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HISTORY AND EVOLOTUION OF MICROBIOLOGY

MICROBIOLOGY

Microbiology is the study of organisms that are the small

that cannot clearly seen by our naked eye.

MICRO ORGANISMS

Organisms with a diameter of 1mm or less which cannot be

seen by the unaided human eye are called microorganisms.

These include bacteria, fungi, algae, virus, and protozoa.

DISCOVERY OF MICRO ORGANISMS:

1. ROBERT HOOKE (1635- 1703)

Robert hooke spent many hours using his compound
microscope to examine various materials. He published
his observations in a book named “micrographia” in 1664.
This book contains detailed drawings of insects, insect
larvae, seeds, eggs, feathers, hairs, rocks and corks.
In his observation , Hooke described the fungi
commonly called as molds. In this first reported
observation of molds, he compared the observed knobs to
the spore bearing caps of mushrooms.

2. ANTONIE VAN LEEUWENHOEK (1632- 1723)

Antonie van Leeuwenhoek was a draper and haberdasher
and he is the owner of a dry goods business in delft,
Netherland. He enjoyed a comfortable living; selling silk,
wool, cotton, buttons and other supplies. He was the head
of the city council, inspector of weights and measures and
court surveyor.
In his spare time, he developed the ability to grind
glass lenses and to mount them into brass contraception
that he called microscopes. Samples are plot on the
adjustable needle mounted above the lens. He was able
to see the specimens on the needle by holding the needle
mounted above the lens. He was able to see the
specimens on the needle by holding the needle close to
his eye and looking through the lens at an angle of 45
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degree to the light source. This created a type of dark

field microscopy in which microbes appear as bright
objects against a dark background. His lenses were very
small, but could magnify an object 50-300 times. Initially,
he used the microscope to inspect the quality of cloth, but
later he observed a large number of samples.
Under the recommendation of Regnier de graaf, the
members of the Royal society in London wrote to Antonie
Van Leeuwenhoek to report his findings in 1673. So,
Antonie Van Leeuwenhoek started spending his
illustrations and observations for the next 50 years.
In September 1674, Van Leeuwenhoek looked at water
samples from the freshwater river. For the first time in the
human history he observed small micro-organisms which
he named ‘animalcules’. This made him observe many
other samples such as materials from his own teeth and
faces.
In 1676, in his 8th letter to the royal society, he sent his
1st detailed description of the microorganisms when he
was experimenting why pepper tasted hot, he observed
bacteria. In 1683, he described rod shaped bacteria as
bacilli, spherical bacteria as cocci, bacteria with spiral
bodies resemble corkscrews are called spirillum or
spirochete .
Many of his observations were unconfirmed to make
microscope of good quality. Thus after his death in 1725,
the science of microbiology by dormant for many years.
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VAN LEEUWENHOEK ‘S DESCRIPTIONS OF BACTERIA.

A and B - Rod forms

C and D - Indicating pathway of motion
E - Spherical form
F - Longer type of clusters
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THE RISE AND FALL OF THE SPONTANEOUS

GENERATION – THE ORIGIN OF MICRO ORGANISMS:
In the 17th and 18th centuries, many people including
Aristotle believed in the ability of living creatures to arise
from non-living matter such as dead animals, meats or
broths made from meat, hay etc., this theory is known as
the theory of spontaneous generation or abiogenesis.

3. FRANCECO REDI (1626- 1697)

Francesco Redi, an Italian priest was one of the first
to dispute the theory of abiogenesis. Redi knew that
maggots developed in unprotected meat, however he was
of the opinion that the maggots did not arise
spontaneously. He thought that they originated from the
flies that landed on the meat. In the 1670‘s Redi
performed a series of tests in which he placed meat in 3
contours; one was uncovered, a second was covered with
paper and the 3rd was covered with a fine gauze that could
exclude flies. Flies are attracted to the meat on the jars,
but they did not develop on the covered meat. These
simple experiments showed that maggots grew from fly
eggs and are not able to develop spontaneously from
meat.

4. JOHN NEEDHAM (1713- 1781)

John need ham, an English priest performed
experiments in 1767 with mutton gravy that supported his
belief in spontaneous generation. He placed hot mutton
gravy in flashes, sealed them with corks to except air and
then heated them. The flasks were cooled and then
exposed for day’s air. Needham observed microorganisms
in all his flask and claimed that they arose spontaneously
from the mutton gravy. He thought that the organic
matter contained a vital force that could confer the
properties of life on non-living mater.
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5. LAZZARO SPALLANZANI (1729-1799)

Lazzarospallanzani, an Italian priest set up 4 groups
flasks:
 Group 1 was made air tight by melting the glass neck
of the flask.
 Group 2 was stoppered with cotton.
 Group 3 was stoppered with wooden corks to
resemble need ham’s experiment.
 Group4 was left open to air.

All the flasks were filled with seeds and vegetative

matter and then heated for one hour at the start of
the experiment. Then they were set aside for 25
days before observing them. The open flasks
contained numerous life forms; the corked flask does
not have any microbial growth, but the flasks that
were stoppered with cotton and the air tight sealed
flasks contained only a few animalcules.
Spallanzani’s contained that the cotton of
animalcules in a flask increased with exposure to the
air. The animalcules in need ham’s corked flasks
must have entered from the air and this could be
prevented with an air tight seal.
But many people contradicted his theory. They
charged that organisms need air to live and could not
originate in Spallanzani’s sealed flasks.

6. THEODORE SCHWANN (1810-1882)

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Schwann allowed air to enter a flask containing a sterile

nutrient solution alfter the air had passed through a red
hot tube . Then the flask remained sterile.

7. SCHRODER AND VON DUSCH’S EXPERIMENT:

George Friedrich Schroder and Theodore von dusch

allowed air to enter a flask of heat sterilised medium after
it had passed through sterile cotton wool. No growth
occurred in the medium even though the air has not been
heated.
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THE GOLDEN AGE OF MICROBIOLOGY.

The science of microbiology blossomed in 1857 and this
period lasted for 60 years. This period is known as the golden
age of microbiology. During these years, numerous branches
of microbiology were established.
8. LOUS PASTEUR
Louis Pasteur, a French organic chemist was a major
contribution for many findings in the field of microbiology.
His interest in microbiology as the causative agents of
diseases, due to the death of his 3 daughters has led to
the openings in various aspects in microbiology, which
were till then a mystery to human population. His major
contributions are given below.

a. GUN COTTON EXPERIMENT:

Louis Pasteur first demonstrated that air contains
microscopically observable organizing structures. He
passed a large quantity of air through a tube that
contained plug gun cotton to serve as a filter. The gun
cotton was then removed and dissolved in mixture of
alcohol-ether and the sediment is examined
microscopically, he found that his sediment contained
not only organic matter but also a large number of
small row structures which were identified to
microorganisms.

b. SWANN NECK FLASK EXPERIMENT:

By a set of experiments, Pasteur finally brought
an end to the theory of spontaneous generation. In the
early 1800’s he prepared broth in a series of swan neck
flasks, so name because of their S-shaped necks
resembled a Swan’s neck. Pasteur boiled the flasks of
broth, then left them open to the air. However, the S-
shaped curvature of the neck trapped air born dust and
micro-organisms and prevented their entry to the flask.
No microorganisms is identified in the broth. When the
neck was later cut off, however, air born organisms
quickly fell into the broth and growth appeared within
hours.
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c. PASTEURAISATION AND FERMENTATION:

Generally people thought that wine fermentation
resulted from the chemical break down of grape juice to
alcohol and no living thing seemed to be involved.
Pasteur made an intensive study of the beer and wine
manufacturing processes and cause of souring and
storage of wines. His observations showed a large
number of yeast cells. He believed that the yeasts
played a major role in fermentation. To prove his
theory, he removed all traces of yeast from a sample of
grape juice and set the juice aside to fermentation
which results nothing. Next he added yeast bacteria to
the juice and soon the fermentation was proceeding
normally.
Pasteur also found that wine spoilage was caused by
the growth of undesirable contaminating microbes. The
solutes to the problem was in preventing the grow of
these undesirable organisms. After considerable
experimentation Pasteur showed that wine did not
undergo spoilage. It was held for few minutes at 50
degree to 60 degree. IN the same way been could also
remain unspoiled by heating to 50 degree to 55 degree.
This gave rise to the new process of preserving wine,
fruit juices, milk etc., and was called pasteurization.
This short heating process killed pathogenic spoilage
micro-organisms.
During his life time, Pasteur studied a number of
other fermentation processes. He showed that each
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type of fermentation is carried out by a specific type of

micro-organism under specific environmental
conditions.

d. PASTEUR’S CLASSIFICATION OF MICROBES BASED

ON OXYGEN REQUIREMENTS:
During the studies on butyric acid fermentation,
Pasteur discovered the existence of life in the absence
of oxygen. He paved a current of air through the
butyric fermentation fluid and found that this resulted in
the complete arrest of the fermentation. He concluded
that some micro-organisms could live only in the
absence of oxygen. The he named life in the presence
of oxygen as aerobic and in the absence of oxygen as
anaerobic.
Pasteur also found that many other micro-organisms
including yeasts grow either in the presence or absence
of oxygen. These are termed as facultative anaerobes.

e. DEVELOPMENT OF VACCINES:
During his study on chicken cholera, a disease that
was caused by pasteurellamultocida . Pasteur grew the
bacterium in cultures repeatedly for increasingly long
periods and found that the bacteria from fresh culture
was weakened. He named the later cultures as
attenuated. (I.e. weakened in ability to cause disease).
The inoculation of healthy chickens with attenuated
stains of pasteurellamultocida would prevent chicken
cholera. He published his results in 1881 which paved
way towards the treatment of disease with vaccines.
Pasteur next prepared rabies vaccine by a different
approach. The pathogen was attenuated by growing it
in the rabbit. After infected rabbits had dies, their brains
and spinal cords were removed and dried whichlet to
the attenuation of the rabies virus. On July 6, 1885, he
injected the 1st of 14 daily doses of rabbit spinalcord
suspensions into the boy named Josephmeister and the
boy survived.

9. JOHN TYNDALL (1820-1893):

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IN 1877, Ferdinand cohn discovered the existence of

certain bacteria in the form of endospores. They are
extremely heat resistant that were not killed by
pasteurization.
The solution to this problem was presented by the
English physician John tyndal in 1877. Tyndall observed
that hay infusions contained heat resistant spores. He
noted that the spores germinated into heat sensitive
vegetative cells following a brief exposure to heat.
Consequent boil killed the newly formed vegetative cells.
From these observations, he devised on alternative
methods called as tyndallisation as intermitted
sterilization. In this method, he boiled the infusions
repeatedly with an interval in bacteria spoilage recurred.

BEGINNING OF MEDICAL MICROBIOLOGY:

Although Fracastor and a few other had suggested that
visible organism produce disease, most believed that
disease was the one which causes such as super natural
forces, poisonous vapours, etc., support to the germinate
theory of disease began to accumulate in the early 19 th
century. Agostinobassi (1775-1856) first showed a micro-
organism could cause disease by demonstrating in 1835
that a silkworm disease was due to a fungal infection. In
1845 M.J. barkeley proved that potato blight was due to a
fungus. Pasteur showed that particular diseases of
silkworms was due to a protozoan parasite.

10. JOSEPH LISTER (1827-1912) ( FATHER OF

ANTISEPTIC SURGERY)
During the 19th century, surgical treatment of human
diseases had come into practise. Most patients who
underwent surgery development severe cares of sepsis
whose cause was not known. Joseph lister, an English
surgeon believed that the microbes entered the wound
through air, hands, surgical instruments or band aids, and
cause sepsis. He argued that one should be able to
prevent sepsis by applying the principle of sterilization
which would destroy these micro-organisms. He sterilized
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the surgical instruments used for surgery by boiling in

water and conducted surgery under a spray of carbolic
acid (phenol). The results were remarkable and lead to a
great reduction in the number of sepsis causes.
The earliest method for the pure culture isolation of
bacteria by serial dilution was also proposed by Lister.
He proposed that if a solution containing a mixture of
micro-organisms is diluted serially in such a way that the
final dilution will contain either one or none of the
organisms, then it should be possible to isolate a pure
culture. To prove his point, he first determined the number
of micro-organisms in a diluted sample of curdled milk
microscopically. Then he diluted the milk with sterile
water in such a way that the final dilution contained either
one or none of the micro-organisms. The inoculated as
separate glasses of milk with this final dilution and
incubated the milk for several days. He found that only in
one of the glasses, the milk had curdled. On microscopy
examination of the curdled milk, he found that it
contained only one type of rod-shaped bacteria which he
called as bacteria lactis.

11. ROBERT KOCH (1843-1910):

a. ANTHRAX
Anthrax, a fatal disease affects domestic sheep
and cattle caused a great economic loss in Germany.
Koch took blood form an infected sheep that died of
anthrax and injected it into a series of mice. When
one of the mice died; he took his blood and injected it
intoa healthy mouse. Koch repeated this experiment
many times and each time the mouse died from
anthrax. Examination of the dead mice revealed
swollen spleen and the presence of bacilli in both
spleen and blood.
Koch placed a drop of aqueous humor. (liquid
from eyeball)on a thin glass coverslip and inoculated
it with a piece of infected spleen. Koch inverted the
coverslip.

Four criteria that were established by Robert Koch to

identify the causative agent of a particular disease, these
include:
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1. the microorganism or other pathogen must be present in

all cases of the disease
2. the pathogen can be isolated from the diseased host
and grown in pure culture
3. the pathogen from the pure culture must cause the
disease when inoculated into a healthy, susceptible
laboratory animal
4. the pathogen must be re-isolated from the new host
and shown to be the same as the originally inoculated
pathogen
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PREPARATION OF MEDIA

 There are some naturally occurring substances which are

used for cultivation of bacteria. E.g.: Milk, but skimmed
milk used rather than whole milk.
 Such natural materials are taken in tubes or flask and
sterilized before milk is used.
 Preparation of nutrient broth or nutrient agar is done by
adding ingredients to water.
 Practically all media are available commercially in
powdered form.

Preparation of media involves following steps.

 Each ingredient is dissolved in an appropriate volume of

distilled H2o in conical flask and mixed well.
 pH is checked with pH meter and adjusted if necessary.
 If solid medium is required, agar is added and the medium
is boiled to dissolve the agar.
 Then medium is sterilized by autoclaving.

PHYSICAL CONDSITION REQUIRED FOR GROWTH:

 For cultivation of bacteria, there are several physical

condition are required for growth. They are as follows
1. Temperature
2. Gaseous conditions
3. pH

TEMPERATURE:

 Growth of microorganism is dependent on chemical

reaction and is influenced by temperature.
 The temperature that allows the most rapid growth during
a short period of time is known as optimum growth
temperature.
 On basis of temperature relationship to bacteria, bacteria
can be divided into
a. Psychrophiles  0.c- 20.c
b. Mesophiles  25.c – 40.c (pathogenic bacteria)
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c. Thermophiles  above 45.c

GASEOUS CONDITIONS:

 The main gases that affect bacterial growth are oxygen

and carbon dioxide.
 Bacteria have a wide variety of responses to free oxygen
and are divided into four groups.
a. Aerobic bacteria
b. Anaerobic bacteria
c. Facultative anaerobic bacteria
d. Micro aerobic bacteria

AEROBIC BACTERIA

 They require oxygen for growth and can grow when

incubated in an air atmosphere. 21% oxygen.

ANAEROBIC BACTERIA

 They do not require oxygen for their growth and oxygen is

toxic to them and organism will not grow when incubated
in an air atmosphere.
 Some can tolerate low level of oxygen and grow but some
can’t grow they may die even they are strict anaerobic or
stringent bacteria.

FACULTATIVEANAEROBIC BACTERIA:

 They do not require oxygen for growth, although they may

use it for energy production if it is available.
 They are not inhibited by oxygen but usually grow well
under an air atmosphere.

MICRO AEROBIC BACTERIA:

 They grow at low levels of oxygen but can’t tolerate

thelevel ofoxygen present in air atmosphere.
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CULTIVATION OF AEROBIC BACTERIA:

 To grow aerobic or facultative bacteria in tubes or small

flask incubation of the medium is under atmosphere
condition is generally satisfactory.
 When aerobic organism is grown in large quantities it is
advantageous to increasethe exposure of themedium to
the atmosphere.
 This is done by dispensing the medium in shallow layer ina
special container.
 Aeration is increased by constant shaking the inoculated
liquid culture.

CULTIVATION OF ANAEROBICBACTERIA:

 Stringent anaerobic bacteria or strict anaerobic bacteria

can be grown only by taking special precautions to
exclude all atmospheric oxygen from the medium. This
can be done with the following methods

PREREDUCED MEDIA:

 In this method, the culture medium is boiled for several

minute to bringout the dissolved oxygen.
 Then a reducing agent is added to lower the oxygen
content, reducing agent is cysteine.
 Oxygen-free N2 is bubbled thorough the medium to keep
it anaerobic.
 Then the medium is transferred to the tubes which are
flushed with oxygen free N2 and closed tightly and
sterilized by autoclaving.
 Such tubes can be stored for many months before being
used.

CULTIVATION OF BACTERIA

 Bacteria are usually cultivated and studied under

laboratory conditions. There are numerous media that
have been developed for cultivation of bacteria.
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 Bacteria exhibit wide difference with respect to physical

conditions favoring the growth such as
1. Temperature
2. pH
3. Gaseous environment

NUTRITIONAL REQUIREMENTS:

 All forms of life from microorganism to human beings

share certain nutritional requirement for growth and
normal functioning.
 The diversity of nutritional types found among bacteria
are
1. All organisms require a source of energy some rely on
chemical compounds for their energy and are called as
chemotrops. Some organism used radiation energy as
energy source (light) are called as phototrops.Some
organism requires both chemical and light.
2. All organisms require a source of electrons for their
metabolism.Some organism use reduced inorganic
compounds as electron donors are termed as lithotrops.
Some are chemolithotrops and some may be
photolithotrops.Some organism use organic compounds as
electron donors are called as organotrophs. Some may be
chemo organotrophs and some may be photo
organotrophs.
3. All organisms require carbon for use in synthesizing cell
components. All organisms require a least amount of
co2.Some organism use co2 as their major or even sole
source of carbon and such organism are called as
autotrophs.Some organisms require organic compound as
their carbon source are called as Heterotrophs.
4. All microorganisms require nitrogen for cell component
eukaryotes and some bacteria use atmospheric
nitrogen.Some organism grow rich on inorganic nitrogen
compound such as nitrates, nitrites or ammonium salts
some organism get from organic compound such as amino
acids.
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5. All organisms require oxygen, Sulphur and phosphorus for

cell components.Oxygen is provided in forms of water,
components of various nutrients or molecular
oxygen.Sulphur is need for synthesis of certain amino
acids such as cysteine, cysteine and methionine. Some
bacteria use inorganic Sulphur and some use elemental
Sulphur.Phosphorus is supplied in forms of phosphate
which is essential component of nucleotides, nucleic
acids,phospholipids, teichoic acids.
6. All living organism require metal ionssuch as potassium,
calcium, magnesium, and iron for their normal
growth.Other metal ions which are required in trace
amount are zinc, copper, manganese, nickel.
7. Most bacteria donotrequire sodium but some certain
marine bacteria and cyanobacteria and photosynthetic
bacteria require it.
8. All living organism contains vitamins and vitamin like
compounds as a building blocks for co-enzymes or either
co-enzyme or several enzymes.
9. All living organism require water and it require for bacteria
all nutrients must be in aqueous solution before they enter
the cell.

BACTERIOLOGICAL MEDIA:

 Chemically defined media are needed for the cultivation of

autotrophs and are useful for defining the nutritional
requirements to heterotrophs.
 For routine cultivation of heterotrophs, chemical defined
media are not used. Instead complex raw materials such
as peptone, meat extract, and yeast extract are used and
resultingmedia supports the growth of heterotrophic
bacteria.
 Agar is a solidifying agent when a solid medium is
designed. Agar is a non-nutritious substance. Agar is a
long chain polysaccharide.
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 The media that support the growth of the heterotrophic

organism are nutrient broth and nutrient agar.2% of agar
is used for solid media.
 Yeast extract contains several of B-vitamins and other
growth promoting substance. Agar is obtained from
seaweed Red algae. Other complex supplements such as
bovine sumen fluid, animal blood, blood serum, or extracts
of plant and animals tissue are required for the cultivation
of certain fastidious (fastidious-organism which grow with
particular nutrients) heterotrophs.

TYPES OF MEDIA

 There are many special media which are needed to

facilitate recognition, enumeration, and isolatecertain
types of bacteria.
 To meet there needs microbiologists has given numerous
media on their application or function and classified as,
1. Solid media,liquid media, semi-solid media.
2. Simple media, complex media, syntheticor definedmedia,
semi definedmedia and special media.

Special media is further classified,

 Enriched media,
 Enrichment media,
 Selective media,
 Indicator media,
 Differential media,
 Sugar media,
 Transport media.
3. Aerobic media, anaerobic media.
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BACTERIAL GROWTH CURVE

 When bacteria is inoculated into a suitable liquid medium

and incubated its growth follows a definite course.
 During the growth the rate of growth can be plotted at
intervals related to time and a growth curve can be
obtained.
 The curve will show various phase such as
1. Lag phase
2. Log phase (logarithmic phase) or exponential phase
3. Stationary phase
4. Decline phase

Stationary phase

No. of
cells

Decline phase

Log phase

Lag phase

Time

LAG PHASE:
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 In this phase, when organism is inoculated into the culture

medium there is no increase in number.
 In this period of time the cells get adopted to the new
environment.

LOG PHASE OR EXPONENTIAL PHASE:

 In this phase, the cells starts dividing and their number

increase exponentially or by geometric progression with
time.
 In this phase the viable count is plotted against time and a
straight line will be obtained.

STATIONARY PHASE:

 In phase, cell division stops due to depletion or decrease

of nutrients and accumulation of toxic products.
 The cells remains stationary as an equilibrium exists
between the dying cells and new cells are formed.

DECLINE PHASE:

 In this phase the population decreases due to cell death.

 Nutritional values are exhausted and toxic accumulation
and cell death occurs by autolytic enzymes.
 Based on these phase only the growth curve is plotted.
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STAINING

 Generally, bacteria lack colour in their cytoplasm. So,

microbiologists commonly stain bacteria to study about
them.
 In order to study about the bacteria they perform a special
technique called staining.
 Staining is done with large number of coloured organic
compounds called dye.
 Dye is nothing but a coloring agent which gives color to
the bacterial cells.
 Dye can be classified into
 Triphenylmethane dyes
 Oxazine dyes
 Thiazine dyes.
 Cytologist, based on the chemical behavior they classify
dyes as
 Acidic dye
 Basic dye
 Neutral dye.

ACIDIC DYE: (Anionic)

 A dye in which the charge on the dye ion is negative.

BASIC DYE: (Cationic)

 A dye in which the charge on the dye ion is positive.

NEUTRAL DYE:

 It is a complex salt of a dye acid with dye base.

Example: eosinate of methylene blue.

 Acid dyes generally stain basic cell components.

 Basic dyes stain acidic cell component.
 Staining is a process which involves ion-exchange reaction
between the stain and cell.
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 There are certain chemical groupings of cell proteins or

nucleic acids may involve in salt formation with positively
charge ions such as Na+ and k+
 So cell carrying a negative charge in connection with
positive charge ion.

Example: (bacterial cell-) (Na+)

 In a basic dye like Methylene Blue the colored ion is

positively charged and represented as MB+.
 The dye which is Methylene blue chloride may be
represented as MB+ Cl+.
 So the ion exchange which take place during staining can
be represent as,

(Bacterial cell-) (Na+) + (MB+) (Cl-) (Bacterial cell-) (MB+)+

(Na+Cl-)

 Bacteria may be stained in line state; routine methods for

staining a bacteria involves-Fixing film, drying and
procures that kill them.
 They are various staining techniques used by
microbiologist and bacteriologist, they are as follows,
 Simple staining,
 Differential staining,
 Negative staining.

SIMPLE STAINING:

 The coloration of bacteria by applying a single solution of

stain to a fixed smear is termed as simple staining.
 In this staining the cells are uniformly stained and provide
same colour.
 Examples for simple stain are Methylene Blue or basic
fuchsine.
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DIFFERENTIAL STAINING:

 The coloration of Bacteria by applying different solution of

stain to a fixed smear is termed as differential staining.
Differential staining is classified as,
 Glom’s staining,
 Acid fast staining.

GRAM’S STAINING:

 It is a differential staining.
 It was 1st presented by CHRISTIAN GLON in the year 1884
a Danish physician.
 It is a differential technique because it differentiates
bacteria into two group such as gram positive and gram
negative.

 The strains used here are

 Crystal violet
 Gram’s iodine
 Acetone or alcohol
 Saffranin
 In gram staining procedure, the smear is 1 st stained with a
primary stain i.e. crystal violet goes and combines or
binds with the negatively (-ve) charged structure of
bacteria (i.e.) is the cell wall.
 Next it is treated with gram’s iodine, (1 min or 60 sec and
washed with water) which is a mordant. Mordant fix the
color to the bacterial cell.
 Mordant increase the affinity or attraction and forms as
CVI complex. So that the cell is stained more strongly.
 In G +ve bacteria, CVI complex binds tightly to
magnesium ribonucleic acid compound of cell forming Mg-
RNA-CV-I complex.
 Then the smear is treated with alcohol or acetone which is
a decolorizing agent.
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 Acetone or alcohol generated the differential aspects of

gram staining such as G=ve bacteria retain the crystal
violet and G-ve bacteria lose the crystal violet and
becomes colorless.
 Acetone acts as lipid solvent activity and protein
dehydration agent.
 Acetone increases the porosity and send out CVI complex
out.
 Finally the smear is counter stained with a basic dye
different in color from crystal violet.
 The counter stain used here is saffranin.
 When CVI complex is been gained by the bacterial cell it
becomes G+ve.
 When CVI complex is lost by the bacterial cell it becomes
G-ve.

ACID-FAST STAINING:

 It is a differential staining.
 It is called as Ziehl-Neelsen method.
 This staining techniques is especially used to identify Acid-
fast bacteria such as
 Tuberclebacilli
 Leprosybacilli
 Clubs associated with the presence in human or
animal tissue of actinomyces.
 These groups of bacteria are characterized by their
relative resistance to ordinary dye stuff and fact of stained
by Ziehl-Neelsen technique.
 These bacteria resist decolonization by strong mineral
acids.
 Their degree of acid-fastness values, Tubercle bacilli will
retain the red carbol fuchsin when challenged with a 25%
of H2SO4.
 Leprosy bacilli will withstand discoloration by 57% H 2SO4.
 Actinomycetes clubs in 1%.
 26

 Acid fast staining do not bind with simple stains. It must

be stained by a harsher treatment (i.e.) heating with a
mixture of basic fuchsin and Phenol.
 Only basic fuchsin has the capacity to penetrate into the
cell with help of heat and phenol.
 Acid fast cells are not easily decolorized by acid alcohol
treatment and hence it remains red color.
 The red color is due to high lipid content of acid-fast cell
walls particularly the Mycolic acid which is a group of
branched chain hydroxyl lipids which is responsible for
acid-fast bacteria.
 Nonacid fast bacteria are decolorized by acid alcohol and
thus stained by Loffler Methylene Blue counter stain.
Example: Mycobacteriumtuberculosis
 Mycobacteriumleprae
 Mycobacteriumbouis
 Mycobacteriumsegmatis
 Staining is of 2 types
 Positive stainingto detect part of bacteria such as
flagella and endospore.
 Negative staining is to see capsule.

NEGATIVE STAINING:

 Negative staining is ofstaining specific structures of

bacterial cells.
 This specific structure is structured with the help of light
microscope.
 Negative staining is a technique that reveals the presence
of capsules surrounding bacteria.
 Capsules are the outer side of the cell wall of bacterial
cell.
 Capsules are highly virulence (able to cause disease) part
of bacteria.
 Reduction of virulence is known as attenuation.
 The process of enhancing virulence is Exaltation.
 Capsules are resistant structure capable of surviving for
long periods in an unfavorable environment.
 27

 Capsules are made up of polysaccharides, polypeptides.

 In polypeptides they have D-glutamic acid.
 Capsules are easily demonstrated by negative staining
technique.
 Negative stain requires the use of an acidic stain such as
Nigrosin or India ink.
 In this technique simple stain is used which does not stain
the surrounding medium.
 Acidic stain with its negatively charged chromogenic will
not penetrate the cells because of the negative charge on
the surface.
 The unstained appear transparent against the dark back
ground.
 Negative staining is advantageous since cells appear less
shriveled or distorted because no heat fixation is done and
capsulated bacteria that are difficult to stain can be
stained.

Glass Slide Glass slide thin smear of

India ink + culture

 

India ink + loop of culture India ink +culture

(20% copper sulphides

Is added) air dry View under the

microscope.

Under microscope
 28

E.g. Bacillusanthrax, Streptococcipneumonia,

Klebsiellapneumonia, Rhizobium

ENDOSPORE STRAINING

 Spore is a resistant body formed by certain

microorganism.
 Spores are 2 types;
 Endospore.
 Exospore.
 In order to find out such spores a special staining
technique is required called as endospores staining.
 An endospore is a structure that is found inside the
bacterial cells.
 The spores are formed when the environmental condition
become unfavorable for the growth of the organism so
they undergo sporogenesis and give rise to a new
intracellular structure called endospores.
 The dipicolinic acid present in the spore coat is
responsible for resistance to heat, freezing, radiation and
chemical agents.
 The spore coat will not allow the dyes to penetration into
the spores.
 Malachite green is a week basic dye. It has a week positive
charge and it will not bind strongly to surface of the cell.
 During heat treatment, the dye penetrates the spore coat
and is very difficult to remove. So vegetative cell and
spore will appear green.
 Then the stain is washed off with water and the primary
stain will be lost and becomes colorless.
 Finally counter stain saffranin is used so the vegetative
cells will take the counter stain and spore with retain the
color of primary stain.
 29

2-5 min cooled 1 min

View under
  microscope

Malachite green washed in H20 saffranin

*H2o acts as a decolorizing agent

Example: Bacillus, Clostridium, Sporosarcina and

disulphatomaculum.

 It is an intracellular granules of polyphosphate found in

certain bacteria.

GRANULAR STAINING:

 Granular staining is a techniques used to find out the

granules present the bacteria.
 This technique was given by Albert and the stain used is
Albert stain.
 Albert technique is used to stain the volutin or
metachromatic granules of corny bacterium diphtheria.
 Granules or metachromatic granules are energy storing
granules from at the end of the rods.
 The stains used are
 Toluidine blue Malachite green-solution A
 Albert stain- solution B
 In this technique, the dried smear is fixed with alcohol.
 Then the smear is stained with solution-A. Toluidine blue
malachite green for 3-5 minutes and then washed with
water.
 Then the smear is colored with solution-B Albert stain for 1
minute and washed and air dried and viewed under
microscope.
 30

 When viewed under microscope, bacterial cells are green

color and granules are green black color.

H20 H2o View under

microscope
=  

Solution-A (:Toluidine Solution-B (Albert stain)

Blue malachite green) Granules

Bacterial
cell
 31

BACTERIA – FINE STRUCTURE OF MORPHOLOGY.

 Bacterial cells major characteristics are based on the size,

shape , structure and arrangement.
 These characters only constitute the morphology of cell.
 Bases on the species or individual cells may be spherical ,
rod like or helical.
 They may be pair, cluster, chains, trichomes and
filaments.
 The internal structure of Bacteria was discovered with the
help of electron microscope.
 The structure of a bacterial cell differ from one another in
their physical feature and chemical characteristic and
their functions.

SIZE
 Bacterial are very small and measures 0.5 – 1.0 in
diameter.
 The largest bacteria is the Thiomargaritanamibiensis
 The smallest known bacteria is Mycoplasmas

SHAPE AND ARRANGEMENT.

 The shape of the bacterium is governed by a rigid cell

wall.
 Typically bacteria are of various shape, They are as
follows,

1. Spherical- cocci (singular,) coccous (Plural)

2. Straight rods- bacilli, bacillus
3. Helically curved – spirilla (singular), Spirillum (Plural).

 Some of the bacteria are pleomorphic which can exhibit a

variety of shapes.
 32

 Bacterial cells are usually arranged in a mannered

characteristic to their particular species.
 Cocciappear in several characteristic arrangement
depending on the plane of cellular division and the
daughter cell stay together.

 But bacilli are not arranged in patterns as complex as

those of cocci . They occur singly or in pair called as
diplobacilli.
 33

 some species such as bacillus subtilis, from chains –

(streptobacilli).
 Other such as Beggiatoa and Saprospira species, form
tirchomes, which are similar to chains but have a much
larger area of contract between the adjacent cells.
 In other bacillus species, such as
Corynebacteriumdiphtheria, the cells are lined side by
side like matchsticks (Palisade arrangement) and at
angles to one another.
 Some species from long branched, multinucleate
filaments called hyphae collectively form a mycelium.
 There are some bacteria which are curve shaped which
are less than one twist are called vibriod shaped example:
Vibriocholera.
 Those bacteria with more than one or more complete
twist are helical shape.
 In addition to there common shape, bacteria occur in Peas
shaped cells example: Pasteuria , lobed species ,
example : sulfobobus.
 Rods with squared rather than hemisphere end, eg.
Bacillusantracis
 Disks arrangement like stacks of coins eg. Caryophanon.

BACTERIAL STRUCTURES:

There are various components of bacterial structures they are

as follows…

1. External to cell wall

2. Internal to cell wall.

STRUCTURE EXTERNAL TO CELL WALL:

 The structures which are external to bacterial cell wall are

a. Flagella
 34

b. Pili (fimbriae)
c. Capsules
d. Sheaths
e. Cell wall

FLAGELLA AND MOTILITY:

 Flagella are hair like, helical appendages protrude outside

the cell wall which are responsible for swimming and
motility.
 Flagella of prokaryotes are much thinner than flagella of
cilia of eukaryotes.
 Flagella are 0.01-0.02 micrometer in diameter.
 They are located at various position depending on the
bacterial species.
 They may be polar or bacterial arrangement .
 In polar arrangement, the flagella are attached to one or
both the ends of the cell.
 They are 3 sub types of this patter , they are as follows..
(trichous means hair)
1. Monotrichous – with a single flagellum. Eg,
pseudomonas
2. Lophotrichous – with small bunches or tuff of tuft
eg.Spirillium flagella emerging from one end.
3. Amphitrichous- a single flagellum can extend from
both ends of the cells
4. Peritrichous- multiple flagella may be randomly
distributed over the entire bacterial cell.

STRUCTURE OF FLAGELLA:

 Flagellum is composed of 3 parts . they are

1. Basal body – it is associated with cytoplasmic
membrane and cell wall.
2. Hook – short, curved segment.
3. Filament – they are long, several times longer than
cell , acts as flexible couples.
 Some gram negative bacteria have a sheath surrounding
the flagellum.
 35

 This sheath is continuous with the outer membrane of

gram negative cell wall.
 The chemical composition of the basal body is unknown.
 The hook and filaments are composed of protein subunits
known as monomers which are arranged in helical fashion
 The Protein of the filament is called flagellum
 The molecular range of flagellin is 30,000 – 60,000
 The filament ends with a protein called capping protein.
 Some bacteria have surrounding their flagella.

Eg, Vibriocholerae has lipopoly saccharide sheath.

 Hooks and basal body are quite different from filament

because they are wider than the filament .
 The hook is made of different protein subunits flagellum.
 In gram negative , the body has 4 rings connected to a
central rod.
1. L-ring
2. P- ring , these 2 rings are associated with
lipopolysaccharides and peptidoglycan.
3. M-ring - inner m ring contact with plasma.
4. S- ring.
 Gram positive have only 2 basal body rings, an inner ring
connected to the plasma membrane and outer one
attached to peptidoglycan.

FLAGELLAR SYSTHESIS (process of building a separate

element)

 Synthesis of flagellar is a complex process because it

involves 20 – 30 genes.
 10 or more genes code for hook and basal body present.
 Remaining genes code for the control of flagella
construction or function.
 In is not known how the cells regulates or detects the
exact location of flagella.
 Bacteria can be deflagellated and regenerated
flagellatefilament.
 36

MECHANISM OF FLAGELLAR MOVEMENT:

 Prokaryotic flagella operate differently from eukaryotic

flagella.
 Filament of bacteria is in the shape of rigid helix and
bacterium moves in helix rotates.
 Flagella act like a propellers of Boat.
 The flagellar motor can rotate vary rapidly. Example: E.
coli rotates 270 revolutions per second and Vibrio
alaginolyticus rotates 1,100revolutions per second.
 Flagellar rotation determines the nature of bacterial
movement.
 Monotrichous, polar flagella rotate in counterclockwise,
during normal forward movement and all itself rotates
slowly in clockwise direction.
 Peritrichous flagellate bacteria operate in similar way. To
move forward , the flagella rotates counterclockwise and
bend their hooks.
 According to one hypothesis, a flagellum rotates because
of interaction between its
S ring and M ring.
 S ring is attached to the cell in gram positive cells and
does not rotate.
 P and L ring of gram negative bacteria would act as
bearing for the rotating rod.
 The basal body rotation mechanism is not clear so far.
 Bacteria can move by mechanism other than flagellar
rotation. Example: Spirochetes are helical bacteria that
travel through viscous substance such as mucus or mud
by flexing and spinning movements caused by a special
axial filament.
 Very different type of motility is gliding motility.

CHEMOTAXIS

Movement towards chemical attractant and move away

from repellents is known as chemotaxis.
 37

 Bacteria do not always swim aimlessly,they are been

attracted by nutrients such as sugars and ammino acids.
 Chemotaxis are demonstrated by observing bacteria in the
chemical gradient produced when there capillary tube is
filled with an attractant and lowered into a bacterial
suspension.
 Bacteria swim up to tube.
 Number of bacteria within the capillary ofter a short time
shows the strength of attraction and rate of chmotaxis.
 Positive and negative chemotaxis was been studied with
Petridis culture
 A bacteria are placed in the center of the plate of agar
containing an attraction the bacteria will show the swim
outwards following the attractant gradient created by
them and show expanding ring of bacteria.
 Attraction and repellent are detected by chemoreceptor.
 Chemoreceptor are special proteins that binds chemicals
and transmit signal to the other components of chemo
sensing system.
 There are about 20 chemoreceptor and 10 chemoreceptor
are for repellents have been discovered.
 Chemoreceptor protein are found in periplasma space or
plasma membrane.
 Some receptors participate in the initial stage of sugar
transport into the cell.
 Chemotactic behavior of bacteria is studied with tracking
microscope which has as a moving stage which helps
individual bacterium in virus.
 A bacteria travels in a straight or slightly curved line.
 It runs for few seconds and stops and tumbles or twiddle
and then runs in different direction.
 38

 Chemotaxis mechanism is done in E.coli

 E. coli has four different chmoreceptor which recognize..
i. Serine
ii. Aspartate and maltose
iii. Ribose and galactose
iv. Dipeptides
 These receptors are called as Methyl-acceptors
chemotaxis proteins (MCPs) , which are found in patches
the end of rod-shaped cells like E.coli.
 MCPs do not directly influence flagellar rotation but act
through a series of proteins.
 The whole process is carried out in less than 200
milliseconds.
 Chemoreceptor are buried in the plasma membrane on
both side.
 The cytoplasmic side on MCP intracts with 2 proteins.
Che W protein – bind to MCP
Che A protein _ helps to attach.
 The full complex in composed of
MCP dimer
2 che W monomer
1 cheA dimer
 When MCP is not bound to attractant, it stimulate che A to
phosphorylate itself by using ATP and this process in
called Auto phosphorylation.
 Phosphorylated che A can done its phosphate to one of
two receptor protein che Y and cheB
 When che Y is phosphorylated by cheA it move the
flagellum and interacts the switch protein at the base and
this cause the flagellum to rotate at clockwise direction.
 The phosphate is removed from che Y withing 10 sec and
that will be aided by che Z protein.
 In short time of phosphorylated che Y is responsible to
change in attraction concentration.

II PILI (pilus– singular)

 39

 They are hollow, nor helical, filamentous appendages.

 They are shorther, thinner and more in number than
flagella.
 They are non motile but found on the motile speris.
 There are several types of function associated with
different types of pili known as F pili or sex pili.
 F pili or sex pili serves as a port entry of genetic material
during bacterial mating.
 In human, this pili gets attached to pathogenic bacteria in
intestine respiratory tracts, genitourinary tract. This
attachment helps the bacteria from being washed away by
flow of mucous …
 Pili are found in gram negative bacteria such as Neisseria
gonorrhea.
 Composition of pili is pilin which is a protein.
 Size is 0.007 – 0.008 micro meter with and 0.5 – 2 micro
meter length.
 Pili function as attachment and conjucate.

III CAPSULE (composed of polysaccharide)

 Capsule is an envelope or slime layer surrounding the cell

wall of certain bacteria.
 When seen by light microscope using special staining is
called as capsule.
 When not seen by microscope it is called as
microcapsule.
 Capsules are not soluble in water.
 Capsule serves as a different function depending upon
the species.
1. Protection against temporary drying by binding
water molecules.
2. They block the attachment of bacteriophages
3. Acts as antiphogocytic.
4. Promote the stability of bacteria.
 Capsules are composed of polysaccharides.
 40

 Capsules composed of single sugar are called as

homopolysaccharide synthesized outside the cell from
disaccharide by exocellular enzymes.
 Capsules composed of several kinds of sugars is termed as
heteropolysaccharides which are synthesized from sugar
precursors that are activated within the cell. Eg. Klebsiella
pneumonia.
 Few capsules are polypeptides eg. B. anthracis having
poly s glutamic acid

S-LAYER

 Many gram positive and gram negative have a regularly

structured layer called as S- layer on the surface.
 S- layer has a pattern something like floor tiles.
 S-layer is composed of protein of glycoproteins.
 In gram negative bacteria, S-layer adheres directly to the
outer membrane.
 In gram positive bacteria S-layer adheres to peptidoglycan
which protects the cell against ion and ph fluctuation,
osmotic stress, enzymes….
 S-layer helps to maintain the shape and envelop rigidity of
some bacterial cells.

SHEATH

 Some species of bacteria, from freshwater and marine

environments form chains or trichomes which are
enclosed by a hollow tube called a sheath.
 Sheath are sometimes impregnated with ferri or
manganese hydroxides which strengthen them.

CELL WALL.

 Beneath the external structures such as flagella, capsules,

sheath, there is an another external to cytoplasmic
membrane , is the cell wall.
 Cell wall is rigid structure which gives shape to cell.
 The main function of the cell wall is to prevent the cell
from expanding and bursting due to intake of water since
most bacterial live in hypotonic environment.
 41

 Hypertonic environment is environment having a lower

osmotic pressure.
 Most bacteria retain their original shapes during and after
hypotonic reaction.
 The cell wall can protect a cell from toxic substance and
action of several antibiotics.
 Christian gram developed the gram staining in 1884 who
divided bacterian into 2 groups based on the gram
staining procedure.
 Gram positive bacteria stained purple.
 Gram negative bacteria stained pink.
 This two differentiation is based upon the cell wall
composition.
 Gram positive cell wall has 20-80 nm thick .homogeneous
peptidoglycan or murinlyines outside the plasma
membrane.
 Gram negative cell wall has 2-7 nm. Peptidoglycan layer
surrounded by 7 – 8 nm thick outer membrane.
 Gram negative and positive are different in structure and
chemical composition.

PEPTIDOGLYCON STRUCTURES:

 Peptidoglycan or murine is an enormous polymer

composed of may identical subunits.
 42

 These polymer consists of 2 sugar derivatives

1. N-Acety glucosamine acid (NAG)
2. N-Acetylemuramic acid (NAM)

And several ammino acids.

 The peptide chain of 4 alternating D and L amino acids is

connected to the carboxyl group of N- acetylmeuramis
acid.
 Linked peptidoglycan subunits are joined by cross links
between peptides.
 Often the carboxylegroupu of the terminal D vebonie is
connected directly to the amino group of diaminopimelic
acid, but a peptide interbridge may be used instead.
 Grame negative cell was peptidoglycan lacks , the peptide
interbridge.
 This cross linkage results in an enormous peptidoglycan
sac which are dense interconnect network.
 These dense interconnected network is formed in gram
positive bacteria and strong enough to retain their shape
and integrity.

GRAM POSITIVE CELL WALL.

 43

 Gram positive cell wall is a thick, homogeneous cell wall

composed of peptidoglycan which contains a peptide
interbridge (penta glycine).
 Gram positive cell wall also contains large amount
ofteichoic acids.
 Teichoic acids is a polymer of glycerol or ribitiol joined by
phosphate groups.
 Amino acids such as D-alanine or sugar like glucose are
attached to glycerol or ribitol group.
 Teichoic acid are connected to peptidoglycan itself by a
covalent bond with 6 hydroxyl of N-acetylmuranic acid or
to plasma membrane lipids and later they are called as
lipoteichoic acids.
 Teichoic acid extend the surface of peptidoglycan because
they are negative charged and give gram positive bacteria
wall negative charge.
 The function of molecules are known but are important in
maintaining the structure of the wall.
 Teichoic acid is not present in gram negative bacteria.

GRAM NEGATIVE CELL WALL.

 gram negative cell wall are more complex than gram

positive cell wall.
 Thin peptidoglycan layer next to plasma membrane is only
5-10% of wall weight.
 Peptidoglycan is 2 nm thick and contains only 1 or 2 layer
or sheet of peptidoglycan.
 Peptidoglycan will be in the form of gel and not as
compact layer.
 The most abundant membrane protein is the braun
lipoprotein or murein lipoprotein which lies outside the
peptidoglycan.
 Braun lipoprotein is small lipoprotein which is covalently
joined to peptidoglycan.
 The outer membrane and peptidoglycan are firmly linked
by this lipoprotein.
 44

 The most constituents of outer membrane is

lipopolysaccharides .
 Lipopolysaccharide are large complex molecules which
contains both lipid and carbohydrates and consist of 3
parts….
1. Lipid A
2. Core polysaccharide.
3. O side chain.
 Lipid A contains 2 glucosamine sugar derivative each 3
fatty acids and phosphate or pyrophosphate.
 Core polysaccharide is joined to lipid A. In salmonella it is
constructed of 10 sugars.
 O side chain or O antigen is a short polysaccharide chain.
It has several peculiar sugars.
 LPS is important for several reason other than the
avoidance the avoidance of host defenses.
 Polysaccharide usually contains charged sugars and
phosphate.
 LPS contributes negative charge on the bacterial surface.
 Gram positive bacteria can form a special resistant,
dormant structure called an endospore.

BACTERIAL ENDOSPORE:

SPORE

Spore are resistant body formed by certain bacteria.

 Spores are formed for the survival of adverse condition

because of its heat and dessication.
 Spores are usually unicellular.
 Spores are thick walled and highly refractive bodies that
are produced by Bacillus, Clostridium, Sporosarcina.
 Spores are endospores and exospores.

ENDOSPORES
 45

 Endospores are developed within the bacteria cells

(vegetative cell).
 Endospores are resistant to environmental stresses such
as heat, ultra violet radiation chemical disinfectant and
desiccation.
 Endospore can remained viable for 500 yrs.
 Several species of endospore forming bacteria are
dangerous pathogen.
 In the environment, endospores aid in survival when
moisture or nutrients are scarce.
 Endospores can be examined with both light and electron
microscope.
 Spores are impermeable to most stains b staining we can
visible them clearly.
 Spores can be located centrally, close to one end so called
sub terminal and at the end called terminal spores.

STRUCTURE:

 Endospore structure is a complex one.

 The spore is surrounded by a thin, delicate covering called
exosporia.
 Spore coat is present beneath the exosporia which is
composed of several protein layers and are fairly thick.
 Spore coat is impermeable and responsible for spore’s
resistance to chemicals.
 46

 Cortes occupy as much as half the spore volume which

lies beneath the spore coat cortex is made up of
peptidoglycan less cross linked than vegetative cell.
 Core wall (spore cell wall) is inside the cortex and the
protoplast or core has the normal cell structures such as
ribosomes and nucleoid.
 Endospore is so resistant to heat and other lethal is still
more not known.
 15%of spores dry weight contains dipoclonic acid which
contains calcium ion.
 Endospore heat resistance probably is due to several
factors such as
a. Calcium dipincolinate
b. Acid-soluble DNA binding protein stabilization.
c. Protoplast dehydration.
d. Stability of cell proteins for growth as high
temperature.

ENDOSPORE FORMATION

CONTROL OF MICROORGANISM
 47

 Sterilization means the process of making sterile or the

killing of all forms of life.
 Sterilization is done to control. Control means reduction in
number or activity of total microbial flora.
 The principle reason behind this is for controlling
microorganism to prevent the transmission of disease and
infection.

DISEASE:

 It is a deviation or interruption of thenormal structure or

function of any part of the body which is characteristic set
of symptom or sign.

INFECTION:

 Infection means a pathological condition due to growth of

microorganism.
 Control of microorganismsalso done to prevent
contamination by undesirable microorganism and to
prevent deterioration and spoilage of microorganism.

CONTAMINATION:

 Contamination means entry of undesirable microorganism

into some material or object.
 Microorganism can be removed, inhibited orkilled by
various physical agent, physical process andchemical
agents.
 In Latinsterile means unable to produce offspring.
 A sterile object should be totally free of viable
microorganism, spores and other infectious agents.
 When sterilization is achieved by a chemical agent is
called as sterilants.

DISINFECTANTS:

 They are chemical agents used to carry out disinfection

and they are used only on inanimate objects.

DISINFECTION:
 48

 It is killing, inhibition or removal of microorganism which

cause disease.

SANITIZATION:

 Reduction of microbial population an inanimate object to

level judged safe by public health standards.

ANTISEPSIS:

 In Greek anti-against; sepsis-decompose putrefaction. It is

the prevention of infection or sepsis with the help of
antiseptic.

ANTISEPTIC:

 Antiseptic is a chemical agent acting against sepsis

putrefaction or decay by either preventing or averting the
growth of microorganism.

(Or)

 Chemical agent applied to tissue to prevent infection by

killing or inhibitory pathogen growth.
 Substances that kill microorganism have suffix ‘cide’ [cide-
kill in Latin]
 GERMICIDE that kills pathogen and non-pathogen but
not the spores.
 BACTERICIDE an agent that kills bacteria.
 FUNGICIDE an agent that kills fungi
 ALGICIDE an agent that kills algae.
 VIRICIDE an agent that kills virus.
 There are other chemicals which do not kill organism but
they prevent growth and names end in static which means
causing to stop.
 For example: bacteriostatic, fungi static.
 The antimicrobial agent should have the following kinds of
action to kill or inhibit the microorganism.
1. Damage the cell wall or inhibition of cell-wall synthesis.
2. Alteration of permeability of the cytoplasmic
membrane.
 49

3. Alteration of the physical or chemical state of proteins

and nucleic acids.
4. Inhibition of enzyme action.
5. Inhibition of protein or nucleic acid synthesis.
 Microorganism is controlled by two methods.
 Physical sterilization
 Chemical sterilization

PHYSICAL STERILIZATION

 There are various methods which are employed to kill or

inhibit microorganism but physical sterilization. They are
1. Temperature- high or low
2. Desiccation
3. Osmotic pressure
4. Radiation
5. Filtration

TEMPERATURE:

 Microorganism can grow in a wide range of temperature-

low temperature is characteristic by Psychrophiles 0 .c-
20.c. High temperature is characteristic by thermophiles
(above55.c). Moderate or optimum temperature is
characteristic by mesophiles (20-45.c).
FromGreek period, fire and boiling water has been used
for sterilization and disinfectant andheating is still one of
the most popularways to destroy microorganism.

HIGH TEMPERATURE:

 Microorganismcanbe controlledwith the help of high

temperature.
 High temperature combines with high moisture which is
more effective method of killing.
 High moisture is distinguish between dry heat and moist
to control microorganism.
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 Moist heat kills microorganism by coagulating their

protein.
 Dryheat destroys microorganism by oxidizing their
chemical constituents.

THERMAL DEATH TIME AND DECIMALREDUCTIN TIME:

 Thermal death time refers to the shortest period of time to

kill a suspension of bacteria at a prescribed temperature
and specific conditions.
 Decimal reduction time is the time in minutes to reduce
the population by 90% to the thermal death time and
decimal reduction time are extremely important in many
application of microbiology.
 The killing action of heat in time temperature
relationshipthe heat employed can be conveniently
divided into2 categories
 Moist heat
 Dry heat

MOIST HEAT:

 Moist heat is used for inhibiting or destroying

microorganismby various methods.

STEAM UNDER PRESSURE:

 Heat is in the form of saturated steam under pressure

which is most practical and dependable agent for
sterilization.
 Advantages of steam under pressure are
 Rapid heating
 Penetration
 Moisture in abundance.

These are 3 factors deals in coagulation of proteins.

Example for steam under pressure is autoclave.

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AUTOCLAVE:

 It is a double-jacket steam chamber whichpermits the

chamberto be filled with saturated steam andmaintain
temperature and pressure.
 The air in the chamber is completely replaced by
saturated steam. If air is present it will reduce the
temperature obtain by the chamber.
 Pressure does not kill the microorganism only temperature
of the steam kills.
 Autoclave is essential equipment in every microbiology
laboratory for sterilization of manymedia, solution,
discorded cultures and contaminated materials.
 Autoclave is operated at a pressure of 15lbs/in12 at 121 ºc.

FRACTIONAL STERILIZATION:

 Some microbiological media, solution of chemicals

andbiological materials can’t be heated above 100ºc.
 It can be done with fractional sterilizationwhich is also
called as tyndallization.
 Tyndallization is a method which involves heating the
material at 100.c on 3 successive days.

BOILING WATER:

 Contaminated materials on exposed to boiling water

cannot be sterilized but vegetative cells will be destroyed.
 Boiling water can’t be used in laboratory as a method of
sterilization.

PASTERURIZATION:

 The process of heating a liquid food or beverage to a

controlled temperature to enhance the keeping quality
and destroy harmful microorganism.
 Pasteurization is done at 62.8ºc for 30 minutes (145.F) and
71.7ºc for 15 seconds (161.F).


DRY HEAT:
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 It is a n apparatus used for sterilization with electric or gas

oven.eg. Hot air oven.
 It works at temperature of 160ºc for 2 hours.
 It is used to sterilize oils, glass wear, sharp instruments,
and metals.

INCINERATION:

 Destruction of microorganism by burning in practiced

routinely in the laboratory.eg. Heating of loop.
 When loop is shown in the flame care should be taken that
spattering does not occur.
 Incineration is used for destruction of carcasses, infected
laboratory animals and other infected materials and care
to be taken that flame does not carry microorganism into
atmosphere.

LOW TEMPERATURE:

 Temperature below the optimum for growth depresses the

rate of metabolism and when temperature is sufficiently
low growth and metabolism is ceased.
 Low temperature is useful for prevention of cultures.
 Agar slant of some bacteria fungi, yeast and molds are
stood for longer period of time at refrigeration
temperature of about 4-7ºc.
 Many bacteria and viruses can be maintained at deep
freeze at -20 to -70ºc, liquid nitrogen at -196ºc.

DESICCATION:

 Desiccation of the microbial cells causes a cessation of

metabolic activity followed by a decline in the total viable
population.
 The time survival of microorganism after desiccation
varies depending on the following factor
1. The kind of microorganism.
2. The material in or on when the organism are dried.
3. The completeness of the drying process.
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4. The physical condition to which the dried organisms are

exposed e.g. light, temperature, humidity.
 Some G-ve organism such as gonococci and Meningococci
are very sensitive and in a matter of hours.
 Streptococci are more resistant and survive after being
dried.
 Tubercle bacilli after dried in spectrum survive for longer
period of time.
 In the process of lyophilization, organism are subjected to
extreme dehydration in frozen state and sealed in a
vaccum. In this culture of microorganism remain viable for
many years.

OSMOTIC PRESSURE:

 The pressure build up within the cell as a result of water

intake is called osmotic pressure.
 When a cells are exposed to solution with higher
concentration and the water will be drawn out of the cell is
called plasmolysis.
 When cells are exposed to solution with low concentration
and the water will be intake by the cell is called
plasmoptysis.

RADIATION:

 Energy transmitted through space in a variety of form is

called radiation.
 It is used to control air born infection and surface
disinfection.
 Electromagnetic radiation, of light and x-rays are some
example.
 Electromagnetic radiation has a dual property of
continuous wave phenomenon and discontinuous particle
phenomenon.
 These particles are called as packets or quanta of energy.
Sometimes called as photons which vibrate at different
frequencies.
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 Radiation can be described as wavelength (λ) which is

measured in Angstroms.
 10,000 Ẵ= 1µm.
 Gamma rays and x-rays are called ionizing radiation (10
eV).
 Radiation damage cells and microorganism.

ULTRA VIOLET LIGHT:

 It has radiations from 150-3900 Ẵ.

 Wavelength of 2600 Ẵ has the highest bacteria efficiency.
 Many lamps are able to emit a high concentration of
ultraviolet light with a wavelength of2600-2700 Ẵ.
 These lamps reduce the microbial population.
 They are used in hospitals, operation theatre, laboratory,
aseptic filling rooms, and pharma industry and in food
industries for treatment of contaminated surface.
 They destroy microorganism on the surface only and uv
light are susceptible to destruction.

MODE OF ACTION:

 They act on nucleic acid.

 Alteration in the formation of pyrimidine dimer.
 DNA replication can be inhibited and mutation occurs.

X-RAYS:

 They are lethal to microorganism and higher forms of lie.

 They are impractical to control microbial population is
because of
1. Very expensive to produce
2. Difficult to utilize efficiency.
 It is used to produce microbial mutant.

GAMMA RAYS:

 They are high energy radiation emitted from certain

radioactive isotopes such as 60CO isotopes are potential
source of gamma radiation.
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 Gamma rays are similar to x-rays but are shorter wave

length and higher energy.
 They are lethal to all life including microorganism.
 Because of their great penetrating power and microbicidal
effect they are used in commercial sterilization e.g.
packed foods and medical device.

FILTRATION:

 Filtration is a process which is available to microbiologist

to remove microorganism from liquids and gas with the
help of filters.
 These filters are made of different materials such as,
1. Asbestos pad in Seitz filter.
2. Diatomaceous earth in Berkefield filters.
3. Porcelain in chamberland-pasteur filter.
4. Sintered glass disks in other filters.
 The diameter of the pore in the filter ranges from 1 to
several micrometers.
 Fillers are available in several grades based on the size of
the pores.
 Mechanical sieves or porosity alone are not responsible for
preventing the passage of organism.
 Electric charges of filter, electric charge carried by
organism and the nature of fluid used influence the
efficiency of filters.
 Later new types of filters were used named as membrane
or molecular filter.
 Membrane or molecular filters are composed of biological
inert cellulose esters.
 They’re circular membrane of size 150µm thickness and
contain millions of microscopic pores.
 The porosities range from 0.01-10 µm.
 Membrane filters are used in laboratory industries to
sterilize fluids and in hospitals.
 Precautions must be taken to prevent contamination while
transferring of materials o other container.
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 For that a special type of filter is develops and called as

HEPA- High Efficient Particulate Air filter.
 This filter is used to disinfect air.
 This air filter is used in laminar air flow chamber which is
used to produce bacteria free air and dust free air.
 HEPA filter can remove 99.97% of 0.3µm particles.
 In filtration, there are 2 types of filter
 Depth filters
 Membrane filters.

DEPTH FILTERS:

 It consists of fibrous or granular materials that have been

bonded into a thick layer filled with twisting channels of
small diameter.
 Depth filters are,
 Diatomaceous earth (Berkefield filter)
 Chamberlain filter
 Asbestos.

MEMBRANE FILTER:

 Membrane filters have replaced depth filters for several

reasons.
 Membrane filters are made up of cellulose acetate,
cellulose nitrate, polycarbonate, polyninhydrine fluoride.