-negative rods, the coli-titer is calculated. The titer of BGKP should
not be lower than 0.3 g.
 Identification of coagulase-positive staphylococci

First day of research

0.1 g of the melted cream is inoculated on milk-salt agar and placed in a thermostat
at a temperature of 37 ° C for 24 hours. In parallel, 0.5 ml is inoculated into 2 test
tubes with 5 ml of 6.5-10% saline broth (accumulation medium) followed by
subculture on yolk-salt agar.
Second day of research
Seeing crops. If there is growth, the plates with crops are left at room temperature
for 24 hours to detect the pigment. From the accumulation medium, seeding is
carried out on yolk-salt agar. Incubate at 37°C for 18-24 hours.
Third day of research
They study the colonies grown on the cups. Colonies suspicious of staphylococci are
studied: the enzyme coagulase is determined (see Chapter 14).
There should not be more than 500 coagulase-positive staphylococci in 1 g/ml.

Control questions

1. What is determined in cream and cream products?

2. How is the titer of plasmacoagulating staphylococcus determined?
 Sanitary and bacteriological study of sausage and meat
products of industrial production - F. K. Cherkes
Meat and meat products can be contaminated with various microorganisms. Ways of
infection of meat:
1) contamination of meat is primary (in vivo) as a result of a weakening of the
animal's body (hunger, trauma), i.e. in a state when the barrier functions of the
intestine are violated, and microbes from it enter the organs and blood;
2) microbes can seed meat, spreading throughout the body, in case of animal
diseases, such as salmonellosis;
3) microorganisms can get into meat when animals are slaughtered and carcasses
are cut (the most common case).
Meat products become infected with insufficiently strict observance of the
technological mode of production, violation of the storage regime, etc.
Sausage products and meat products of industrial production are examined in
accordance with GOST 9958-74, which regulates the definition of:
1) the total number of bacteria in 1 g of the product;
2) the presence of bacteria of the Escherichia coli group in 1 g;
3) salmonella in 5 g of the product, as well as proteus and clostridia.
Sampling . From each batch of sausages, at least two samples are taken from
different places. Of these, they make up the general sample that is being
investigated. From sausages, sausages make up a common sample of several
copies. From such products as jelly, pate and other products without casing, samples
are taken from 2-3 places in the amount of 200-250 g each.
General samples of each product are packed separately in sterile parchment. All
samples taken are sealed and labelled. The label says:
1. Product name.
2. Pickup time (date, hour).
3. Place of taking.
4. The purpose of the study.
5. Where the material is directed.
6. Signature of the person who took the sample.
Attention! Products should be transported at a temperature not higher than 6-8 ° C,
and the study should be carried out no later than 4 hours from the moment of
sampling.
Preparation of samples for research . The surface of solid products (loaf) is wiped
with a swab moistened with alcohol and fired. The loaf is cut and a sample is taken
from different places with a sterile knife. From smoked meats and other meat
products, sections are cut out, preferably closer to the bone.
From products without a shell, swabs are first taken from the surface with a
moistened swab and inoculated on the HB, Heifetz or Kessler medium. Then the
surface moistened with alcohol is fired, cut off and pieces of 20 g are cut out with a
probe.

Determination of the number of microbes in 1 g of

the product

First day of research

From each test sample, 2 inoculations of 0.1 and 0.01 g are made. To do this, 2
tenfold dilutions are made and 1 ml of each is added to 2-3 sterile Petri
dishes. Seeded cups are poured with 12-15 ml of agar melted and cooled to 45 °
C. After the agar has solidified, 4-5 ml of hungry agar is poured onto it - to prevent
the growth of the proteus. The cultures are incubated at 37°C for 48 hours.
Second day of research
The cups are removed from the thermostat, the number of grown colonies is
counted and multiplied by the dilutions made. Having determined the number of
bacteria in 1 g of the product, they compare it with GOST.

Determination of BGKP in 1 g of the product

The definition is based on the ability of intestinal bacteria to ferment mannitol to

form acidic products that change the color of the indicator in HB and Heifetz medium
(double concentration), and to break down glucose to form acid and gas (in Kessler
medium with floats).
 First day of research
In each of the above media add 5 ml of the first and second dilutions. The
inoculations are incubated at 43°C for 18-20 hours.
Second day of research
Take out the crops from the thermostat. In the presence of Escherichia coli growth,
the HB and Heifetz medium turns yellow (from blue), and gas bubbles form on the
Kessler medium. The identification of grown bacteria is carried out according to the
method described above (see Chapter 18).

Salmonella detection

First day of research

25 ml of the resulting suspension is inoculated into a vial containing 100 ml of the
accumulation medium (Muller, Kaufmann or selenite broth). Crops are incubated in a
thermostat at a temperature of 37 ° C.
Second day of research
From the accumulation medium, seeding is done on Endo media and bismuth-sulfite
agar in Petri dishes, and then the study is carried out according to the generally
accepted scheme (see Chapter 19).

Proteus Definition

First day of research

0.5 ml of the suspension is added to the freshly cut agar condensate (Shukevich's
method).
Second day of research
The presence of Proteus is determined by the presence of a creeping veil-like plaque
(it is microscopically examined and mobility is determined).
Control questions
1. When can bacterial contamination of meat and meat products occur?
2. How are samples of meat products taken?
3. What bacteriological studies are carried out in meat products?
4. What method determines the presence of a protein?
Nutrient media
"HB" (quinosol bromcresol purple medium) . In 1 liter of tap water dissolve 10 g of
peptone, 5 g of sodium chloride, 5 g of mannitol. The prepared mixture is boiled for
15-20 minutes, the pH is adjusted to 7.4-7.6, filtered through a paper filter, the
filtrate is boiled for 10 minutes, cooled to 60 ° C, after which 30 ml of yeast dialysate,
15 ml of bile, 10 ml of chinosol solution are added. and 10 ml of 1.6% alcohol
solution of bromcresol purple. The medium is poured into sterile tubes of 7-8 ml.
Heifetz Wednesday . In 1 liter of tap water (for double concentration take 500 ml)
dissolve 10 g of peptone, 5 g of sodium chloride, 5 g of mannitol. After adjusting the
pH to 7.4-7.6, the medium is heated to boiling, filtered, 1 ml of a 5% alcoholic
solution of rosolic acid and 2.5 ml of a 0.1% solution of methyl blue are added. The
medium is sterilized by boiling for 5 minutes. Pour into test tubes of 8-10 ml. The
color of the medium is red-violet.
 Sanitary and bacteriological examination of canned
food - F. K. Cherkes
The microflora of canned food can be represented by aerobic and anaerobic
microorganisms that can get into canned food when the original product is infected
or as a result of improper sterilization.
Sampling . Produced in accordance with GOST 8756-070. Initial samples are taken
from each batch of canned food, from which an average sample is prepared. From
canned food packaged in glass, tin or polymer containers with a capacity of up to 1
liter, a sample is taken from three packaging units. When packing with a capacity of
1-3 liters, 1 unit of packing is examined. From the products in barrels and boxes, take
500 g.
Preparation for the study . The banks to be examined are inspected: are there any
deformations of the box, smudges, rust. Then their tightness is determined and
checked for bombing.
The tightness is checked by immersing the cans in a pot of water heated to a
boil. The amount of water should be 4 times the capacity of the jar, and the layer of
water above the jars should be at least 5 cm. The jars are kept in water for several
minutes. Air bubbles appearing in the water indicate a lack of tightness.
Bomb check. Banks are placed in a thermostat and kept at a temperature of 37 ° C
for 5-6 days. The presence of bombage is judged by swelling of the lid or bottom of
the jar.
Banks with a lack of tightness and the presence of bombing are not subject to
research (they are rejected).
Preparation of jars for bacteriological research . The study is carried out in a box
under aseptic conditions.
The box should contain punches, glass tubes (sterile), nutrient media: broth with 1%
glucose, Tarozzi medium.
Banks are freed from labels, the data on them is recorded and washed with hot
water and soap, rinsed and wiped dry. The surface of the jar is burned with cotton
wool moistened with alcohol. The tip of a flambéed punch is brought under the
burning cotton at an angle of 30-40 ° C. A hole is punched and a glass tube 8-9 mm in
diameter, closed on one side with cotton, is inserted into it, and the test material is
collected into it. After taking the material, the hole made is closed with the lid of a
sterile Petri dish.
Sowing . To identify mesophilic aerobes (GOST 10.444.3.75), 2 cm 3 of the samples taken are inoculated into
2 test tubes with 5-6 ml of broth with 1% glucose. The inoculations are incubated at
37° C. for 5 days, observing the appearance of growth daily. In the presence of
growth, smears are made, stained according to Gram. Canned food should not
contain aerobic flora.
To detect mesophilic anaerobes (GOST 10.444.4.75), 2 cm 3 of the test product are
inoculated into 2 test tubes with 10-13 cm 3Tarozzi medium, preheated in a water
bath for 20 minutes, and quickly cooled to 40 ° C. The inoculations are placed in a
thermostat at 37 ° C for 5 days. In the presence of growth, material is taken from the
bottom of the tube, smears are made and a sample is put on catalase. If gram-
positive microbes are found in the smears, and the catalase test is negative, then 2
ml of the resulting culture is transferred to a sterile Petri dish and filled with agar
with 1% glucose. A sterile glass slide is placed on the frozen surface of the agar so
that there are no air bubbles under it. The dish is incubated upside down at 37°C for
48 hours. When bacteria grow under the glass or gaps in the agar appear, receding 3-
4 mm from the edge of the glass, anaerobes are considered to be present in the
inoculation.
According to sanitary and epidemiological indications, regardless of the type of
canned food, studies are carried out for the presence of: pathogens of coagulase-
positive staphylococci (see Chapter 14), pathogens and toxins of botulism (see
Chapter 36), Clostridium perfringens, cereus bacteria, thermophilic aerobes and
anaerobes, yeast and mold.
Canned food should not contain pathogenic microorganisms. The presence of non-
pathogenic spore-forming microbes is allowed with normal organoleptic qualities of
the product.
Nutrient media
MPB with 1% glucose . See chapter 7.
Wednesday Tarozzi . See chapter 34.

Control questions
1. How canned food is processed before taking material for research?
2. How are cans checked for leaks and bombing?
3. How is a study carried out to detect anaerobes?
 Sanitary and bacteriological examination of swabs - F.
K. Cherkes
To assess the sanitary and hygienic condition of public catering establishments, food
industry enterprises, medical and preventive and children's institutions, a study is
carried out on washings from the hands of personnel and environmental objects.
Depending on the purpose of the study, determine:
1. The presence of BGKP.
2. Presence of S. aureus.
3. The total number of bacteria.
Studies on pathogenic microflora are carried out only according to epidemiological
indications.
In food service establishments and childcare facilities, testing is usually limited to
detection of CGB (as an indicator of faecal contamination) and S. aureus.
In surgical departments (operating rooms, intensive care units, intensive care units,
etc.), in addition to the above indicators, quantitative contamination with
microorganisms, the presence of Pseudomonas aeruginosa and Proteus are
determined.
Sampling . Sampling is carried out by the method of washings. Cotton swabs are
used (a stick with cotton wrapped around it is inserted into the test tube) or napkins
5 × 5 cm, which are captured with sterile tweezers. Swabs and wipes are moistened
by placing them in test tubes with 2 ml of isotonic sodium chloride solution.
Note. Gauze napkins, previously wrapped one by one in paper bags, and cotton
swabs placed in test tubes, are sterilized in a sterilization cabinet for 1 hour at 160 °
C.
Washes from the hands are done in the following sequence: they start from the left
hand, from areas of less contamination - they wipe the back of the hand from the
hand to the fingers, then the palmar side, between the fingers and under the nail
bed. With the same swab, in the same sequence, washouts are made from the right
hand.
Washouts from household items during the control of large surfaces are made from
several places. The investigated areas are limited by a stencil frame with an area of
50×50 or 100×100 cm 2 . The stencil is made of wire and burned over the flame of a
burner before use.
Note. Washouts, as a rule, are taken from clean, prepared for work items, and from
used ones - only according to epidemiological indications.

Study at BGKP

First day of research

The swabs taken are inoculated on the Code medium. With the growth of Escherichia
coli, the medium changes color. When the color of the medium changes, the test
material is inoculated onto the Endo medium.
Second day of research
The colonies are studied on Endo's medium. Suspicious colonies are smeared and
microscopically examined. Further research is carried out according to the usual
scheme.

Detection of S. aureus

The resulting washings are inoculated on yolk-salt agar in a Petri dish and in parallel
on 6.5% saline broth (accumulation medium). Yolk-salt agar can be inoculated with a
swab. The broth is preliminarily poured into test tubes of 5 ml and 0.2-0.3 ml of wash
is inoculated into each. Crops are incubated at 37°C for 24 hours. Further research is
carried out according to the generally accepted method.

Determination of the total number of bacteria

First day of research

To 2 ml of swabs taken, add 8 ml of isotonic sodium chloride solution. It turns out a
dilution of 1:5. Swabs are thoroughly washed by shaking. 1 ml is inoculated into a
Petri dish and poured into 12 ml of melted and cooled to 45 ° C agar. The plates are
incubated in a thermostat at 37°C for 24 hours.
Second day of research
The crops are removed from the thermostat, the number of grown colonies is
counted and recalculated per 1 cm 2 of the surface under study.
Detection of Pseudomonas aeruginosa (see Chapter 24).
Proteus detection (see chapter 32).
Attention! Detection of pathogenic flora in washings is carried out only according to
epidemiological indications.

Control questions

1. What is the purpose of testing swabs from hands and household items?
2. What are the main studies carried out?
3. How is the total number of bacteria determined?
4. On what medium are swabs inoculated for:
a) allocation of BGKP?
b) isolation of S. aureus?

Tasks

1. Prepare swabs.
2. Wash each other's hands and inoculate on Endo medium (wash before and after
hand washing).
3. On the second day, consider the results of the crops.
 Sanitary and bacteriological examination of dressing
and surgical material for sterility - F.K. Cherkes
The inoculation of the test material is carried out in a box, observing the rules of
asepsis. They reveal aerobic and anaerobic microflora.
For crops you need:
1) a set of sterile instruments (scissors, forceps, anatomical tweezers);
2) sterile 10% hyposulfite solution;
3) sterile distilled water;
4) Nutrient media: Hottinger's sugar broth, Sabouraud's medium and thioglycol
medium.
The material for research is sent on the day of its sterilization in closed and sealed
biks.
Subject to investigation: bandages, tampons, cotton balls, gauze pads and suture
material (catgut and silk).
The material to be studied is removed from the bix with sterile tweezers, pieces are
cut out from different parts over the burner flame, placed in sterile Petri dishes, and
each sample is inoculated into 2 test tubes of sugar broth and Sabouraud's medium.
Suture material . Catgut is stored in an alcoholic solution of iodine. To neutralize
iodine, catgut is placed for 24 hours in a jar with 10% hyposulfite solution and for 24
hours in sterile distilled water. Silk is preserved in alcohol, and before sowing, a skein
of silk is kept for 24 hours in sterile distilled water.
The suture material prepared in this way is removed from distilled water with sterile
tweezers, placed in a Petri dish, cut into pieces 2-5 cm long with sterile scissors.
Individual pieces are inoculated into 2 test tubes of each of the above media.
Crops are placed in a thermostat at a temperature of 37 ° C, incubated for 12-14
days, looking at them every day (crops on Sabouraud's medium are incubated at 20-
22 ° C). In the presence of growth in test tubes, the material is considered non-
sterile.

Control questions
1. What material is tested for sterility?
2. What media are used?
3. Under what conditions and how is sowing done?
 Sanitary and bacteriological study of soft drinks - F. K.
Cherkes
Methods for the study of soft drinks are regulated by GOST 13273-75.
For research, drinks are delivered to the laboratory in factory packaging, sealed with
corks.
Before the study, the neck and cork are wiped with a sterile swab, burned, the cork is
removed and the bottle is closed with a sterile cotton-gauze stopper.
To remove gas, the bottle is kept at 43 ° C for 1 hour.
Acid drinks are neutralized with sterile 10% sodium bicarbonate before inoculation.
The study and evaluation of beverages is carried out in the same way as the study
and evaluation of drinking water. Beverages obtained by fermentation are not tested
for general bacterial contamination, since they contain a large amount of yeast and
specific microflora.
To determine the coli-titer, drinks are inoculated on Kessler or GPS medium in 2
volumes of 100 ml and 10 volumes of 10 ml. Syrups are pre-diluted 10 times. Kvass
and beer as more contaminated to determine if the titer is sown in volumes of
10; 1; 0.1 and 0.01 ml Inoculation is carried out on Kessler medium. The further
course of the study is similar to the study of water.
The titer of Escherichia coli of carbonated soft drinks should be at least 300, non-
carbonated - at least 100, bread kvass - at least 10.
To identify mucilaginous bacteria (leukostock) that cause food spoilage, 1 ml of a
sugar-containing drink is inoculated into yeast water with 10% sucrose and
chalk. The crops are grown at 22-30° C for 48 hours. The presence of mucilaginous
bacteria is determined morphologically (gram-positive cocci, often located in
pairs). On medium with sucrose, they form large slimy capsules. Mucous colonies
grow on nutrient media.
 Brief Dictionary of Microbiological Terms - G. I. Katz
Aggressins are substances produced by pathogenic microorganisms that ensure their
introduction and reproduction in the macroorganism.
Adhesion - sticking.
An allergen is a substance of an antigenic or hapten nature that sensitizes the body
and causes an allergy.
Allergy is a state of altered reactivity of the organism in the form of increased
sensitivity to foreign substances or components of its own tissues
(allergens). Anaphylaxis is a type of allergy, a state of increased sensitivity of the
body to the repeated introduction of a foreign protein. Atopy is the general name for
diseases that develop in people with hypersensitivity to certain substances (bronchial
asthma, hay fever, etc.). Idiosyncrasy - a type of atopy, characterized by intolerance
to certain nutrients in individuals with altered sensitivity. Infectious allergy is a state
of increased sensitivity of the body to repeated contact with microorganisms or their
metabolic products.
Anabiosis is a temporary reversible almost complete suspension of life processes in
the absence of visible external manifestations of life.
Anatoxin is a bacterial exotoxin that has lost its toxicity, but retained its
immunogenic properties. Preparation for active immunization.
Anaphylaxis - see allergy.
Anaphylactic shock - see anaphylactic shock.
Anaerobes are microorganisms that can exist and multiply in the absence of free
oxygen in the environment. Obligate anaerobes - die in the presence of free oxygen
in the environment. Facultative anaerobes - able to live and reproduce both in the
absence and in the presence of free oxygen in the environment.
Antibiotics are substances produced by microorganisms, higher plants and animals
that have the ability to selectively inhibit the development and cause the death of
microorganisms and tumor cells.
Antigenic determinant - see antigenic determinant.
Antigens of microorganisms - antigens found in a microbial cell. H (flagellated) -
thermolabile antigen associated with flagella. K (capsular) - associated with the
capsule and shell of the microbial cell. O (somatic) - antigen of the lipopolysaccharide
layer of the cell wall of a microbial cell. Vi (surface) is a polysaccharide antigen of
some Gram-negative microorganisms.
Defective antigens - see haptens.
Complete antigens are substances that are genetically alien to the body, causing an
immune restructuring of the body and entering into a specific reaction with the
resulting antibodies.
Protective antigens are antigens produced by pathogens in the host organism.
Antiseptics - a set of measures aimed at the destruction of microorganisms in order
to prevent diseases (in a wound, on environmental objects, etc.).
Antibodies are immunoglobulins that are produced by the immune system under the
influence of an antigen and enter into a specific reaction with it. Normal - antibodies
present in the serum of people and animals that have not previously suffered an
obvious infectious disease and have not been immunized with the corresponding
antigen.
Antitoxins - antibodies formed under the influence of a toxin or toxoid and neutralize
the toxin.
Anthroponoses are infectious diseases caused by microbes that, under natural
conditions, parasitize only in the human body.
Asepsis - a system of measures that prevent microbial contamination of an object
(surgical field, cultures of microorganisms, etc.).
Atopy - see allergy.
Aerobes are microorganisms that live and reproduce only in the presence of free
oxygen.
Bacteremia (microbemia) is the presence of viable microorganisms in the blood. The
general (generalized) form of infection (for example, with typhoid fever). Viremia -
the circulation of viruses in the blood. Sepsis (septicemia) is one of the forms of
bacteremia, in which microorganisms accumulate in large numbers, stay for a long
time and even multiply in the blood. Septicopyemia (pyemia) - sepsis, accompanied
by the formation of purulent foci (metastases) in various organs.
Bacteriostasis (bacteriostatic action) - a temporary cessation of bacterial
reproduction.
Bactericidal - the ability to destroy bacteria.
Phage plaque - see phage colony negative.
A vaccine is a preparation for creating artificial active immunity.
Vaccination is a method of creating active immunity.
Viremia - see bacteremia.
Virogeny - see symbiosis.
Virulence - the degree of pathogenicity of a microorganism in relation to a specific
host with a specific method of infection.
Haptens are substances that have specificity and are able to enter into serological
reactions with ready-made antibodies of the same specificity, but are not able to
induce the synthesis of antibodies in the body. When combined with a protein, the
hapten acquires the properties of a full-fledged antigen.
Generalized infection - the spread of pathogens throughout the body.
Hyposensitization is a set of measures that prevent or weaken allergic reactions.
Homeostasis is the relative dynamic constancy of the internal environment of the
body (blood, lymph, tissue fluid) and the stability of basic physiological functions
(circulation, respiration, temperature, etc.).
Homology - the similarity in the structure of the organs of various types of
organisms, due to their origin from the same rudiments.
Humoral - pertaining to the liquid internal environments of the body.
Disinfection is the destruction of pathogenic microorganisms in the environment.
Desensitization - see hyposensitization.
An antigenic determinant is a portion of an antigen molecule that determines its
specificity and interacts with an antibody.
Dissociation of bacteria - the emergence in a population of bacteria that differ from
the original in some morphological, cultural, antigenic, virulent properties.
The dose is lethal. Minimum (DLM) - the minimum dose of a pathogen or toxin that
causes the death of the majority of experimental animals of the same species, weight
and sex with a certain route of administration. Absolute (DCL) - the minimum dose
that kills all animals taken in the experiment. Average DL 50 is the dose that causes
the death of 50% of experimental animals in a fixed time.
Identification of microorganisms - a system of studies (microscopic, microbiological,
serological, etc.) aimed at establishing signs that determine the type, type, genus of a
microorganism.
Idiosyncrasy - see allergy.
Immunization is the creation of artificial immunity. Active - with the introduction of
vaccines; passive - with the introduction of sera or immunoglobulin.
Immunity - the body's immunity to genetically alien antigens, including
microorganisms and their toxins. Natural - occurs after an infection or upon receipt
of ready-made antibodies from the mother. Artificial - develops during
immunization. Active - occurs as a result of the body's immune response to the
introduction of an antigen. Antibacterial - against bacteria. Antitoxic - against a
toxin. Humoral - due to the presence of protective substances in the blood, lymph
and other body fluids. Hereditary (specific, congenital constitutional resistance) -
immunity inherent in representatives of this species and inherited. Acquired -
developing during immunization or after an illness. Cellular - due to the activity of
cells (phagocytes, etc.). Non-sterile (infectious) - due to the presence of a living
infectious agent in the body and is lost when the body is released from the
pathogen. Passive - due to the introduction of immune sera or the intake of ready-
made antibodies from the mother (through the placenta or with milk). Post-
vaccination - occurs as a result of active immunization. Sterile - immunity that
persists after the release of the body from the pathogen.
Immune system - cells, tissues and organs that carry out protective specific reactions
of the body, maintaining the constancy of the internal environment (lymphoid
system of the body).
Immunoglobulins - globulins, mainly gamma globulins, that carry the function of
antibodies. Preparations for passive immunization obtained by purification and
concentration of immune sera.
Impregnation (in microbiology) - impregnation of microscopy objects with solutions
of metal salts.
Invasiveness - the ability of microorganisms to overcome the protective barriers of
the host, penetrate into his body and spread in it.
Inhibitors are substances that slow down or completely suppress biological
processes.
An ingredient is a component of a compound or mixture. The incubation period is
the latent period of an infectious disease from the moment of infection to the
appearance of the first clinical symptoms.
Interferon is a low molecular weight species-specific protein synthesized in the body
and cell cultures that inhibits the reproduction of viruses.
Infectious allergy - see allergy.
Infectious disease is a clinically expressed infectious process.
Infectious process - see infection.
Infection (infectious process) - a set of processes that develop in a susceptible
organism during its interaction with a pathogenic microorganism, occurring under
certain environmental conditions.
The capsid is the protein coat of the virion.
Capsomere is a protein macromolecule that makes up the capsid.
Capsular antigen - see antigens of microorganisms.
Plasma cell (antibody-forming) - cells of lymphoid tissue that produce antibodies.
Clone - see pure culture of microorganisms.
Colony - see pure culture of microorganisms.
Negative phage colony - foci of lysis formed around individual phage particles on
dense media with a bacterial lawn.
Commensalism - see symbiosis.
Complement is a complex protein of blood serum, lymph and tissue fluid - a factor of
nonspecific defense of the body.
Consistency is the degree of density of a substance.
Cultivation - the cultivation of microorganisms, animal and plant cells, tissues and
organs in artificial conditions.
Culture of microorganisms - microorganisms grown on a nutrient
medium. Population - a set of microorganisms of the same species that have grown
in a given volume of the environment for a certain time. Culture pure - see pure
culture of microorganisms.
Lysogeny is the coexistence of the genome of bacteria and a temperate phage as a
single chromosome, in which the phage reproduces together with the bacterium, but
retains the ability to release the phage genome and cell lysis.
Lysozyme is an enzyme that breaks down the cell wall of bacteria. Factor of
nonspecific protection of the organism.
Lymphocyte is a cell that takes part in immunological reactions. B-lymphocyte -
differentiates from a B-precursor at an unspecified location. Causes the reaction of
humoral immunity. T-lymphocyte (thymus-dependent) - differentiates in the
thymus. Causes reactions of cellular and humoral immunity.
Frontal lens - the outer lens of the microscope objective.
The line of animals is pure - animals with the most homogeneous heredity, obtained
by long-term (at least 20 generations) closely related crossing.
Lyophilization is a method of dehydration by freezing followed by vacuum drying.
Luminescence - the ability of some substances to emit light after absorbing energy
(light of a shorter wavelength, x-rays, etc.). Fluorescence is a type of luminescence
characterized by attenuation within 10 -9 - 10 -8 s after excitation ceases.
Metabiosis - see symbiosis.
Minimum lethal dose (DLM) - see lethal dose.
Mutualism - see symbiosis.
Normal antibodies - see antibodies are normal.
Opsonic index - the ratio of the phagocytic index of the test serum to the phagocytic
index of normal (not immune) serum.
Obligate anaerobes - see anaerobes.
Focal infection - an infectious process localized in one place.
Parasitism - see symbiosis.
Pasteurization - see sterilization.
Pathogenicity - the ability of microbes of a certain species to cause disease in certain
types of macroorganisms.
Peplos - the second, outer shell of some viruses, consisting of individual protein
molecules (peplomers).
Pigment formation (in microorganisms) - the ability to produce coloring substances
(pigments).
Pyemia (septicopyemia) - see bacteremia.
Plasma cells - see plasma cells.
Polymorphism is the existence of different morphological forms within one species.
Microbial population - see microorganism culture.
Preparation - a biological object prepared for macro- or microscopic examination,
native (not fixed), fixed and stained.
Prokaryotes are single-celled organisms that have one (usually ring-shaped) strand of
DNA, without a limited nucleus and mitochondria.
Protective antigens - see protective antigens.
Proteolytic properties of microorganisms - the ability to enzymatically break down
proteins, peptones and polypeptides.
Recombination - the formation of mixed offspring due to the exchange of genetic
material.
Saprophytes are microorganisms that feed on dead organic matter. Some are
pathogenic to humans.
Saccharolytic properties - the ability to break down carbohydrates.
Sensitization is an increase in the body's sensitivity to the action of a factor.
Symbiosis is a type of interaction between two biological species. Virogeny is a
symbiosis of a virus and a cell. Commensalism - one organism lives at the expense of
another without harming it. Metabiosis - the waste products of one species serve as
a source of nutrition for another species. Mutualism - brings mutual benefits to both
participants. Parasitism - one organism (parasite) lives at the expense of another
(host), bringing harm to it. Synergism - strengthening the functions of the members
of the association.
Somatic antigen - see antigens of microorganisms.
Sterilization is the complete release of substances and objects from microorganisms
(defertilization). Pasteurization - the destruction of non-spore forms of
microorganisms by heating to a temperature below 100 ° C. Tyndalization - fractional
sterilization at a temperature of 56-60 ° C for 30 minutes 3 days in a row.
Sterile spots - see phage colony negative. Tyndalization - see sterilization.
Tinctorial properties - the ability of microorganisms to stain with various colors.
Toxigenicity - the ability to produce exotoxin.
Microbial toxin - a toxic substance formed as a result of the vital activity of
microorganisms and causing disease or death of a person or animal. Exotoxin -
released into the environment. Endotoxin is a component of a microbial cell that is
closely associated with it.
Toxemia (toxinemia) - the presence of a toxin in the blood.
Toxicity - the ability to cause death or poisoning of the body.
Transduction is the transfer by a temperate phage of a piece of DNA from one
bacterium (donor) to another (recipient).
Transformation of bacteria is the inclusion in the chromosome or plasmid of a
bacterium (recipient) of a DNA fragment of another bacterium (donor) as a result of
the transfer of its isolated DNA.
Phage (bacteriophage) is a virus capable of infecting a microbial cell, reproducing in it
and causing its death or transition to a lysogenic state. Vegetative - located inside the
bacterial cell. Virulent - causes lysis of a microbial cell with the release of numerous
phage particles into the environment. Moderate - is able to exist in a microbial cell as
a prophage and under certain conditions can turn into a virulent phage.
Phagocytic index - the average number of microorganisms absorbed by one
phagocytic cell.
Phagocytosis - active capture and absorption of microorganisms, various cells and
foreign particles by special cells of the macroorganism (phagocytes).
Facultative anaerobes - see anaerobes.
Phytoncides are biologically active substances secreted by some plants (onions,
garlic, bird cherry, pine), which retard the development and cause the death of
microorganisms.
Fluorescence - see luminescence.
Fluorochromes are organic substances that have the ability to fluoresce.
Frontal lens - see frontal lens.
Chemotherapy is the treatment with chemicals that have a specific antimicrobial or
antitumor effect.
A pure culture of microorganisms is a culture containing microorganisms of the same
species. Clone - a genetically homogeneous population of microorganisms obtained
from a single microbial cell by direct isolation. A colony is an isolated accumulation of
microorganisms on a dense medium. Strain - a pure culture of microorganisms
isolated from a specific source at a specific time.
Anaphylactic shock is a generalized reaction of a sensitized organism to repeated
administration of a foreign protein.
Strain - see pure culture of microorganisms.
Exotoxin - see microbial toxin.
An experiment is an experiment carried out under exactly the right conditions.
Extract - an extract from the tissues of animals or plants.
Endotoxin - see microbial toxin.
Eukaryotes are organisms that have a well-formed nucleus and chromosomes.