Introduction to Medical

Microbiology

1
 Outline of lecture
• Introduction
• Historical perspectives
• The microbial cell structure
• Introduction to microbial genetics
• Classification of bacteria
• The growth, survival and death of microorganisms
• Normal microbial flora
• Isolation and identification of microbes
• Sterilization and Disinfection

2
Introduction

• Microbiology is a subject which deals with living

organisms that are individually too small to be seen
with the naked eye.
• It considers the microscopic forms of life and deals
about their:
– Morphology,
– reproduction,
– physiology,
– Beneficial & harmful r/ship with humans
– Diagnosis, treatment, prevention

NB: These organisms include bacteria, algae, protozoa, fungi, and

virus
3
Different disciplines in Medical Microbiology:

▪ Bacteriology – which deals with bacteria

▪ Mycology- which deals with fungi
▪ Virology –studies about viruses
▪ Immunology-The study of immune system or
immunity

4
 Distribution of microorganisms in nature
• Microorganisms can be found nearly everywhere as
normal inhabitants of the earth (biosphere).

• They exist in soil, water, air, in our food, in our clothing, in

our body etc.

• Microorganisms can also survive in most unlikely

environment like in cold air, in hot springs at temperatures
of 900C.

• Microorganisms inhabit the surface of living human and

animal bodies and grow abundantly in the mouth and
intestinal tract.
5
 Distribution of microorganisms in nature…

• Actually only a small percentage microbes are

pathogenic, few are able to cause disease.

• The others are considered beneficial or harmless, or

they cause disease only if they accidentally invade the
wrong place at the right time such as when the host
immunity is low. These microbes are considered
opportunistic.

• Most of this Microorganisms that live on the human body

with out causing disease and apparent physiological
response comprise the normal flora.

6
 Why we study microorganisms?

1. Beneficial to man
• Production of :- wine, beer, soft drinks
• Used as food
• Recycling material (decomposition of dead bodies)
• Commensals in our body are important in preventing
disease
• Research :- because they are easy to grow in laboratory
(multiply rapidly, need small space)
• Drug production

7
 2. Enemy of human kind (Pathogenic)

• Responsible for the loss of many lives

• Spoil food
• So, in order to eradicate them from this world, first we
have to have information about them.
• “KNOW YOUR ENEMY” and Act

8
 Historical perspectives
– Man kind has always been affected by diseases
which were originally believed to be visitations by
the gods and meant to punish evil doers.

– Hippocratus, father of medicine, observed that ill

health resulted due to changes in air, winds,
water, climate, food, nature of soil and habits of
people.

9
– Fracastorius (1500 G.C.) proposed that the agents of
communicable disease were living germs, that could
be transmitted by direct contact with humans and
animals, and indirectly by objects ; but no proof because
of lacking experimental evidence.

– Antony Van Leeuwenhoek (1632-1723 G.C.), father of

Microbiology, observed “animalcules” using his simple
microscope with one lens.

– He was the first who properly described the different

shapes of bacteria.

– Question raised - where did they originate?

10
• On the bases of this observation, two major theories
were formulated.

• Theory of Abiogenesis deals with the theory of

spontaneous generation; stating that living things
originated “spontaneously” from non-living things.

• Aristotle (384-322 BC): The founder of a theory

spontaneous generation.
– He observed spontaneous existence of fishes from
dried ponds, when the pond was filled with rain.

11
• Biogenesis: - States that life comes from pre existing
life
• Francesco Redi (1626-1697): He is the scientist who
first tried to set an experiment to disprove spontaneous
generation.

12
Francisco Redi
• Introduced experimental procedure to disproof spontaneous
generation
• Performed experiments that disproved theory of
Spontaneous Generation for more complex forms of life
(began approx.1668).
• Utilized jars containing meat. Some were covered, some
were not.
• Maggots appeared in uncovered jars. And conclude that
maggots did not emerge spontaneously but from the eggs
laid on the meat by the fly.
• The controversy on spontaneous generation took 200 years.

13
John Needham (1749)
• Performed experiments similar to Redi’s on the origin of life
in microscopic organisms
• Introduced the first culture medium for microbial growth.
• Utilized infusion broth prepared by boiling meat, grain, etc.
to extract nutrients.
• Broth put in flasks, some were sealed with corks, and some
were not.
• All flasks became cloudy, result different from Redi’s
experiment.
• He suggested that life originate spontaneously from
nonliving matters
• The spontaneous generation opponents didn’t accept his
conclusion, they said it could be due to entrance organisms
from air or flasks, improper seal.

14
Lazzaro Spallanzani (1776)
• Repeated Needham’s experiments to disproof
spontaneous generation in microscopic life.
• Boiled broth after placing in flasks.
• Sealed flasks by plugging with solid stopper.
• Results more consistent with Redi’s.
• Occasionally sealed flask → cloudy.
• Not accepted by spontaneous generation supporters,
because they said that heating may have destroyed,
degraded “vital force” and air was not allowed to enter.

15
• Louis pasture (1822- 1895) was the scientist who
disproved the theory of abiogenesis once and for all.
– Performed experiment to disprove Theory of
spontaneous generation.
– He designed a large curved flask/swan-necked
(pasture goose neck flask) and placed a sterile
infusion broths.
– Flasks remained sterile unless tilted or neck broken.

16
* In ‘A’ air freely moved through the tube, but dust particles were
trapped in the curved portion of the flask. And no microbial
growth was observed.
.

17
Therefore, Pasteur proved that microorganisms entered to
the broth with the air and micro organisms did not evolve
spontaneously
18
 Other major contribution of Louis Pasteur

• Microbial theory of fermentation

• Principles and practices of sterilization and
pasteurization
• Development of vaccines against anthrax and rabies
• Discovery of streptococci.

19
 The Germ Theory of diseases

• Pasture has also developed the germ theory of

diseases, which states that “a specific disease is caused
by a specific type of microorganism”.

• Robert Koch, in 1876 established an experimental

procedure to prove the germ theory of disease, which
states that specific disease is caused by specific
pathogen. The scientific procedure is known as Koch’s
Postulate.

20
 Koch’s Postulate: - proof of germ theory of disease
• A Micro-organism can be accepted as a causative agent of
an infectious disease only if the following conditions are
satisfied.
1. The microorganism should be found in every case of the
disease and under conditions, which explain the
pathological changes and chemical features.

2. It should be possible to isolate the causative agent in pure

culture from the lesion

3. When such pure culture is inoculated in to appropriate lab

animal, the lesion of the disease should be reproduced.

4. It should be possible to re-isolate the bacterium in pure

culture from the lesion produced in the experimental
animal.

21
Fig. Koch’s postulate
22
 Exceptions to Koch’s postulate

• Many healthy people carry pathogens but do not exhibit

symptoms of the disease. E.g. HIV carrier

• Some microbes are very difficult or impossible to grow in

vitro (in the laboratory) in artificial media. E.g. Treponema
pallidum.

• Many pathogens are species specific. E.g. Brucella

abortus cause abortion in animals but not in humans.

• Certain diseases develop only when an opportunistic

pathogen invades imuno-compromised host.

23
 Major achievements of Robert Koch

• Discovery and use of solid medium in bacteriology

• Discovery of causative agents of tuberculosis and
cholera
• Koch’s postulate

24
 Microbiology In Future
❖ Aging population coupled with socio economic and
medical advances see fall
o in problems posed by infectious disease
o decreased in death from the cause
❖ population growth in developing countries continuing
changes in life style will result in increase infectious
disease
❖ In developed countries
o increase drug resistance
o decreasing development of new anti-microbials and
vaccines will create problem in control
❖ Bioterrorism –with possible spread of previously rare or
eradicated disease ,anthrax and small pox
❖ Climate change-will increased the incidence of vector
borne infections
25
 Structure Of Bacteria

❖ Micro-organisms are classified under kingdom Protista,

further divided into
1.prokaryotes
2.eukaryotes
❖ Bacteria grouped under prokaryotes

❖ All other organism are eukaryotes

❖ But, viruses are not cells –they do have DNA/RNA

lacking of cell membrane, cytoplasm and machinery for
synthesing macromolecule

26
Eukaryotic Cell
 Eu- true
 Karyote- nucleus
❖ The eukaryotic cell has a true membrane bound nucleus,
usually containing multiple chromosomes, a mitotic
apparatus, a well defined endoplasmic reticulum and
mitochondria
Prokaryotic Cell
 Pro- primitive
 Karyote- nucleus
The prokaryotic cell possesses naked DNA without
associated basic proteins, divides amitotically by binary
fission and bounded by a semi rigid cell wall.
27
❖ Bacterial structure is considered at three levels.
1. Cell envelope proper:
➢ Cell wall and cell membrane.

2. Cellular element enclosed within the cell envelope:

➢ Mesosomes, ribosomes, nuclear apparatus, and
cytoplasmic granules.

3. Cellular element external to the cell envelope:

➢ Flagellum, Pilus and Glycocalyx

28
The distinguishing features between Eukaryotic cell and
Prokaryotic cell

 Features Prokaryotic cell Eukaryotic cell

 Size 1m 10m
 Nuclear membrane absent Present
 Chromosome Single Multiple
 Nucleolus absent Present
 Histones absent Present
 Sexual reproduction absent Present
 Mitochondria absent Present
 Endoplasmic reticulum absent Present
 Lysosomes absent Present.
 Peptidoglycan present absent
 Cell membrane composition
Phospholipids &Protein Sterols

29
1. Cell envelope proper:
A. Cell wall
Multi layered structure and constitutes about 20% of the
bacterial dry weight.
Average thickness is 0.15-0.5 µm
Main structural component – Peptidoglycan
 Peptidoglycan (Back bone of the cell wall gives rigidity )
 repeating disaccharide units
 polypeptides
Components of cell wall of Gram negative bacteria
 Peptidoglycan (thin)
 Lipoprotein
 Phospholipid
 Lipopolysaccharide
Components of cell wall of Gram positive bacteria
 Peptidoglycan (Thick)
 Teichoic acid
 Lipotechoic acid
30
31
 The acid fast cell wall
• Resemble Gram positive cell wall
• Acid-fast bacteria have a cell wall with a relatively
impermeable cell wall containing a waxy lipid called mycolic
acid.
• Mycolic acids confer
resistance to
– Desiccation
– Most antibiotics
– Phagocytosis
– Contributes to pathogenicity

32
 Functions of cell wall

 Provides shape to the bacterium

 Gives rigidity to the organism
 Protects from environment
 Provides staining characteristics to the bacterium
 Contains receptor sites for phages
 Site of action of antibody
 Contains components toxic to host

33
Grouping based on cell wall composition

1. Gram positive cells (e.g. Bacillus subtilis)

2. Gram negative cells (e.g. E. coli)
3. Bacteria with chemically unique cell walls
– (e.g. Mycobacterium tuberculosis)
4. Bacteria without cell walls (e.g. Mycoplasma)

34
B. Cell membrane
 Also named as cytoplasmic membrane
 It accounts for 30% of the dry weight of bacterial cell.
 It is composed of 60% protein, 20-30% lipids and 10-20%
carbohydrate
Function of cell membrane
 Regulates the transport of nutrients and waste products into
and out of the cell.
 Synthesis of cell wall components
 Assists DNA replication
 Secrets proteins
 Carries on respiration
 Captures energy in ATP
35
2. Cellular element enclosed within the cell envelope:
A. Mesosomes
 Convoluted invagination of cytoplasmic membrane often at sites of
septum formation.
 It is involved in DNA segregation during cell division and
respiratory enzyme activity.
B. Ribosomes
 Cytoplasmic particles which are the sites of protein synthesis.
 It is composed of RNA (70%) and proteins (30%) and constitutes
90% of the RNA and 40% of the total protein.
C. Cytoplasmic granules
 Represent accumulated food reserve
D. Nuclear apparatus
- well defined nucleus and nuclear membrane, discrete chromosome
and mitotic apparatus are not present in bacteria;
- so nuclear region of bacteria is named as nuclear body, nuclear
apparatus and nucleoid
36
 3. Cellular element external to the cell envelope
A. Glycocalyx (capsule and slime layer): Coating of
bacteria, external to the cell wall, made of sugars
and/or proteins. 2 types
▪ Capsule is gel firmly adherent to cell envelope,
composed of polysaccharide.
▪ Slime is gel easily washed off from cell envelope.
❖ All bacteria have at least a thin slime layer
Features of capsule
1. Usually weakly antigenic.
2. Attachment to other bacteria or host tissues
3. Endow virulence.
4. Protects from phagocytosis.
5. Visualized by negative staining and capsule staining
37
B. Flagellum
➢ It is the organ of locomotion in bacterial cell.
 Size: 3-20m in length and 0.01-0.013m in diameter.
 It is composed of protein named as flagellin.
 The flagellar antigen in motile bacterium is named as H
antigen
➢ consists of three parts. These are.
Filament
hook and
basal body

➢ The basal body and hook are embedded in the cell

surface while the filament is free on the surface of
bacterial cell.
38
Importance of Flagella
– Are not essential for survival of the cell
– Attachment to surfaces (epithelial tissue)
– Motility
– as a virulence factor
Flagellar arrangements
 Atrichous: Bacteria with no flagellum. E.g. Shigella
 Monotrichous: Bacteria with single polar flagellum. E.g.
Vibrio cholerae
 Lophotrichous: Bacteria with bunch of flagella at one
pole. E.g. Helicobacter pylori
 Amphitrichous: Bacteria with flagella at both poles. E.g.
Campylobacter jejuni
 Peritrichous: Bacteria with flagella all over their surface.
E.g. Escherichia coli, Proteus mirabilis 39
40
C. Pili (fimbriae)
 It is hair like structure located on bacterial surface and
important for adherence to bacterial cell surface.
Two types
 Common pili: The structure for adherence to cell surface.
 Sex pili: The structure for transfer of genetic material
from the donor to the recipient during the process of
conjugation.

41
D. Spores
• Structures which are capable of surviving under adverse
environmental conditions like heat, drying, freezing,
action of toxic chemicals and radiation.
• Bacterial spore is smooth walled and oval or spherical in
shape.
• It does not take up ordinary stains.
• It looks like areas of high refractivity under light
microscope.
• It is significant in spread of disease and sterility of
materials.

42
 Bacterial Genetics

 Most of the genetic information in a bacterial cell is

contained within chromosome.
• A single molecule of DNA arranged as a double
in closed loop.
 Bacterial inherited characteristics are encoded in DNA.
These are:
• Chromosome
• Extra chromosome (Plasmid)

43
 Chromosome- Bacterial chromosome is circular double
stranded DNA attached to bacterial cell membrane

 Plasmids: are self replication extra chromosomal DNA

molecules.

 Plasmids are not essential to the life of the cell but they
may have selective advantage for these organism
 R-factor (Resistance)
 Extra cellular toxin

44
Genetic variation in Bacteria

Genetic Variation in Bacteria can occur by

 Mutation :Bacterial mutations occur when the information
in a bacterial chromosome may alter by different means
 There are two types of mutation:
Spontaneous mutation
Induced mutation
 Gene-Transfer: The transfer of genetic information can
occur by either of the three methods:
 Conjugation
 Transudation
 Transformation
45
➢ Conjugation
 A process where by DNA is transferred from one
bacterium to another by cell to cell physical contact
 Plasmids are the genetic elements most frequently
transferred by conjugation

46
Transduction
 Is a method of gene transfer in which a virus (phage) acts
as a vehicle for carrying DNA from a donor bacterium to
recipient bacterium.

Fig. Transduction of a chromosomal DNA sequence (a) and

a plasmid (b). 47
• Transformation
 A process by which a bacterium acquire DNA fragments
or genes from surroundings.
 Usually this occurs in microbial culture.

48
 Classification of Bacteria
Classification of Bacteria-Taxonomic classification
• Taxonomy-a science of classification of living organism
which consists three separate things, but inter related
➢ classification
➢Nomenclature
➢identification
❖ Classification
• A number of criteria have been employed to
group bacteria
➢ energy sources
➢ phototrophic
➢ chemotrophic
➢ autotrophic
➢ heterotrophic
49
• as nutrient requirement
-simple
-complex

• Optimum temperature requirement

i. psychrophylic (cold loving)
ii. Mesophilic (25-40 c)
iii. Thermophilic (inc T loving)

• O2 requirement(aerobic, anaerobic)

50
 Biological classification
• Based on observable characteristics:
1. Morphology
2. Staining
3. Motility
4. Growth
5. Biochemical and metabolic activity
6. Pathogenicity

51
1. Morphology
▪ When bacteria are visualized under light microscope, the
following morphology are seen.
1. Cocci - Round or oval bacteria measuring about 0.5-1.0
mm in diameter. They are found in single, pairs, chain or
cluster
– Micrococci:- Cocci occurring single.
– Diplococci:- arranged in group of two
– Streptococci :- chain forming cocci
– Staphylococci:- bunches of cocci or irregular groups
or grapes of cocci

52
 Spherical Bacteria

53
• 2. Bacilli(Rod shaped):- stick- like bacteria with
rounded- tapered square with a site measuring 1-10mm
in length by 0.3 - 1.0 mm in width

54
3. Spiral shaped bacteria
 It is a regular or irregular distance between twisting
➢ Borrelia, Treponema, leprospira

Spirochete:

burgdorferi
Borrelia

55
2. Staining
 Bacteria staining are the process of coloring of colorless
object using stains (dyes)
Uses of staining
 To observe the morphology size and arrangement of
bacteria
 To differentiate one group of bacteria from the other
group.

56
I-Simple staining method
• It is a type of staining method in which only a single dye
is used.
Two kinds of simple stains
A. positive staining:- The bacteria or its parts are stained
by the dye
E.g. Methylene blue stain, Crystal violent stain
B. Negative staining: - the dye stains the background and
the bacteria remain unstained
E.g. India ink stain
57
2. Differential staining method
▪ Multiple stains (dyes) are used to distinguish different
group of bacteria
▪ React differently with different types of bacteria
▪ Two Most Common
 Gram Stain
 Acid-Fast Stain

58
 A. Gram stain
Gram Stain: Named after Christian Gram who invented it
 Most bacteria are differentiated by their gram reaction
due to difference on their cell wall structure.
Principle:
 Bacteria are stained with Crystal violet and Iodine,
then subjected to decolorization with alcohol/acetone.

• Bacteria that retain the dye are: Gram-positive

• Bacteria that loose the dye are: Gram-negative

59
Principle of staining technique:
1- Crystal violet: all cells are stained violet.

2- Iodine acts as a mordant (fixes the dye) all cells remain

violet.

3-Alcohol acetone decolorizes gram negative cells only:

Gram-positive remains violet, gram-negative becomes
colorless.

4-Counter stain with Safranin: Gram-positive remains

violet while gram negative becomes red.

60
 Gram-Positive

Gram-Negative

61
 II. Ziehl- Neelson staining Method

▪ Ziehl-Neelsen stain (Acid fast stain) is used to stain

mycobacterium and other acid fast organisms which
cannot be stained with gram stain.

▪ Principle- once the mycobacterium is stained the primary

stain it can not be decolorized with acid. So named as
Acid fast bacteria

62
1. Carbolfuchsin (Red)
2. Acid Alcohol
3. Counterstain with Methylene Blue
• Acid - Fast Cells Red
• Non Acid - Fast Blue
Result
➢ Acid fast bacilli . . . . Red
➢ Back ground . . . . Blue

63
III. Cultural characteristics
➢ EXAMPLE: - colorless white, pink and pigmented colony
Smooth, (Mucoid) and shinny rough colony

IV. biochemical reaction

➢ catalase positive/neg, oxdase pos/neg…, lactose
fermenter/lactose nonefermenter

64
 Nomenclature

• Binomial system of nomenclature

– The generic (genus) name followed by the species
name
– Generic part is capitalized, species is lowercase
– Both are italicized or underlined if italics aren’t
available
– Example: - Streptococcus pneumoniae

65
 Bacterial Growth
 It is an orderly increase in all the components of an
organism.
 It is an increment in biomass.
Generation time
 It is the time taken for the size of a bacterial population to
double.
➢ time required for a cell to divide

 bacteria divide into two equal daughter cells and double

the number
 The generation time varies with
– the species
– the amount of nutrients, the temperature, the pH, and
other environmental factors.
66
 Bacterial growth phases
1.Lag phase
 The period of adaptation with active macro molecular
synthesis like DNA, RNA, various enzymes and other
structural components.
 It is the preparation time for reproduction; no increase in cell
number
2. Exponential (log) phase
 The period of active multiplication of cells.
 Cell division proceeds at a logarithmic rate, and determined
by the medium and condition of the culture.
• The number of bacteria during log phase growth can be
calculated by the following equation
• Nt= No x 2 t/d
• Nt = is the number of bacteria after time (t),
• t/d = is the amount of time divided by the doubling time
67
• No = the initial number of bacteria
3. stationary phase
• The period when the bacteria have achieved their
maximal cell density or yield.
• There is no further increase in viable bacterial cell number.
• The growth rate is exactly equal to the death rate.
❖ A bacterial population may reach stationary growth when
one of the following conditions occurs:
• The required nutrients are exhausted
• Inhibitory end products are accumulated
• Physical conditions do not permit a further increase in
population size

68
4. Decline phase
 The period at which the rate of death of bacterial cells
exceeds the rate of new cell formation.
 There is drastic decline in viable cells.

 Few organisms may persist for so long time at this

period at the expense of nutrients released from
dying micro-organisms.

69
Fig. Bacterial growth curve
70
 Normal microbial flora
• Normal microbial flora (normal microbiota) :
– the population of microorganisms that inhabit the skin
and mucous membranes of healthy normal persons

• The genomes of these microbiota are collectively defined

as the microbiome

• members of the normal flora vary in both number and

kind from one site to another

71
• Acquisition starts just after birth (sterile in utero)

• Normally sterile parts

– central nervous system, blood, lower bronchi and
alveoli, liver, spleen, kidneys, and bladder are free of
all but the occasional transient organism.

• Can be altered by antimicrobial therapy

• dynamic state (reflect age, nutrition, environment)

72
 Normal flora can be

• Residents : present for invariable period

• Transients : establish itself briefly , excluded by host
defense or competition from residents
• Carrier state : potentially pathogenic
[ eg. In upper respiratory system : Streptococcus
pneumoniae, Neisseria meningitides]

73
74
 Beneficial effects

• Normal microbiota :-
– Provide the first line defense against microbial
pathogens
– Assist in digestion
– Play a role in toxin degradation
– Contribute to the maturation of the immune system

75
 Unfavorable role
• Can be source of opportunistic infection (endogenous
source)
– Ineffective immune system (e.g. HIV)
• Can cause disease when displaced from normal sites
– During : intestinal perforation /broken skin
– tooth extraction (viridans Streptococci)
– organisms from the perianal area ascend the urethra and
cause UTI (E.coli)
• cause of nosocomial infections
• Cause infection during changes in the composition of the
normal flora
– antimicrobial treatment, increases of the local
environment such as increase in stomach or vaginal pH
76
• Present as diagnostic challenge in the laboratory
• cross reaction with normal tissue components
– eg, antibodies to various ABO group arise because of
cross reaction between intestinal flora and the
antigens of A &B blood substances

77
78
 Isolation and identification of microbes

• The aims of the microbiology laboratory are:

1. To provide accurate information about the presence or
absence of microorganisms in a specimen that may be
involved in a patient's disease process;
2. To provide information on the antimicrobial susceptibility
of the microorganisms isolated.
• Identification is achieved by detecting
– the microorganism or
– its products or
– the patient's immune response

79
Methods in bacterial identification

• Microscopy
• Culture and identification
• Serology
• Molecular techniques
• Detection of metabolites Etc.

80
➢ Culture media are artificially prepared media containing
the required nutrients used for propagation of micro
organisms.
Once the bacteria are grown we can:
– Identify them either by presumptive lab diagnosis like
Gram stain or by definitive lab diagnosis like
biochemical test
– Test the antimicrobial sensitivity of the bacteria (drug
testing).
• This helps to know whether the bacteria are sensitive or
resistant to known antimicrobial drugs.

81
 Types of culture media
The main types of culture media are:
1. Basic media: support the growth of micro organisms that do not
have special nutritional requirements. E.g. Nutrient Agar, Nutrient
broth
2. Enriched or enrichment media: for growth of organism with
extra nutritional requirements such as H. influenza, Neisseria spp.,
and some streptococcus species E.g. Blood Agar, Chocolate agar
3. Selective media: contain substances which inhibit the growth of
one organism to allow the growth of the other to be more clearly
demonstrated. e.g. Salmonella and Shigella agar
4. Indicator (differential) media: dyes or other substances are
added to differentiate micro-organisms .E.g. MacConkey agar
5. Transport media: E.g. Amies transport medium

82
83
 Sterilization and Disinfection

Sterilization: Destruction of all forms of microbial life

including spores.
Disinfection: Disinfection of microbes that cause disease;
may not be effective in killing spores
Antiseptic: destruction or inhibition of microorganisms in
living tissue there by limiting or preventing the harmful
effect of infection.
• The agents, which are used for sterilization and
disinfection, can be divided into two broad groups:
I. Chemical mean's of sterilization & disinfection
II. Physical mean's of sterilization & disinfection

84
I. Chemical Means' of sterilization and disinfection
▪ These chemical agents destroy any type of microbes
with out showing any form of selectivity unlike antibiotics
▪ These chemical agents destroy any type of microbes
with out showing any form of selectivity unlike
antibiotics.
▪ The efficacy of these agents depends on the following
factors:
– Concentration of the agent
– Time of exposure
– PH of the medium
– Temperature
– Nature of the organism
– Presence of extraneous materials
85
Classification of Chemical Mean’s of Sterilization and
Disinfection
1. Chemical agents that damage the cell membrane
 Phenols
 Organic solvents ( Alcohol e.g. Ethyl alcohol, Isopropyl
alcohol).
2. Chemical agents that denature proteins
 Acids and alkalis (like benzoic acid, citric acid and
acetic acid
3. Chemical agents that modify functional groups of
proteins and nucleic acids
 Heavy metals (mercuric chloride, Silver nitrate)
 Oxidizing agents (Chlorine, iodine)
 Alkylating agents (Formaldehyde, Glutaraldehdyde)
86
 II. Physical methods
1. Heat
– It is the most reliable and universally applicable
method
• Types
A. Dry heat
B. Moist heat
A. Dry heat
– It is less efficient and requires high temperature and
long period heating than moist heat.
a. Incineration: It is an efficient method of sterilization
and disposal of contaminated needles, syringes and
cover slips at high temperature.
87
 Dry heat …

b. Red heat: Inoculating wires, loops and points of

forceps are sterilized by holding them in the flame of
a Bunsen burner until they are red hot.
c. Flaming: Scalpels and neck of flasks, bottles and
tubes are exposed for a few seconds, but it is of
uncertain efficacy.
d. Hot Air Sterilizer (Oven): it is essential that hot air
should circulate between the objects being sterilized.
It is done by applying 1600c for 1 hour.

88
 B. Moist heat
• It is preferred to dry heat due to more rapid killing.
• Moist heat can be used by the following methods.
1. Boiling: It is not reliable method of sterilization.
It is done by applying 1000c for 30 minutes.
• Used for sterilizing catheters, dressing
2. Tyndallization: Intermittent steaming
Steaming of the material is done at 1000c for 30 minutes on
three consecutive days.
– Principle is that spores which survived the heating
process would germinate before the next thermal
exposure and then would be killed.
– It is used for sterilizing heat sensitive culture media
containing materials such as carbohydrates, egg or
serum. 89
 Moist heat …

3. Pasteurization:
– It is the process of application of heat at temperature of
620c for 30 minutes or 720c for 15 seconds followed by
rapid cooling to discourage bacterial growth.
Uses:
– Pasteurization of milk
– Preparation of bacterial vaccines.
4. Autoclave: Steam under pressure
– It is based on the principle that when
microorganism is boiled at increased pressure, hot
saturated steam will be formed which penetrates
and gives up its latent heat when it condenses on
objects.
90
 Autoclaving …

• Uses: Sterilize solid and fluid culture media, gowns,

medical and surgical equipment.
• Time-Temperature-Pressure Level Relationship in moist
heat sterilization (Autoclaving)
Temperature Time Pressure level
1210c 15 minutes 15 Ib/inch2
1260c 10 minutes 20 Ib/inch2
1340c 3 minutes 30 Ib/inch2

91
2.Filtration
 Removes microorganisms from solutions that might be
damaged by heat
➢ culture media
➢ Enzymes
➢ Vaccines
➢ antibiotics

92
3. Radiation
1. Ionizing Radiation
 gamma rays & x-rays
 penetrates most substances
Used on substances that could be damaged by heat
 plastic petri dishes
 plastic syringes
 catheters
 surgical gloves
 . Non-Ionizing Radiation
 UV Light
 does not penetrate plastic, glass or proteinaceous
matter
 Used to reduce microbial populations
 hospital rooms
 nurseries
 operating rooms 93