MICROBIOLOGY AND PARASITOLOGY
Kathleen Jan M. Los Banez
BIO 300N LAB
PRELIM REVIEWER
INTRODUCTION TO MICROBIOLOGY
MICROBIOLOGY
 is the study of all living organisms that are too
small to be visible with the naked eye.
 This includes bacteria, archaea, viruses, fungi,
prions, protozoa and algae, collectively known as
'microbes'.
MEDICAL MICROBIOLOGY
 deals with the study of microorganisms responsible
for various diseases of low to high intensity
 it can be divided into:
BACTERIOLOGY
 which deals with study of bacteria.
VIROLOGY
 which facilitates study of viruses.
PARASITOLOGY
 which involves unicellular to multicellular
parasites.
MYCOLOGY
 that deals with various fungal microorganisms
IMPORTANCE OF MICROBIOLOGY IN NURSING
PRACTICE
 Role of a nurse in relation to the condition of the
patient, disease stage, diagnosis, treatment module,
and hospital environment in a microbiological
perspective.
 The role of a nurse is varied, and it is important for
a nurse to multitask intellectually with fundamental
knowledge, decisive thinking, and application.
 Evolving nurses with a conceptual clinical
approach towards the following:
TO IMPLEMENT IMMUNIZATION SCHEDULE IN
o Asepsis, sterilization, and disinfection
o Recognition of infection
o Infection control—pathogenesis and
transmission
o Nosocomial infection
o Immune system
o Clinical thought process.
WHY MICROBIOLOGY IS NEEDED IN NURSING?
 Nurses are involved in managing all aspects of
patient’s health and infection control in the
hospitals.
 Nurse must know microbiology to take care of
patient and to protect oneself from pathogenic
microorganisms.
 Nurses utilize concepts of microbiology while
giving patient care or doing procedures
TO PREVENT SPREAD OF INFECTION
 Nurses should have knowledge about the mode of
spread of infection.
 Some of the infections are spread by contact
(touch), air (air-borne), droplets (sneezing,
coughing), some by eating contaminated food or
drink (food borne), sexual contact (STDs), by
arthropod bite (vector born) and others by
contaminated blood transfusion, etc.
 the knowledge would help a nurse to use particular
measures to save community and hospital spread of
infection
TO MAINTAIN STERILE FIELD
 A nurse must know procedures used to create and
maintain a sterile field in the hospitals and these are
based on the knowledge of microbiology
 The knowledge of microbiology can further help a
nurse to use sterile equipment which are a
necessary part of invasive procedures done on
patients
 The principles of asepsis are based on
microbiology. The knowledge of sterilization
techniques is mandatory for a nurse
 The proper disposal of biomedical waste is equally
important
TO COLLECT SPECIMENS
 Nurse must recognize the importance of proper
collection of specimens to be sent for
bacteriological examination to obtain accurate
results.
 For instance, she has to be familiar with the various
infectious disease and their route of infection so as
to collect clinical specimen from a proper site like,
pustule or blood or stool etc.
HOSPITALS
 A nurse also plays an important role in
immunization to control threats of various diseases
like diphtheria or MMR etc.
 So they must have knowledge of various antisera
and vaccines used in preventing the dreadful
diseases.
 The immunization schedules and the cold chain
used to deliver the vaccines from the production to
the administration should be known to a nurse.
  Knowledge of immunology makes a nurse well
prepared for vaccination and protection of vaccines
by using cold chain
CONTRIBUTION OF THE FOLLOWING
SCIENTISTS IN THE FIELD OF MICROBIOLOGY
VAN LEEUWENHOEK
 (24 October 1632 – 26 August 1723)
 He is commonly known as “The Father of
Microbiology", and one of the first microbiologists.
 Van Leeuwenhoek is best known for his pioneering
work in microscopy
 Dutch businessman and scientist in the Golden Age
of Dutch science and technology. A largely self-
taught man in science.
 In the 1670s, he started to explore microbial life
with his microscope. This was one of the notable
achievements of the Golden Age of Dutch
exploration and discovery (c. 1590s–1720s).
 Using single-lensed microscopes of his own design
and make, van Leeuwenhoek was the first to
observe and to experiment with microbes.
FRANCESCO REDI
 (18 February 1626 – 1 March 1697)
 Italian physician, naturalist and biologist.
 He is referred to as the "founder of experimental
biology", and as the “Father of Modern
parasitology".
 He was the first person to challenge the theory of
spontaneous generation by demonstrating that
maggots come from eggs of flies.
SPONTANEOUS GENERATION
 He said simpler life forms such as worms and
maggots need no parents – they emerge alive from
the earth and from rotting organic matter.
 This idea had been accepted for over 2,000 years.
 Again, Redi used experiments to research this
subject.
o He observed that flies laid eggs on meat.
o These eggs hatched into maggots.
o If the meat was protected from flies, no eggs
were laid and no maggots appeared.
 He described his work in 1668 in Experiments on
the Generation of Insects.
LOUIS PASTEUR
 French chemist and microbiologist renowned for
his discoveries of the principles of vaccination,
microbial fermentation, and pasteurization.
 He is regarded as one of the founders of modern
bacteriology and has been honored as the “Father
of bacteriology"
 Pasteur was responsible for disproving the doctrine
of spontaneous generation and his experiment
demonstrated that in sterilized and sealed flasks,
nothing ever developed.
 Conversely, in sterilized but open flasks,
microorganisms could grow.
 For this experiment, the academy awarded him the
Alhumbert Prize carrying 2,500 francs in 1862.
ROBERT KOCH
 German physician and microbiologist. As the
discoverer of the specific causative agents of
deadly infectious diseases including tuberculosis,
cholera, and anthrax.
 He is regarded as one of the main “founders of
modern bacteriology”.
 He was the first to use oil immersion lens,
condenser and microphotography in microscopy.
 In appreciation of his works, he was appointed as
government advisor at the Imperial Health Office
in 1880, promoted to a senior executive position
(Geheimer Regierungsrath) in 1882, Director of
Hygienic Institute and Chair (Professor of hygiene)
of the Faculty of Medicine at Berlin University in
1885.
 The methods Koch used in bacteriology led to
establishment of a medical concept known as
Koch's postulates, four generalized medical
principles to ascertain the relationship of pathogens
with specific diseases.
 For his research on tuberculosis, he received the
Nobel Prize in Physiology or Medicine in 1905
EUKARYOTIC AND PROKARYOTIC CELL
PROKARYOTIC
 Nucleus Absent.
 Cell Type Usually unicellular.
 True Membrane bound Nucleus absent.
 Example: Bacteria and Archaea.
 EUKARYOTIC
 Nucleus present.
 Cell Type usually multicellular
 True Membrane bound Nucleus present
 Example: Animals and Plants.
BASIC PROPERTIES OF VIRUS
 The size of virus ranges from (20-300) nm in
diameter.
 The overall shape of virus varies in different groups
of virus.
 Two basic symmetry are recognized in virus, they
are helical symmetry and icosahedral symmetry.
STRUCTURE AND CHEMICAL COMPOSITION OF
VIRUS
GENOME
 Viral genome or nucleic acid contains either DNA
or RNA but not both.
CAPSID
 Composed of capsomere. Capsid protects the
nucleic acid and also helps in attachments on host
cell surface during infection.
ENVELOPE
 Some virus contains phospholipid bilayer known as
envelope.
GLYCOPROTEIN SPIKE
 Envelope of some virus contains viral coded spike
projected outside the envelope called glycoprotein
spike or peplomers.
ENZYMES
 Some virus possess their own enzymes. Retrovirus
possess reverse transcriptase
VIRAL REPLICATION
 Virus only replicates inside host cell.
METABOLISM
 Viruses are metabolically inert outside host cell.
They are also called as obligate intracellular
parasite.
RESISTANCE
TEMPERATURE
 Most viruses are heat labile.
COLD
 Viruses are stable and resistant to cooling.
 Virus can be stored for long duration at -40°C to -
70°C by lyophilization or freeze drying.
RADIATION
 Both non-ionizing and ionizing radiation can kill
virus.
ORGANIC SOLVENT
 Chloroform, ether and bile salt can destroy all
viruses by lipid solution.
DISINFECTANT
 Most viruses are destroyed by oxidizing agents
such as chlorine, H2O2, iodine etc.
ANTIBIOTICS
 Viruses are resistant to antibiotics
LIST BASIC NUTRITIONAL REQUIREMENTS OF
MICROORGANISM
 In order to get energy and to maintain cellular
biosynthesis, every organism must be provided
with the essential substances needed for growth
from its environment.
 Most bacteria can be grown away from their natural
habitats in laboratories if suitable nutrients are
provided in the form of culture media.
 Some are obligate intracellular parasites of other
cells. The host cells must satisfy the nutritional
requirements of mutualisms and parasites as these
are the residents of the host
MAJOR ELEMENTS
 The nutritional requirements of elementary level.
 The cell’s elementary composition consists of C,
H, O, N, S, P, K, Fe, and Ca and traces of Zn, Ni,
Mn, Co, Cu, and Mo.
 Some bacteria uptake these elements as water, as
some inorganic ions, microelements, and macro
elements. These elements serve as either functional
or structural units of the bacterial cell
CLASSIFY BACTERIA ON THE BASES OF THEIR
NUTRITIONAL REQUIREMENT AND
MORPHOLOGY
MICROBIAL NUTRITION
 In order to get energy and to maintain cellular
biosynthesis, every organism must be provided
with the essential substances needed for growth
from its environment.
 These essential substances required for bacterial
growth are referred to as ‘nutrients’
AUTOTROPHIC BACTERIA
 Based on their nutrition, the bacteria are classified
into the following:
 The bacteria that can synthesize organic food from
inorganic substances are called autotrophs.
 Autotrophic bacteria are of two types, which are
mentioned below.
PHOTOAUTOTROPHIC BACTERIA
 These bacteria contain photosynthetic pigments in
thylakoids that utilize the solar energy to synthesize
food.
 Bacterial photosynthesis is completely different
from that of green plants. As a result, this process is
known as an oxygenic photosynthesis.
 CO2 + H2 S + Sunlight → Sugar + Sulphur +
Water
CHEMOAUTOTROPHIC BACTERIA
 Chemoautotrophs construct organic compounds
from inorganic substances.
 Here, while the oxidation of inorganic substances
occurs, energy gets liberated, which in turn is used
to construct food, that is, organic compounds.
 Example Nitrifying Bacteria: These bacteria derive
energy by oxidizing ammonia into nitrates (e.g.,
Nitrosomonas and Nitrobacteria).
 NH4 + + 2O2 → NO2 + 2H2 O + Energy
HETEROTROPHIC BACTERIA
 Heterotrophic bacteria cannot synthesize their own
food they are always dependent on external
sources.
 The heterotrophs are of different forms, which are
mentioned below:
SAPROPHYTIC ORGANISMS
 Bacteria that obtain their nutritional requirements
from dead and decaying matter.
DECOMPOSITION
 During it, aerobic breakdown of organic matter
takes place
FERMENTATION
 Here anaerobic breakdown of organic matter
occurs.
 Fermentation reactions are incomplete and always
release foul gases.
PUTREFACTION
 which is the breakdown of protein molecules, is
also done by heterotrophs.
SYMBIOTIC BACTERIA
 Some bacteria can live with other organisms in
such a way that both are not harmed by each other
but rather are benefitted by one another.
 The cellulose-digesting bacteria that live in the
alimentary canal of ruminant mammals such as
cows and goats are another example.
 The relationship observed here is the same mutual
relationship as can be seen in E. coli residing in the
human alimentary canal
PARASITIC BACTERIA
 Synthesize their own food and always need a host
for their survival.
 These bacteria obtain their food from their hosts
such as animals and plants.
 The majority of parasitic bacteria are pathogens
and are responsible for several chronic diseases in
the host by exploiting them
BACTERIAL MORPHOLOGY
 Bacteria are microscopic living organisms that are
structurally very simple.
 They are strictly unicellular and thrive solitarily.
However, some bacteria are found living in groups.
 Bacteria range from 1 mm in diameter (largest end
of the scale) to 200 nm in length (smallest end of
the scale).
SHAPE OF BACTERIAL CELL
 Bacterial cells vary in shape and fundamentally are
of four groups, which are as mentioned below.
SPHERICAL TYPE
 Cells are circular in shape. This type of cells are
termed as ‘cocci’ are of different forms as
mentioned below:
o MICROCOCCUS (if the coccus is single)
o DIPLOCOCCUS (if the coccus lives in pairs)
o STREPTOCOCCUS (if the coccus exists in
chains)
o STAPHYLOCOCCUS (if the coccus occurs
in clusters, i.e., grape-like)
o SARCINA (cubical packets of eight or more)
 BACILLUS TYPE
 The cells are elongated and rod-like, and such cells
are called ‘bacilli’.
 Bacilli may again be of different types.
 They might be single, in pairs, or in groups to form
a chain (streptobacilli).
SPIRILLUM TYPE
 The cells are spirally coiled and hence referred to
as spirillum.
 Some of the important genera that fall under this
type are Spirillum and Microspira.
VIBRIO TYPE
 The cells are comma-shaped.
 The rod-shaped cell is curved at one end and hence
appears like a ‘comma’. Vibrio cholerae is an
important species of this type.
Some bacteria tend to change their shape according to
environmental changes.
Such bacteria fall under two categories, which are as
follows:
MONOMORPHIC
 Here, the bacterium exhibits only a single shape.
POLYMORPHIC OR PLEOMORPHIC
 The bacterium exhibits different shapes as the
environmental or physiological condition gets
changed.
 Examples of polymorphic bacteria are
Corynebacterium diphtheriae and Mycopla
 STAINING TECHNIQUES
 As bacteria consist of clear protoplasmic matter,
differing but slightly in refractive index from the
medium in which they are growing, it is difficult
with the ordinary microscope, except when special
methods of illumination are used, to set them in the
unstained condition.
 Staining, therefore, is of primary importance for the
recognition of bacteria.
 Staining may be simple staining and differential
staining.
DYES
BASIC DYES
 Methylene blue, Basic fuchsin, Crystal violet,
Safranine, Malachite green have positively charged
groups (usually from penta-valent nitrogen) and are
generally sold as chloride salts.
 Basic dyes bind to negatively charged molecules
such as nucleic acids, many proteins and surfaces
of bacterial and archeal cells.
ACIDIC DYES
 Eosin, Rose Bengal and Acid fuchsin possess
groups such as carboxyls (-COOH) and phenolic
hydroxyls (-OH).
 Acidic dyes, in their ionized form, have a negative
charge and bind to positively charged cell
structures.
SIMPLE STAINING
 These show not only the presence of organisms but
also the nature of the cellular content in exudates.
 A single stain is used.
 Examples are Loeffler’s methylene blue,
polychrome methylene blue, dilute carbol fuchsin.
 Simple staining is of positive staining and negative
staining.
POSITIVE STAINING
NEGATIVE STAINING
 Negative staining procedure helps to study the cell
shape, cell breakage, refractable inclusion bodies
and spores besides poly- hydroxybutyrate granules.
 It is useful for those bacteria which are difficult to
stain.
 Very slender bacteria like spirochaetes that are not
detectable by simple staining methods can be
viewed by negative staining.
PRINCIPLE
 Negative staining requires the use of acidic stains
such as Indian ink and nigrosin.
 The acidic stain with its negatively charged
chromogen will not penetrate the cell because of
the negative charges on the surface of the bacteria.
 So unstained cells are differentiable against the
dark background.
 Since heat fixation is not required, the cells are not
subjected to the destaining effect of chemicals and
heat, their natural size, shape and arrangement can
be seen by this method. It is possible to observe
bacteria that are difficult to stain.
PROCEDURE
 A loopful of undiluted Indian ink was placed on
one end of clean slide.
 A loopful of inoculum was transferred into the drop
of stain.
 By using a second dirt-free slide with smooth edge
over the suspension, it was spread uniformly along
the edge.
 The suspension was spread to the end of the slide
so as to form a uniform smear.
 The slide was then air dried and observed under oil
immersion objective.
 Colorless bacteria are seen against a dark
background.
OBSERVATION
DIFFERENTIAL STAINING
 This type of staining is to differentiate two
organisms.
 Mainly used differential  Safranin (counterstain) for 30-60 seconds. Water
rinse. Blot dry.
 Gram positive cells remain purple. Gram-negative
HANS CHRISTIAN GRAM cells appear red.

 Introduced gram’s staining

GRAM’S STAINING

 Gram Stain is developed in 1884 by the Danish

physician Christian Gram, is the most widely used
method in bacteriology.
 It is first and usually the only method employed for
the diagnostic identification of bacteria in clinical
specimens.
OBSERVATION

PRINCIPLE

 Violet dye and the iodine combine to form an

insoluble, dark purple compound in the bacterial
protoplasm and cell wall.
 This compound is dissociable in the decolorizer,
which dissolves and removes its two components
from the cell.
 But the removal is much slower from Gram-
positive than from the Gram-negative bacteria, so
that by correct timing the former stay dark purple
whilst the latter become colorless. PAUL ERHLICH
 The difference between the two types of bacteria is
that the Gram positive have thicker and denser  First identified Mycobacterium tuberculosis
peptidoglycan layers in their cell walls, which
makes them less permeable to the stain than those
of the Gram negative bacteria. ACID – FAST STAINING
 The iodine has a critical role in enhancing this
 This is also known as Ziehl – Neelsen staining.
difference.
 This method is a modification of Ehrlich’s (1882)
 It seems to bind temporarily to the peptidoglycan
original method for the differential staining of
and make it even less permeable to the dye.
tubercle bacilli and other acid-fast bacilli with
aniline-gentian violet followed by strong nitric
acid.
PROCEDURE
 Stain used consists of basic fuchsin, with phenol
STEP 1 added.

 Crystal violet (primary stain) for 1 minute. Water

rinse.
PRINCIPLE
 Cells stain purple.
 Acid fast bacteria retain primary stain (carbol
STEP 2
fuchsin) even after washing with a strong acid.
 Iodine (mordant) for 1 minute. Water rinse.  It appears red while non-acid fast bacteria are
 Cells remain purple. decolorized on washing with acid and takes the
color of the counter stain (methylene blue).
STEP 3  The property of acid fasting appears due to the
presence of mycolic acid in their cell walls.
 Alcohol (decolorizer) for 10-30 seconds. Water
 Mycolic acid is a group of branched chain hydroxy
rinse.
lipids.
 Gram-positive cells remain purple. Gram negative
 It is most commonly used to identify
cells become colorless.
M.tuberculosis and M.leprae, the pathogen
STEP 4 responsible for tuberculosis and leprosy,
respectively. These bacteria have cell walls
 containing lipids constructed from mycolic acids, a
group of branched chain hydroxyl fatty acids,
which prevent dyes readily binding to the cells.
 However, M.tuberculosis and M.leprae can be
stained by harsh procedures such as the Zeihl-
Neelson method which uses heat and phenol to
derive basic fuchsin into cells.
 Once basic fuchsin has penetrated, M.tuberculosis
and M.leprae are not easily decolorized by
acidified alcohol (acid-alcohol) and thus are said to
be acid-fast.
 Non acid-fast bacteria are decolorized by acid-
alcohol and thus are stained blue by methylene blue
counterstain.

OBSERVATION

ZN METHODS FOR WEAKLY ACID-FAST

ORGANISMS

PROCEDURE
 A clean sterile glass slide was taken.
 A thin uniform smear was prepared in the slide
using a inoculation loop.
 The smear was allowed to air dry and then heat
fixed.
 The smear was flooded with carbol fuchsin and
heated until steam arises.
 Preparation was allowed to stay for 5-7 min. The
stain must not be allowed to evaporate or dry in the
slide. Pour more carbol fuchsin on slide is
necessary. It is then washed under a low steam of
running water.
 The smear was decolorized with 20% sulphuric
acid.
 The slide is then washed again under running tap
water.
 Counter stain the smear with methylene blue for the
2 min.
 Washed under running tap water.
 Slide was blot dried and examined under
microscope.
LEPROSY BACILLI
 are acid-fast, but usually to lesser degree than the
tubercle bacillus.
 They are stained in films or sections in the same
way as the tubercle bacillus, except that 5%
sulphuric acid is used for decolorization in the
place of 20% sulphuric acid or acid-alcohol.
SECTIONS OF TISSUES CONTAINING ‘CLUBS’
 formed by actinomycetes, mycobacteria and no
cardiae can be stained by ZN stain and decolorized
with 1% sulphuric acid to demonstrate the acid-
fastness of the clubs.
BRUCELLA DIFFERENTIAL STAIN
 Brucellaabortus in infected tissue or exudate may
be distinguished from the latter by its weakly acid-
fast reaction.
 Stain with dilute (1-in-10) carbolfuchsin, without
heating, for 15 min. Decolorize with 0.5% acetic
acid solution for 15 seconds, wash thoroughly with
tap water and counter stain with Loeffler’s
methylene blue for 1 min.

VOLUTIN-GRANULE STAINING
 Volutin granules are a type of cytoplasmic
inclusion bodies found in many bacteria as well as
in some fungi, algae, protozoa.
 These granules are composed mainly of
polyphosphate, RNA and protein.
 These granules are found most prominent in old
cultures before starvation occurs.
 The method of volutin granule staining is known as SPORE STAINING
ALBERT-LAYBOURN METHOD.
 The morphology of bacterial endospores is best
observed in unstained wet films under the phase
contrast microscope, where they appear as large,
refractile, oval or spherical bodies within a
bacterial mother cells or else from the bacteria.
 If spore-bearing organisms are stained with
ordinary dyes , or by Gram’s stain , the body of the
bacillus is deeply colored , whereas the spore is
unstained and appears as clear area in the organism.
 This is the way in which spores are most
commonly observed.
 If desired , however , it is possible by vigorous
staining procedures to introduce dye into the
substance of the spore.
 When thus stained , the spores tends to retain the
dye after treatment with decolorizing agents, and in
this respect behaves similarly to the tubercle
PRINCIPLE bacillus, but is more weakly acid-fast.

 Albert’s stain contains cationic dyes like toludine

blue and malachite green.
 Due to the highly acidic nature of the granules, they
can be selectively stained by acidified basic dyes.
 The toludine blue preferentially stain volutin
granules while malachite green stains the
cytoplasm.
 Later due to application of Albert’s iodine, the dye
molecule are fixed by precipitation.
 Well-developed granules of volutin
(polyphosphate) may be seen in unstained wet
preparations as round refractile bodies within the
bacterial cytoplasm
PRINCIPLE
PROCEDURE
 The spores are thick walled structures and very
 A thin uniform smear of culture was made. It was resistant to physical and chemical agents.
air dried and heat fixed.  The spores have a capacity to survive for long
 Lower the slide with Albert’s stain A and allowed periods even in unfavourable environmental
to react for 3-5 min. conditions.
 The slide was then washed under running tap  The heat resistance by spores is due to the high
water. content calcium- dipicolinicacid.
 Flood the slide with Albert’s Iodine and allowed to  The spores are differentially stained using special
react about 1 min. procedures that help dye to penetrate the spore
 Slide was then washed and blot dried. wall.
 The slide was observed under oil immersion  An aqueous primary stain, malachite green is
objective of a microscope. applied and steamed to enhance the penetration of
the impermeable spore coat.
OBSERVATION  Once stained the endospore does not readily
decolorize even with the application of decolorizer
and they appear, but the cytoplasm of the cell takes
the color of safranine and appears red.
  A modified Ziehl-Neelsen stain in which weak,
0.25% sulphuric acid is used as decolorizer, yields
red spores in blue-stained bacteria. Lipid granules
also stain red, appearing like small spherical
spores.
PROCEDURE
Films are dried and fixed with minimal flaming.
1. Place the slide over a beaker of boiling water , resting it
on the rim with the bacterial film uppermost.
2. When , within several seconds , large droplets have
condensed on the underside of the slide , flood it with
5% aqueous solution of malachite green and leave to act
for 1 min while the water continues to boil.
3. Wash in cold water.
4. Treat with 0.5% safranineor 0.05% basic fuchsin for 30
seconds.
5. Wash and dry. This method colors the spores green and
the vegetative bacilli red. Lipid granules are unstained.
OBSERVATION
CAPSULE STAINING
 Some bacteria secrete a prominent slimy or gummy
material on their surface usually polysaccharide
make up the capsule.
 It is not essential for life, but may serve as a reserve
food.
 It provides protection against dehydration and also
against phagocytosis.
 The chemical composition of capsule varies
according to organism but usually consist of
polysaccharides e.g., glucose, galactose, amino
sugars.
 Capsule are colorless and have low refractive index
and so are difficult to observe without a special
staining technique.
 Moreover the capsule is non-toxic, hence cannot be
stained in the usual manner.
 Techniques like negative staining can be used to
demonstrate the capsule
PRINCIPLE
 Congo red is a negative stain. It stains the back
ground leaving the capsule unstained.
 Acid fuschin fixes congored and reacts with it to
give a blue back ground.
 Acid fuschin also stains the organism pink, leaving
the capsule colorless.
PROCEDURE
 Placed a drop of Manevali’s solution I on a clear
glass slide.
 Sterilised the nichrome loop, cooled it and just
touched to the growth of organism on the slant.
 Mixed the culture on loop with a drop of
Manevali’s solution I on the slide.
 The drop was spread into a thin film.
 The film was allowed to dry completely.
 The smear was covered with Manevali’s solution II
and allowed to react for 1 min.
 The excess stain was discarded and dried in air.
 Observe under oil immersion objective.
OBSERVATION
FLAGELLAR STAINING
 Flagellar staining provides taxonomically valuable
information about the presence and distribution
pattern of flagella on prokaryotic cells.
 Bacterial and archaeal flagella are fine, threadlike
organelles of locomotion that are so slender (about
10 to 30 nm in diameter) they can only seen
directly using electron microscope.
 To observe bacterial flagella with the light
microscope, their thickness is increased by coating
them with mordants such as tannic acid and
potassium alum, and then staining with
pararosalineorbasic fuchsin.
PROCEDURE
 Grow bacteria for 16 – 24 hrs on a non-inhibitory
medium , e.g. tryptic soy agar or blood agar.
 Touch a loopful of water onto the edge of a colony
and let motile bacteria swim into it.
 Then transfer the loopful into a loopfulof water on
a slide to get a faintly turbid suspension and cover
it with a cover-slip.
 The bacterial suspension is thus prepared with a
minimum of agitation , which would detach the
flagella.
 After 5-10 min , when many bacteria have attached
to the surfaces of the slide and cover-slip , apply
two drops of Ryu’s stain to the edge of the cover-
slip and leave the stain to diffuse into the film.
 Examine with the microscope after standing 5-15 LEISHMAN’S STAIN
min at ambient temperature.
 Dry unfixed films are used. The stain is first used
OBSERVATION undiluted, and the methyl alcohol fixes the film.
 The stain is then diluted with distilled water, and
the staining properly carried out.
 Pour the undiluted stain on the unfixed film and
allow it to act for 1 min.
 By means of a pipette and rubber teat, add double
the volume of distilled water to the slide, mixing
the fluids alternately sucking them in the pipette
and expelling them.
 Allow the diluted stain to act for 12 min.
 Flood the slide gently with distilled water ,
allowing the preparation to differentiate in the
PERIODIC ACID-SCHIFF (PAS) METHOD FOR distilled water until the film appears bright pink –
FUNGI IN TISSUE SECTIONS usually about 30 seconds.
 Remove the excess water with blotting paper and
 The polysaccharide constituents of bacteria and
dry in air.
fungi are oxidized by periodate to form
polyaldehydes which yield redcolored compounds
with Schiff’s fuchsin-sulphite;
 the proteins and nucleic acids remain uncolored.
The method may be used to reveal fungal elements
in sections of infected animal tissue; the fungi stain
red, while the tissue material, except glycogen and OBSERVATION
mucin, fails to take the stain.

PROCEDURE

 Bring sections to distilled water.

 Treat for 5 min with a freshly prepared 1% solution
of periodic acid in water.
 Wash in running tap water for 15 min and rinse in
distilled water.
 Stain with fuchsin-sulphite for 15 min.
 Wash two or three times with sulphite wash
solution. GIEMSA STAIN
 Wash with water.
 This consists of a number of compounds made by
 Wash in running tap water for 5 min and rinse in
mixing different proportions of methyleneblue and
distilled water.
eosin.
 Counterstain with dilute aqueous malachite green
 These have been designated Azur I, Azur II and
or with0.1 % light green in 90 % alcohol for 1 min.
Azur II-eosin.
 Dehydrate rapidly in absolute alcohol , clear in
 It is an advantage of Giemsa’s stain over
xylene and mount in Canada balsam.
Leishman’s that fixation is by alcohol instead of by
OBSERVATION undiluted stain which, particularly in the tropics,
may deposit precipitate on the film.
 Rapid and slow methods are preferred for staining
and multiple films of malaria blood and tissue
aspirates.
 PROCEDURE
1. Fix films in methyl alcohol for 3 min.
2. Stain in a mixture of 1 part stain and 10 parts buffer
solution pH 7.0 for 1 hr.
3. Wash with buffer solution, allowing preparation to
differentiate for about 30 seconds.
4. Blot and allow to dry in air.
 This method of staining gives excellent results with
thin blood films for malaria parasites, Schuffner’s
dots being well defined. Trypanosomes are also
well demonstrated.
OBSERVATION
STAINING FOR
SPIROCHAETES
 The large spirochaetes borreliae stain by ordinary
method including Gram’s (giving a negative
reaction), Leishman’sand Giemsa’s.
 The smaller ones, e.g, treponemesand leptospires,
are too thin to be demonstrated by ordinary stains.
They are best observed in unstained wet films
under the dark-ground microscope where their
bright appearance and motility draw attention to
them.
 If a permanent preparation of small spirochaetes is
required, use may be made of a silver impregnation
method which artificially thickness them with a
deposit of silver.
 The classical method are Fontana’s (for films) and
Levaditi (for sections).
 Faulkener and Lille’s modified silver methods has
been recommended for demonstrating leptospires
in sections of tissue.
PROCEDURE
 Treat the film three times, 30 seconds each, with a
fixative.
 Wash off the fixative with absolute alcohol and
allow the alcohol to act for 3 min.
 Drain off the excess alcohol and carefully burn off
the remainder until the film is dry.
 Pour on the mordant, heating till steam rises, and
allow it to act for 30 seconds.
 Wash well in distilled water and again dry the slide.
 Treat with ammoniated silver nitrate, heating till
steam rises, for 30 seconds, when the film becomes
brown in color.
 Wash well in distilled water, dry and mount in
Canada balsam.
 It is essential that the specimen be mounted in
balsam under a cover-slip before examination, as
some immersion oils cause the film to fade at once.
 The spirochaetes are stained brownish-black on a
brownish-yellow background
OBSERVATION
LACTOPHENOL COTTON BLUE STAINING
 Lactophenol cotton blue stain is used for making a
semi-permanent microscopic preparation of fungi.
 It stains the fungal cytoplasm and provides a light
blue background against which walls of the hyphae
can readily be seen.
 It contains four constituents, phenol serves as
fungicidal agent, lactic acid acts as a clearing agent,
cotton blue stains the cytoplasm of fungus and
glycerine which gives the semi-permanent
preparation.
 A permanent preparation is made by incorporating
polyvinyl alcohol in place of glycerine.
PROCEDURE
 A drop of lactophenol cotton blue was placed on a
clean glass slide.
 A small part of fungal colony was taken from
Potato Dextrose Agar (PDA) plate using a sterile
needle and it was placed on the drop of lactophenol
cotton blue that had been taken on the glass slide.
 The fungal elements were spread with sterile
needle.
 A coverslip was placed over the specimen taking
care to avoid the formation of air bubbles.
 Excess stain was wiped off.
 The slide was kept for 5-10 min and was observed
first under 10X then under 45X objective.
OBSERVATION