Pharmacy Exam

Guide
Step II
PHARMACEUTICS-
III
(MICROBIOLOGY &
IMMUNOLOGY)
1st Edition
(p2c4)

-----------------------------------------------------------------------------------------------------------------------------

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 Compiled By:
Abdul Sattar Rashid

Ali Ahsan

Ammara Khalique

Anmol Tahreem

Fareed Ahmed Rang Ali

Hamza Rohail

Laiq Ur Rehman Khan

Mehrab Fatima

Memoon Babar

Muhammad Qasim Yousaf

Ramsha Tahir

Sadia

Salbia Shereen

Sharmeen BaiG

Umair Javaid

Zafeer Naeem
Dedicated to Our Parents

And TeacherS
 Acknowledgement

The future belongs to those who believe in the beauty of their

dreams.
The preparation of this book “Pharmacy Exam Guide” was
just a dream of some students of Doctor of Pharmacy,
University of Central Punjab, which could not be fulfill
without the help and support of our teachers and parents.
We appreciate the tireless efforts of Our Teachers who
encouraged us always to achieve our endeavor, no matter,
how hard they can be.
We are much indebted to Our Parents for inspiring and
motivating us to achieve the great goals in life.
 1

Chapter 1 Introduction ........................................................................................................................ 2

Chapter 2 Bacteriology ........................................................................................................................ 9
Chapter 3 Reproduction and Growth ................................................................................................24
Chapter 4 Viruses ...............................................................................................................................39
Chapter 5 Protozoa ............................................................................................................................47
Chapter 6 Fungi ..................................................................................................................................56
Chapter 7 Normal Flora .....................................................................................................................65
Chapter 8 Microbes of Air..................................................................................................................67
Chapter 9 Microbiology of Water ......................................................................................................69
Chapter 10 Microbiology of Soil ..........................................................................................................72
Chapter 11 Immunology ......................................................................................................................75
Chapter 12 Sterilization .......................................................................................................................95
Chapter 13 Manufacture of Antibodies .............................................................................................113
Chapter 14 Factory and Hospital Hygiene .........................................................................................120
Chapter 15 Vaccine & Sera ................................................................................................................127

Pharmacy Exam Guide

 Introduction 2

Robert Hooke, also spent much of his life working

with microscopes and improved their design and
Chapter 1 INTRODUCTION capabilities

1.1 MICROBIOLOGY 1.5 MICROSCOPY TECHNIQUES

The study of organisms of microscopic size
(microorganism), including their culture, economic
importance and pathogenicity etc.
1.2 MICROBES
Any microscopic organism; a microorganism e.g.
bacteria, fungi, virus, algae and protozoa.
1.3 MICROSCOPE
It is an important scientific tool which is used for
studying small objects that are not visible to naked
eyes. The resolution power of a microscope is its
extent to which it can make information or details
of an object clearly visible. Different microscopes
have different resolution power.
1.4 HISTORY OF MICROSCOPE
Anthony Leeuwenhoek of Holland learned how to
make lenses. By grinding and polishing, he was
able to make small lenses with great
curvatures. These rounder lenses produced
greater magnification. Anthony Leeuwenhoek
(1632-1723) has since been called the "Father of
Microscopy". For his great contributions.
Brightfield Microscopy - the most elementary
microscopy technique but important to
understand and apply correctly.
Oil Immersion Microscopy - when used properly
increases the refractive index of a
sample/specimen. oils increase refraction despite
short focal lengths.
Fluorescence Microscope - study the most used
microscope in medical/biological fields which uses
high powered light waves to provide unique image
viewing options.
Dark Field Microscope - learn more about how
when the light source is blocked off, light scatters
as it hits the specimen and is then able to reveal
details otherwise difficult to see.
Phase Contrast Microscope - learn about an entire
new world that has opened up in the field of
microscopy. Once limited to bright field
illumination phase contrast observation is now a
standard feature on almost all modern
microscopes.
Research Microscope- It is ordinary microscope
with vertical and horizontal scale present on the
stage which can be used for research purposes.

1.6 HISTORY OF MICROBIOLOGY

Year Event
1546 Fracastoro suggests that invisible organism cause disease
1590-1608 Jansen develops first useful compound microscope
1676 Leeuwenhoek discover animalcules
1798 Jenner introduces cowpox vaccination
1838-1839 Schwann and Schleiden proposed Cell Theory
1849 Snow studies the epidemiology of cholera epidemic in London
1857 Pasteur shows that lactic acid fermentation is due to microorganism
1861 Pasteur shows that microorganisms do not arise by spontaneous generation
1867 Lister publishes his work on antiseptic surgery
1876-1877 Koch demonstrates that anthrax is caused by Bacillus antracis
1880 Laveran discovers Plasmodium, the cause of malaria
1881 Koch cultures bacteria on gelatin
Pasteur develops anthrax vaccine
1882 Koch discovers tubercle bacillus, Mycobacterium tuberculosis
1884 Koch postulates first published
Metchnikoff describes phagocytosis
Autoclave developed
Gram Stain developed
1885 Pasteur develops rabies vaccine
Escherich discovers Escherichia coli, a cause of diarrhea

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 Introduction 3

1886 Frankel discovers Streptococus pneumonia, a cause of pneumonia

1887 Petri dish (plate) developed by Richard Petri
1887-1890 Winogradsky studies sulfur and nitrifying bacteria
1890 Von Bering prepares antitoxins for diphtheria and tetnus
1892 Ivanowsky provides evidence for virus causation of tobacco mosaic disease
1894 Kitasato and Yersin discover Yersinia pestis, the cause of plague
1897 Buchner prepare extract of yeast theferments
Ross shows that malaria parasite is carried by mosquito
1902 Landsteniner discovers blood groups
1903 Wright and other discovers antiboides in the blood of immunized animals
1905 Schaudinn and Hoffmann show Treponema pallidum causes syphilis
1906 Wassermann develops complement fixation test for syphilis
1909 Ricketts show that Rocky Mountain spotted fever is transmitted by ticks and caused y
microbe (Rickettsia rickettsia)
1910 Erlich develops chemotherapeutic agent for syphilis
1915-1917 D’Herelle and Twort discover bacterial viruses
1921 Fleming discovers lysozymes
1923 First edition of Bergey’s Manual
1928 Griffith discovers bacterial transformation
1929 Fleming discovers penicillin
1937 Chatton divides living organism into prokaryotes and eukaryotes
1944 Avery shows that DNA carries information during transformation
1946 Lederberg and Tatum describe bacterial conjugation
1949 Enders, Weller and Robbins grow poliovirus in human tissue cultures
1952 Hershey and Chase show that bacteriophages inject DNA into host cells
Zinderand Lederberg discover gernalized transduction
1953 Phase contranst microscope developed
Medawar discovers immune tolerance
Watson and Crick propose the double helix structure for DNA
1955 Jacob and Wollman discover the F factor is a plasmid
1962 Porter propes the basic stuructre of immunoglobulin G
first quinolone antimicrobial (nalidixic acid) sunthesized
1975 Kohler and Milstein develops technique for the production of monoclonal antibodies
1982 Recombinant hepatitis B vaccine developed
1986 First vaccine (hepatitis B vaccine) produced by genetic enginerring approved for human use
1997 Discovery of Thiomargarita namibiensis, the largest known bacterium
Escherichia coli genome sequenced
2000 Discovery that Vibrio cholerae has two separate chromosomes
2002 Genome of malaria parasite, Plasmodium falicaprum, sequenced
 Most of the microbes are beneficent
1.7 CHARACTERISTIC OF ALL BIOLOGICAL  Escherichia coli is present in all our
SYSTEMS intestines, helps in digestion and also
provide us with Vitamins.
1) the ability to reproduce  Minimum one bacterial cell can produce
2) the ability to ingest or assimilate food 1000 products. E.coli can produce 4000
substances and metabolize them for energy and products and maximum of 10,000
growth products have been reported by
3) ability to excrete waste products microbes.
4) ability to react to changes in environment –  Microbes are simply the factories which
irritability provide us with food.
5) susceptibility to mutation.
 No life is possible without Microbes
 Out of all, only few microbes are
1.8 Importance of Microbes pathogenic

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 Introduction 4

 We can use microbes as a tool or factory

to prepare whatever we need e.g. food
 Every life requires oxygen except
bacteria. Bacteria need CO2 and it is
oxygen toxic and certain bacteria produce
Oxygen.
 Products obtained from microbes are
highly pure and with minimum side
effects.
 Biologist, Physician, Scientists, and Pasteur found that fermentation of fruits and
Geologists all use microbes.
grains, resulting in alcohol, was brought about by
 Microbes have changed the course of microbes.
history and are convenient and useful in
the study.
 Can directly check or control the growth 1.11 CELL THEORY (1838-1839)
of other microbes. Schleiden and Schwann proposed the cell theory.
 Other changes the starch and sugars into It states that:
Alcohols of important products and their  All cells arise from existing cells
microbial activity is long and surprising.  Cells are basic structure of organisms
 All living organisms are compsosed of

 It is interesting to know that the harmful microbes can be made harmless by

“Genetic Engineering”.
We can alter the genetic DNA code in the chromosome and grow it then. So we
can use microbes as a tool or factory to prepare anything we want.
 Do you know that 100 brains of sheep are required for “Growth Hormone” and 2
litre flask full of E.coli is equivalent to 100 brains.

cells
1.9 EXPERIMENT TO DISPROVE
ABIOGENESIS THEORY 1.12 GERM THEORY
Pasteur (in 1861) performed experiments that The germ theory of disease states that some
ended arguments for all time. Actually, he did it diseases are caused by microorganisms. These
through a very simple and elegant experiment. He small organisms, too small to see without
took two goose neck flasks (see figure 3 for a magnification, invade humans, animals, and other
visual image) with a rich broth inside. Pasteur living hosts. Their growth and reproduction within
heated both flasks to boiling. Next, he broke the their hosts can cause a disease. "Germ" may refer
neck of one flask, allowing the broth to be exposed to a virus, bacterium, protist, fungus, or prion.
to the air. He left the other flask intact. Dust from Microorganisms that cause disease are called
the air was trapped in the bend of the flask and pathogens, and the diseases they cause are called
could not contaminate the sterile broth in the infectious diseases.
flask. He let the unbroken flask sit for months. KOCH’S POSTULATES (1884)
Nothing ever grew in the unbroken flask. This 1) A specific organism can always be found in
research leads to the Germ Theory. association with a given disease
2) The organism can be isolated and grown in pure
1.10 Fermentation by Louis culture in the laboratory
3) The pure culture will produce the disease when
Pasteur inoculated into a susceptible animal
4) It is possible to recover the organism in pure
culture from the experimentally animal

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1.13 KINGDOM
 Gram +ve bacteria can be transformed to Gram –ve bacteria but Gram –ve bacteria can never be
transformed to Gram +ve bacteria.
 BACILLUS THURINGIENSIS is a Gram-positive, soil-dwelling bacterium, commonly used as a
biological Insecticide
In biology, kingdom is a highest taxonomic rank.
Kingdoms are divided into categories called phyla,
each phylum is divided into classes, each class into
orders, each order into families, each family into
genera, and each genus into species.
SPECIES: A species is often defined as a group of
organisms capable of interbreeding and producing
fertile offspring.
1.13.1 HISTORY
The classification of living things into animals and
plants is an ancient one. Carolus Linnaeus laid the
foundations for modern biological nomenclature,
now regulated by the Nomenclature Codes. He
distinguished two kingdoms of living things:
Regnum Animale ('animal kingdom') for animals
and Regnum Vegetabile ('vegetable kingdom') for
plants.
In 1866, the German investigator Ernst Haeckel
proposed a three-kingdom system of classification.
Haeckel's three kingdoms were Animalia, Plantae,
and Protista. Members of the kingdom Protista
included the protozoa, fungi, bacteria, and other
microorganisms. Haeckel's system was not widely
accepted, however, and microorganisms
continued to be classified as plants (for example,
bacteria and fungi) or animals (for example,
protozoa).
In 1969, Whittaker's classification scheme
recognizes five kingdoms: Monera, Protista, Fungi,
Plantae, and Animalia. The five-kingdom
classification scheme is in general use today.
1.14 FIVE KINGDOM CLASSIFICATIONS
1.15 Prokaryotes
The prokaryotes are a group of organisms whose
cells lack a membrane-bound nucleus.
1.15.1 MONERA
Individuals are single-celled, may or may not
move, have a cell wall, have no chloroplasts or
other organelles, and have no nucleus. Monera are
usually very tiny, although one type, namely the
blue-green bacteria, look like algae. They are
filamentous and quite long, green, but have no
visible structure inside the cells. No visible feeding
Introduction 5
mechanism. They absorb nutrients through the cell
wall or produce their own by photosynthesis.
Examples - bacteria, blue-green bacteria
(cyanobacteria)
1.15.1.1 B ACTERIA
Bacteria constitute a large domain or kingdom of
prokaryotic microorganisms. Bacteria are
unicellular or association of simple cells.
A RCHAEBACTERIA – primitive bacteria which are
less complex, now consider as separate division
called Archea
E UBACTERIA – Complex bacteria which different
from archeabacteria on bases of reproduction
(form spores unlike archea) and chemical
composition of cell wall and cell membrane.
1.16 NON CELLULAR
1.16.1 VIRUSES
Viruses are obligate parasite i.e. an organism that
cannot live independently of its host. They are
small infectious agent.
V IRIOD - An infectious particle, similar to but
smaller than a virus, that consists solely of a strand
of RNA and is capable of causing disease in plants.
1.17 EUKARYOTES
The organisms whose cells do have a nucleus are
called eukaryotes.
1.17.1 PROTISTA
Protists are single-celled and usually move by cilia,
flagella, or by amoeboid mechanisms. There is
usually no cell wall, although some forms may
have a cell wall. They have organelles including a
nucleus and may have chloroplasts, so some will
be green and others won't be. They are small,
although many are big enough to be recognized in
a dissecting microscope or even with a magnifying
glass. Nutrients are acquired by
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 Introduction 6

acquired by absorption. For the most part, fungi

photosynthesis, ingestion of other organisms, or
both.
Examples - amoeba, diatom, euglena,
paramecium, some algae (unicellular), etc
1.17.2 FUNGI
Fungi are multicellular,or may be unicellular, with
a cell wall, organelles including a nucleus, but no
chloroplasts. They have no mechanisms for
locomotion. Fungi range in size from microscopic
to very large ( such as mushrooms). Nutrients are
acquire nutrients from decaying material.
Examples - mushroom, mold, puffball,
shelf/bracket fungus, yeast, etc.
1.17.3 PLANTAE
Plants are multicellular and most don't move,
although gametes of some plants move using cilia
or flagella. Organelles including nucleus,
chloroplasts are present, and cell walls are
present. Nutrients are acquired by photosynthesis
(they all require sunlight).
Examples - multicellular algae, mosses, ferns,
flowering plants (dandelions, roses, etc.), trees, etc
1.17.4 ANIMALIA
Animals are multicellular, and move with the aid of
cilia, flagella, or muscular organs based on
contractile proteins. They have organelles
including a nucleus, but no chloroplasts or cell
walls. Animals acquire nutrients by ingestion.
Examples - sponge, jellyfish, insect, fish, frog, bird,
man

1.18 DIFFERENCE BETWEEN PROKARYOTES AND EUKARYOTES (1937 BY CHATTON)

Features Prokaryotes Eukaryotes

Group Bacteria Algae, fungi protozoa, plants,
animals
Size 1-2 x 1-4 um Greater than 5um in
width/diameter
Genetic system location Nucleoid Nucleus
Chromatin bodies Mitochondria
Nucleus material Chloroplast
Structure of nucleus Not bounded Bounded by nucleus membrane
One circular chromosome (rarely More than one chromosome
more than one chromose as in Chromosome have histone
vibrio cholerae) without histone Mitotic nucleus division
No mitotic division Nucleus is present
Nucleus is absent
Cytoplasmic streaming Absent Present
Pinocytosis Absent Present
Gas Vacuole Can be present Absent
Mesosomes Present Absent
Ribosomes 70S (distributed in cytoplasm) 80S arranged in membrane,
endoplasmic reticulum
70S in mitochondira/chloroplast
Mitochondria Absent Present
Chloroplast Absent May be Present
Golgi bodies Absent Present
Endoplasmic Reticulum Absent Present
True vacuoles membrane Absent Present
bounded
Cell Wall Peptidolycan (Murecin. Absent in protozoa and animal
Mucopeptide) Made up of other substance

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 Introduction 7

Pseudopedia Absent May be present in some cases

1.19 CLASSIFICATION OF MICROBIOLOGY
1.19.1 MEDICAL MICROBIOLOGY
Causative agent of dieseas; diagnostic procedure,
procedures for identification of causative agents;
preventive measures
1.19.2 AQUATIC MICROBIOLOGY
Water purification; microbiological examination;
biological degradation of waste; ecology
1.19.3 AEROMICROBIOLOGY
Contamination and spoilage; dissemination of
disease
1.19.4 FOOD MICROBIOLOGY
Food preservation and preparation; food borne
disease and their prevention
1.19.5 AGRICULTURAL MICROBIOLOGY
Soil fertility; plant and animal disease
1.19.6 INDUSTRIAL MICROBIOLOGY
Production of medicinal products such as
antibiotics, vacines; fermented beverages,
industrial chemical, production of proteins and
hormones by genetically engineered
microorganisms
1.19.7 EXOMICROBIOLOGY
Exploration for life in outer space
1.19.8 GEOCHEMICAL MICROBIOLOGY
Coal, mineral and gas formation; prospection for
deposits of coal, oil and gas; recovery of minerals
from low grades ores

1.21.1 ON BASE OF NAME OF SCIENTIST

1.20 BINOMIAL NOMENCLATURE  Shigella boydii
 Shigella dysenteriae
Binomial nomenclature (also called binominal
nomenclature or binary nomenclature) is a formal  Shigella sonnei
system of naming species of living things by giving  Neisseria meningitides
each a name composed of two parts, both of  Neisseria gonorrhoeae
which use Latin grammatical forms, although they  Yersinia pestis (formerly Pasteurella
can be based on words from other languages. The pestis)
first part of the name identifies the genus to which  Escherichia coli
the species belongs; the second part identifies the 1.21.2 ON BASE OF DISEASE CAUSED BY
species within the genus. For example, humans MICROBE
belong to the genus Homo and within this genus to  Mycobacterium tuberculosis (cause TB)
the species Homo sapiens.  Neisseria meningitides (cause Meningitis)
 Salmonella typhi (cause typhoid fever(
1.21 VARIOUS CRITERIA OF NAMING also known as enteric fever)

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 Introduction 8

 Shigella dysenteriae (cause dysentery)

 Vibrio cholerae (cause Cholera)
 Corynebacterium diphtheria (cause
diphtheria)
1.21.3 NONSENSICAL
 Runella slithyformis

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 Bacteriology 9

Chapter 2 BACTERIOLOGY
2.1 CLASSIFICATION OF BACTERIA

Genus Gram Staining Energy Source Shape Other

Eubacteria
Treponema Spirochetes
Azospirillum Gram Negative Aerobic/ Helical/vibroid
microaerophlic
Helicobacter
Spirillum
Spirosoma Gram Negative Curverd Non Motile
Acetobacter Gram Negative Aerobic/ Rods and cooci
Bordetella microaerophlic
Neisseria
Pseudomonas
Rhizobium
Xanthomonas
Escherichia Gram Negative Facultative anaerobic Rods
Shigella
Salmonella
Vibrio
Yersinia
Bacterioides Gram Negative Anaerobic Straingt curved and
Selenomonas helical
Thermotoga
Desulfovibrio Sulfur or sulfate
reducing bacteria
Veillonella Gram Negative Anaerobic Cocci

Chlamydia Rickettsias and

Rickettsia chalmydias
Rhodospirillum Anoxygenic
Phototrophic
Anabena Oxygenic Phototrophic
Nostoc (Cyanobacteria)
Thiobacillus Aerobic
Nitrobacter Chemolithotrophic
Nitrosomes
Stella Budding and
Appendages
Sphaerotilus Sheathed bacteria
Beggiatoa Nonphotosynthetic Gliding
Non-Fruiting
Chondromyces Fruiting Gliding
(Myxobacteria)
Staphylococcus Gram Positive Cocci
Streptococcus
Cocci Gram Postive Aerobes Rods or Cocci Endospore forming
Bacillus
Clostridium Gram Postive Anerobes Rods or Cocci Endospore forming

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 Bacteriology 10

Lactobacillus Regular rods nonsporing

Cornyebacterium Irreqular nonsporing
Cellumonas (Pleomorphic-change
shape)
Mycobacterium Aerobic rods Non-motile
Nonsporing
acid fast
Particular name:
Mycobacteria
Nocardia Filamentous Particular name:
Streptomyces Actinomycetes
Mycoplasma No cell wall Particular name:
Mycoplasmas
Archaeobacteria
Methanococcus Obtain energy via the Particular name:
Methanobacterium formation of methane Methanogens
Archeaoglobus Sulfur reducing
Halobacterium Extremely Halphilic
aerobic bacteria
Thermoplasma No cell wall
Pyrodicitum Hyperthermophilic,
Sulfolobus Sulphur metabolizer

2.2 IMPORTANCE OF BACTERIA

Genus Importance
Eubacteria
Treponema Syphilis
Azospirillum Nitrogen fixer in soil

Helicobacter Stomach Ulcers

Spirillum Rat bite fever
Spirosoma Soil microbe
Acetobacter Vinegar Production
Bordetella Perusis
Neisseria Gonorrhea
Pseudomonas Pathogen of burns
Rhizobium Symbiotic nitrogen fixer
Xanthomonas Xanthan production
Escherichia Genetic engineering intestinal &
urinary infections
Shigella Dysentery
Salmonella Food posion; typhoid fever
Vibrio Cholera
Yersinia Plague
Bacterioides Normal flora in intestinal tracts
Selenomonas Rumen bacteria
Thermotoga Geothermal marine sediments
Desulfovibrio Sulfur cycle
Veillonella Dental caries
Rickettsia Spotted fever
Chlamydia Trachoma, urogenital infection
Rhodospirillum Anaerobic food chain
Anabena Primary producers in aquatic and

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 Bacteriology 11

Nostoc soil food chain

Thiobacillus Sulfur cycle
Nitrobacter Nitrogen Cycle
Nitrosomes Nitrogen Cycle
Stella Grow in low nutrient in aquatic
environment
Sphaerotilus Filamentous slime in polluted
water; sewage treatment plant
Beggiatoa Sulfur cycle
Chondromyces Complex prokaryote behavior
Staphylococcus Food poisoning, toxic
Streptococcus Scarlet fever, pneumonia
Cocci Sporosarcina
Bacillus Anthrax
Clostridium Tetanus, normal flora of soil
Lactobacillus Food fermentation, normal flora of
intestine
Cornyebacterium Diphtheria
Cellumonas Cellulose degradation
Mycobacterium Tuberculosis
Nocardia Lung infection
Streptomyces Antiobiotic production
Mycoplasma Pneumonia
Archaeobacteria
Methanococcus Sewage treatment
Archeaoglobus Sulfur cycle
Halobacterium Grow in hypersaline
Thermoplasma Contain small genome for a
prokaryotes
Pyrodicitum Has highest optimum temperature
know
Sulfolobus Residents to volcanic habitats

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 Bacteriology 12

2.3 SHAPE AND ARRANGEMENT

Bacteria are typical example of prokaryotes. They
have a definite shape. Cell wall is responsible to
maintain the shape of bacteria, but there a few
classes of bacteria who show absence of cell wall.

2.4 CLASSIFICATION OF BACTERIA

ACCORDING TO THEIR SHAPE

Type Shape Typical Example

Bacilli Rod Gram +ve: Gram –ve:
(Singular: Bacillus subtilis Salmonella typhi
Bacillus) Clostridium difficile Escherichia coli
Lactobacillus acidophilus Shigella dysenteriae
Cocci Spherical Neisseria gonorrhoeae
(Singular: Gaffkya tetragena
Coccus)
Coccobaciilius Show up as coccus at Yersinia pestis
low power
Spirillum Spiral Spirillum volutans
Spirochete Screw Treponema pallidum
Vibrio Comma Vibrio cholerae
Mycelium Fungi like Hyphae Actinomycetes
(Hyphae) structure

Type Arrangement Typical Example Reproduction

Diplobacilli Pair Corynebacterium Remain in pair after
diphtheriae divide
Steptobacilli Chain Bacillus subtilis Fail to separate after
divide and form chain
Palisades Match stick arrangement – Corynebacterium
Parallel and pair diphtheriae (also show
diplobacilli
arrangement)
Filament Filament Streptomyces Species
*filamentous bacteria are also considered to be a
different shape of bacteria.

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 Bacteriology 13

Type Arrangement Typical Example Reproduction

Single (monococci) Single Chlamydia trachomatis
diplococci Pair Neisseria meningitidis Remain in pair after
divide
Steptococci Chain forming Streptococcus Fail to separate after
pneumoniae divide and form chain;
Produce chain by
dividing in one
direction on each side,
the cell once divide
cannot divide again,
because no space is
left
Tetrad Group of four Gaffkya tetragena Fail to separate after
they divide, but
instead remain in
groups of four
forming squares;
Cell divides in two
plane
Sarcinae Group of eight Sarcina species, e.g. Fail to separate after
Sarcina lutea (Sarcina they divide, but
species always form instead remain in
spores) groups of eight
forming cubes; Cell
divides in 3 planes
Stephylococcus Cluster, grapes like Stephylococcus species, Reproduction can be
structures e.g. Staphylococcus at any plane (not fix)
aureus
environmental conditions, or even in the same
2.4.1 MONOMORPHIC culture.
A trait of a bacterium that tends to display the
2.4.2.1 E XAMPLES :
same shape regardless of physiological or
 Corynebacterium diphtheria
environmental conditions.
2.4.2 PLEOMORPHIC [POLYMORPHIC]
A trait of a bacterium that can display different
shapes under different physiological or

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 Bacteriology 14

 Mycoplasma pneumonia namibiensis is the largest known bacteria

Bacillus Vs. Clostridium Vs. Lactobacilli

All of them are genus of bacilli shape bacteria. Bacillus is the biggest genus.
Bacillius are aerobic bacteria while clostridium is anaerobic (toxic to oxygen) and obligate parasite. But both of
them form spores unlike lactobacilli that do no form spores.
Lactobacilli acidophilus always produce acid.
discovered in 1997.
2.5.1 DETERMINATION OF SIZE OF BACTERIA
2.5 SIZE OF BACTERIA Size of bacteria is commonly determined by the
Bacteria are very small, most being approximately help of micrometry. Two types of micrometers are
0.5 to 1.0 um in diameters. An important used
consequence of the small size of microorganism is  Stage Micrometer (Calibrated
that the surface area/volume ration of bacteria is micrometer)
exceedingly high as compared to large organisms  Ocular Micrometer
of similar shape. A relatively large surface through Stage micrometer is used to calibrate the ocular
which nutrients can enter (or waste products can micrometer. Stage micrometer divisions are
leave) compared to a small volume of cell known (usually 0.01mm or 10um). The pure
substance to nourished accounts for usually high culture of bacteria is negatively stained with the
rate of growth and metabolism of bacteria. help of a special dye. The dye carries negative
Moreover because of the high surface are volume charge as the wall of bacteria, which repels each
ration, the mass of cell substance to be nourished other, making the bacteria prominent in a dark
is very close to the surface; therefore, no field. Nigrosine black dye is a common example of
circulatory mechanism is needed to distribute the dye used for this purpose. By the help of ocular
nutrients that are taken in and there is thought to micrometer the divisions are observed and
be little or no cytoplasmic movement within a recorded, which is used to calculate the size of
bacterial cell. Despite these advantages, a high bacteria.
surface area/volume ratio limits the size of
bacteria to microscopic dimensions. Thiomargarita 2.6 PLANT CELL VS. ANIMAL CELL VS.
BACTERIAL CELL

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 Bacteriology 15

Organelles Plant Cell Animal Cell Bacteria

Capsule ✓
Cell Wall ✓ ✓
Centriole ✓
Chloroplast ✓
Chromatin ✓
Cilia, Flagella ✓(cilia) ✓ (flagella)
Cytoplasm ✓ ✓ ✓
Cytoplasmic membrane ✓ ✓ ✓
Golgi apparatus ✓ ✓
Lysosomes ✓ ✓
Microfilament ✓
Microtubules ✓
Mitochondria ✓ ✓
Nuclear envelope ✓ ✓
Nuclear pore ✓
Nucleoid ✓ ✓
Nucleolus ✓ ✓
Peroxisome ✓ ✓
Pilli ✓
Plasmodesmata ✓
Ribosomes ✓ ✓
Rough endoplasmic ✓ ✓
Reticulum
Smooth endoplasmic ✓ ✓
Reticulum
Vacuole ✓ ✓ (small or absent) ✓ (gas vacuole)
 A helical filament – which is usually
2.7 FLAGELLA AND MOTILITY several time longer than the cell body.
According to the motility of bacteria, it can be
classified as following:
 Motile (flagellated) Bacteria
 Non- Motile (non-flagellated) Bacteria
The bacteria which can move through their flagella
are called as motile and others who have no
flagella are called as non-motile bacteria. Gram –
ve bacteria are mostly motile with few exceptions.
 Example of motile bacteria: Escherichia
coli (Gram –ve)
 Example of non-motile bacteria:
Spirosoma linguale (Gram –ve)
Bacterial flagella are hairlike, helical appendages
that responsible for swimming motility. They are
much thinner than the flagella or cilia of
eukaryotes and are much simple in structure.
Flagella arise from cytoplasm, just beneath the
cytoplasmic membrane and cross up through cell
wall. They are wavy in structure and larger the Some Gram –ve bacteria have a sheath
length of the cell. surrounding the flagellum. The chemical
Flagellum is composed of three parts: composition of the basal body is unknown but the
 Basal body – associated with cytoplasmic hook and filament are composed of proteins
membrane and cell wall subunits arranged in helical fashion. Filament of
 A short hook flagella is composed of flagellin (protein).
Unlike hair, flagellum grows at its tip rather than at
the base. Flagellum monomers synthesized within

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the cell are believed to pass along the hollow
center of flagellum and are added to distal end of
filament.
Bacteria propel themselves by rotating their helical
flagella. The principle involved can be illustrated
by imagining the penetration of piece of cork by a
corkscrew. There are two phases of movement
shown by the flagella: running and tumbling
(random movement other than the direction of
previous movement). Bacteria becomes thin
during the movement. Prokaryotes flagella works
as oars of boats which is differs in movement from
eukaryotes flagella which shows whip like
movement.
2.7.1 CLASSIFICATION OF MOTILE BACTERIA
According to number and position of flagella,
motile bacteria can be classified as following:
2.7.1.1 M ONOTRICHOUS
Bacteria having single flagellum. E.g. Vibrio cholera
and Pseudomonas aeruginosa
2.7.1.2 A MPHITRICHOUS
Bacteriology 16
Bacteria having single flagellum at each end of
bacteria or a cluster of flagella at both end. e.g.
Spirillum serpens
2.7.1.3 L OPHOTRICHOUS
Bacteria showing cluster of flagella at one end
only. E.g. Pseudomonas fluorescens
2.7.1.4 P ERITRICHOUS
The flagella are present throughout the body of
bacteria. E.g. Escherichia coli and Salmonella typhi
In solid media i.e. 2% Agar , no penetration occur
but in semi-solid media, i.e. 0.5% agar, only
flagellated bacteria can penetrate.
Bacteria are capable of directed swimming
towards or away from various chemical
compounds – a phenomena called bacterial
chemotaxis. Beside the swimming motility of
bacteria shown by the flagella of bacteria, bacteria
also shows gliding (E.g. Cytophaga species) are
motile only when they are in contact with solid
surface) and Sliding.
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 Bacteriology 17

growth is present in solid media, suspension is

prepared so that they separate from clump with
the help of very light shaking.

2.8.1.2 D IRECT O BSERVATION UNDER

M ICROSCOPE
Special chemicals are used in the method so that it
can stick to the flagella, and stain it making it able
to be observed under microscope.

2.8.1.3 O BSERVATION OF MOTILITY WITHOUT

THE USE OF MICROSCOPE
A semi solid medium is used in this method.
Triphenyl tetrazolium chloride, TTC, is used as
an indicator (it shows growth in red colour).
Growth is placed with the help of needle in a
test tube containing semi solid media, and
TTC. The needle makes a line of inoculum
where the growth is present. If the growth is
kept steady for some time, and still is able to
expand from the line of inoculation then the
growth is said to be motile.

2.9 CELL WALL

2.9.1 STRUCTURE AND COMPOSITION
Cell wall of bacteria is unique in its composition as
it is made up of peptidoglycan
2.8 DETERMINATION OF MOTILITY
2.8.1.1 H ANGING D ROP M ETHOD
In this method a suspension of pure culture of
bacteria is made and is hang in a cavity.
Suspension is prepared separately in a test tube. If

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2.9.2 CELL WALL OF GRAM POSITIVE VS. GRAM NEGATIVE BACTERIA
2.9.3 FUNCTION OF CELL WALL
 Cell wall helps in the maintaining of the
shape of bacteria, i.e. rod shape or cocci
shape etc.
 Cell wall provides the protection.
 Cell wall may also be involved in the
sexual reproduction of bacteria.
2.9.4 DEGRADATION OF CELL WALL
Cell wall can be degraded or dissolved by the
antibodies or lysozymes.
2.10 CAPSULE
Bacteriology 18
Bacterial capsule is the outermost covering i.e.
envelope of the bacterial cell which are considered
as capsulated microbes or bacteria. There are
certain bacteria which can never produce capsule
which can be considered as non-capsulated
bacteria.
 Capsulated Bacteria
E.g. Bacillus anthracis and Streptococcus
pyogenes
 Non Capsulated Bacteria
E.g. Escherichia coli
Capsule provide intense protection to cell, so there
will be difficulty in removing the capsulated
bacteria by defensive system. So the capsule is
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associated with virulence of the microbe.
Streptococcus having capsule is virulent and
without capsule is avirulant. Capsule is slippery
and give a smooth appearance to the cell wall as
the capsulated colony (by the help of this,
capsulated colony can be easily separated from
non-capsulated as capsulated colony is smooth
while non-capsulated colony is rough)
Sometimes the capsule layer may be thin so
known as microcapsule, and may also called as
slime layer.
The capsule is chemically made up of
polysaccharides. Special capsule may be
polypeptide or contain lipid e.g. Corynebacterium
diphtheriae. Bacillus anthracis capsule also has
different composition of capsule, and forms spores
as well.
The capsulated bacteria can be transformed into
non-capsulated and vice-versa.
2.10.1 OBSERVATION OF CAPSULE
D IRECT METHOD – by observing if the colony is
smooth or rough
2.10.1.1 M ANEVAL ’ S STAINING OF CAPSULE
The smear is directly prepared in a solution of
Congored dye after air drying the solution
Maneval’s solution for 1-3 mins and observed
under microscope. Negative staining stains
background only, not bacterium or capsule (if
present ). Manevals stain contains fuchsine,
coloring the bacterium, FeCl3 and phenol to
strengthen the capsule, and acetic or
diluted hydrochloric acid to minimize the pH.
The Congo Red from negative staining becomes
blue in acidic medium and you get a high-contrast
slide with blue background, white capsule and red
bacterium. This contrast is the purpose of
Manevals stain during negative staining.
2.10.1.2 S IMPLE N EGATIVE STAING CAN ALSO
BE USED FOR CAPSULE
2.10.1.3 A NTHONY ’ S M ETHOD
In Anthony method fresh solution of copper
sulphate (2-20%) is used to avoid crystallization,
and Crystal violet (7min) to negative stain the
bacteria.
2.10.2 FUNCTION OF CAPSULE
Capsule can serve number of functions depending
upon bacterial species. Some of them are
mentioned below:
1. They may provide protection against
temporary drying by binding water
molecule
2. They may block attachment of
bacteriophages
Bacteriology 19
3. They may be antiphagocytic – inhibit
engulfment of pathogenic bacteria by
WBCs, thus contributing to virulence
4. They may promote attachment of
bacteria to surface.
5. If capsule are composed of compounds
having an electrical charge such as sugar
or uronic acid, they may promote the
stability of bacterial suspension y
preventing the cells from aggregating and
settling.
2.11 SPORES
Bacterial spores are resistant bodies appeared
during the normal life cycle of spore forming
bacteria, however the culture may show a large
number of spores if these area not germinated
because of depletion of substrate. Endospres are
extremely resistant to desiccation, staining,
disinfecting chemicals, radiation and heat.
Spore requires fresh media/food to germinate
directly into bacterial cell, thus the bacterial spore
is considered as vegetative i.e. without
reproduction. However, each and every bacterial
cell will show sporulation (formation of spores).
Thus the bacterial cell shows reproduction and
then spores. N the other hand fungal spores are
considered as reproductive i.e. prior to germinate,
the fungal spore divide into four, six or eight cells.
After the division the fungal spore will show
reproduction and in this way about 8 cells are
produce.
The bacterial spores usually form and remain
inside the cell and this is labeled as endospore and
these spores are only liberated outside after the
destruction of cell but the ideal condition of spore
forming cell is a bacterial cell having or showing
spore inside
Fungal spore show outside and inside spore i.e.
exospore and endospore respectively.
2.11.1 CLASSIFICATION ON BASIS OF POSITION
OF SPORE
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The position and number of spore help in VARIATIONS IN ENDOSPORE MORPHOLOGY: (1, 4) CENTRAL
ENDOSPORE; (2, 3, 5) TERMINAL ENDOSPORE; (6) LATERAL
classification of bacteria. The normal shape of the
ENDOSPORE
the spore is cylindrical/ spindle.
2.11.2 SPORE STAINING
 The spindle shape spore are usually
There are two common method of spore staining.
present almost in center of the cell e.g.
Bacillus cereus
 The spore sometime remains in centre
2.11.2.1 N ORMAL S PORE S TAINING
but the cell may appear as bamboo like In his method the less acidic dye (e.g. malachite
structure. E.g. Bacillus anthracis green) is flooded on slide containing the smear
and heat is passed through the slide as well so that
 Sometimes the spores may be bigger than
the dye easily penetrate in the spore. After cooling
the diameter of cell. They may be on the
it down, decolorizer is used (e.g. glacial acetic acid)
terminal side and giving an appearance of
after decolorizing, the slide is washed at low
dumbbells shape. E.g. Clostridium tetani
stream and safranin red dye is used. It makes the
 Slightly beneath the pole, spore show
green spore surrounded by red cytoplasm visible
bulging character, it is classified as sub-
under the microscope.
terminal spore. E.g. Clostridium
Second method of spore staining is similar to
subterminale
Normal spore staining but it is aided by the use of
 Some also show central spore but
filter paper.
swalloen e.g. Clostridium perfringens
2.11.3 SPOROLATION
Sporolation is the process of spore formation.
Before the formation of spore the bacterial cell
grow in size.

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2.12 CYSTS
Cysts are dormant, thick walled, desiccation resistant
forms that develop by differentiation of a vegetative
cell and which can later germinate under suitable
conditions. In some ways cysts resemble
endospores; however, their structure and chemical
composition are different and they not have the high
heat resistant of endospore. Azotobacter species
forms cysts.
2.13 PILLI
Pilli are hollow, nonhelical, filamentous appendages
that are thinner, shorter and more numerous than
flagella. They do not function in motility, since they
are found on nonmotile as well as motile species.
There are however, several functions associated
with different types of pili. One type, known as the F
pilius (or sex pilus) serves as the port of entry of
genetic material during bacterial mating. Some pili
play a major role in human infection by allowing
pathogenic bacteria to attach to epithelial cell lining
the respiratory, intestinal or genitourinary tracts.
This attachment prevents bacteria from being
washed away by the flow of mucous or body fluids
and permits the infection to be established.
Escherichia coli are a typical example of bacteria,
which normally do not contain pili but pili appears
on Escherichia coli when there is some protective
effect against a drug. Escherichia coli becomes
pathogen
2.14 SHEATHS
Some species of bacteria, particularly those from
freshwater and marine environments, form chain or
trichomes that are enclosed by hollow tube called
sheaths.
2.15 CYTOPLASMIC MEMBRANE
The plasma membrane or bacterial cytoplasmic
membrane is composed of a phospholipid
bilayer and thus has all of the general functions of
a cell membrane such as acting as a permeability
barrier for most molecules and serving as the
location for the transport of molecules into the cell.
In addition to these
functions, prokaryotic membranes also function in
energy conservation as the location about which a
proton motive force is generated. Unlike eukaryotes,
Bacteriology 22
bacterial membranes (with some exceptions
e.g. Mycoplasma and methanotrophs) generally do
not contain sterols. However, many microbes do
contain structurally related compounds
called hopanoids which likely fulfill the same
function. Unlike eukaryotes, bacteria can have a
wide variety of fatty acids within their membranes.
Along with typical saturated and unsaturated fatty
acids, bacteria can contain fatty acids with
additional methyl, hydroxy or even cyclic groups.
The relative proportions of these fatty acids can be
modulated by the bacterium to maintain the
optimum fluidity of the membrane (e.g. following
temperature change).
As a phospholipid bilayer, the lipid portion of the
outer membrane is impermeable to charged
molecules. However, channels called porins are
present in the outer membrane that allow
for passive transport of
many ions, sugars and amino acids across the outer
membrane. These molecules are therefore present
in the periplasm, the region between the
cytoplasmic and outer membranes.
The periplasm contains the peptidoglycan layer and
many proteins responsible for substrate binding
or hydrolysis and reception of extracellular signals.
The periplasm is thought to exist in a gel-like state
rather than a liquid due to the high concentration of
proteins and peptidoglycan found within it. Because
of its location between the cytoplasmic and outer
membranes, signals received and substrates bound
are available to be transported across
the cytoplasmic membrane using transport and
signaling proteins imbedded there.
2.16 CYTOPLASM AND RIBOSOMES
The cell material bounded by the cytoplasmic
membrane may be divided into
 The cytoplasmic area, granular in
appearance and rich in the macromolecular
RNA protein bodies known as ribosomes on
which proteins are synthesized
 The chormatinic area, rich in DNA
 The fluid portion with dissolved substance
Unlike animal and plant cells, there is no
endoplasmic reticulum to which ribosomes are
bound; some ribosomes are free in the cytoplasm
and other especially those involved in the synthesis
of proteins to be transported out of the cell, are
associated with the inner surface of the cytoplasmic
membrane. When the ribosomes of prokaryotes
undergo sedimentation in centrifuge they have
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 Bacteriology 23

sedimentation coefficient of 70S (Svedberg unit) and  Carboxysomes (involve in carbon fixation)
are composed of two subunit, a 50S and a 30S  Cell signaling substance
subunit. This is in contrasting to the ribosomes of
eukaryotes organism which have sedimentation of
80S and are composed of 60S and a 40S subunit.

2.17 NUCLEAR MATERIAL

In contrast to eukaryotes, bacterial cells contain
neither a distinct membrane-enclosed nucleus nor a
mitotic apparatus. However they do contain an area
near the center of the cell this regarded as nuclear
structure and the DNA of cell is confined to this area.
Because it is not discrete nucleus this nebulous
structure has been designated by such term as the
nucleoid. It consists of a single, circular DNA
molecule in which all the genus are linked. The
nucleoid can be made visible under the light
microscope by Feulgen staining, which is specific for
DNA.

2.18 PLASMID
th
Plasmid is 1/100 part of nucleoid. A plasmid is a
small DNA molecule that is physically separate from,
and can replicate independently of, chromosomal
DNA within a cell. Most commonly found as small
circular, double-stranded DNA molecules in bacteria,
plasmids are sometimes present
in archaea and eukaryotic organisms. In nature,
plasmids carry genes that may benefit survival of the
organism (e.g. antibiotic resistance), and can
frequently be transmitted from one bacterium to
another (even of another species) via horizontal
gene transfer.

2.19 CYTOPLASMIC INCLUSIONS

Cytoplasmic inclusions includes
 Voltuin
Volutin granules are known as
metachromatic granules and are composed
of polyphosphare. They serves as a reserve
source of phosphate (energy). e.g.
Corynebacterium diphtheriae and Yersinia
pestis. Another polymer often foun in
aerobic bacteria, espically under high
carbon, low nitrogen culture condition is
chloroform soluble, lipid like material, poly-
betahydroxybutyrate (PHB), which can
serve as reserve of carbon and energy.
 Magnetosomes (work as compass)
 Gas vacuole (that provide buoyancy)

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Chapter 3 REPRODUCTION AND
GROWTH
When bacteria are inoculated into a suitable
medium and incubated under appropriate conditions
a tremendous increase in the number of cells occurs
within a relatively short time. With some species the
maximum population is reached within 24 h. but
others require a much longer period of incubation to
reach maximum growth. The term growth as
commonly applied to bacteria and other
microorganisms usually refers to changes in the total
population rather than an increase in the size or
mass of an individual organism. More frequently
than not, the inoculum contains thousands of
organisms: growth denotes the increase in number
beyond that present in the original inoculum.
Therefore the determination of growth requires
quantitative measurement of the total population of
cells or cell crops at the time of inoculation and
again after incubation.
Growth can be positive as well as negative while
reproduction is always positive.
3.1 MODE OF REPRODUCTION
3.1.1 BINARY FISSION
The most common, and no doubt the most
important, mode of cell division is the usual growth
cycle of bacterial populations is transverse binary
fission, in which a single cell divides after developing
a transverse septum (crosswall). Transverse binary
fission is an asexual reproductive process. Bacillus
subtilis and Streptococcus faecalis are some example
of bacteria showing binary fission.
3.1.2 CONJUGATION
Special pili are involved in the sexual reproduction,
called conjugation. These pili are named as f-pili or
sex-pili.
3.1.3 BUDDING
Some bacteria, such as Rhodopseudomonas
acidophila, reproduce by budding, a process in which
small protuberance (bud) develops into a new cell
which separates from the parent.
3.1.4 FRANGMENATION
Bacteria that produce extensive filamentous growth,
such as Nocardia species, reproduce y fragmentation
of the filaments into small bacillary or coccoid cells,
each of which give rise to new growth.
Reproduction and Growth 24
3.1.5 FORMATION OF CONIDOSPORES OR
SPORANGIOSPRE
Species of genus Streptomyces and related bacteria
produce many spores per organism by developing
cross walls (septation) at the hyphal tips; each spore
gives rise to a new organism.
3.2 GROWTH DETERMINATION
The most common means of bacterial reproduction
is binary fission; one cell divides, producing two cells.
Thus, if we start with a single bacterium the increase
in population is by geometric progression:
2 3 4 n
1 2  2  2 2 … 2
Where n = the number of generations. Each
succeeding generation, assuming no cell death,
doubles the population. The total population N at
the end of given time period would be expressed:
n
N=1x2
However, under practical sonditions, the number of
bacteria N0 inoculated at time zero is not 1 but more
likely several thousand, so the formula now
becomes:
n
N = N0 x 2
Taking log to base 10 and solving the equation we
get:
n
Log10N = Log10 (N0 x 2 )
Log10N = Log10N0 + n Log102
If we now substitute the value of Log102, which
0.301, in the above equation, we get:
Thus by the use of the above equation, we can
calculate the number of generation (n) that have
taken place providing we know the initial population
and the population after the growth has occurred.
3.3 GENERATION TIME
Certain time period is required for bacteria to divide
into 2 or a population to become double is called
generation time. The generation time is the time
interval required for the cells (or population) to
divide.
G (generation time) = t (time, in minutes or hours)/ n
(number of generations)
G = t/n
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 Reproduction and Growth 25

3.4 EXAMPLE OF GENERATION TIME

Bacteria Generation Time
Escherichia coli (require 12.5 mins ( 17 mins
carbohydrates) max)
Staphylococcus aureus 27-30 mins
Salmonella typhi 23.5 mins
Lactobacillus Species 66 mins (approx. 1
hour)
Mycobacterium 13 hours
tuberculosis
Treponema pallidum 33 hours
Mycobacterium leprae 13 days

3.5 GROWTH RATE

Growth rate is the number of generations form in a
unit time.in biological terms, growth rate is defined
as absolute or relative growth increase, expressed in
units of time.
Growth Rate = n/t
3.6 INOCULATION PERIOD
Inoculation period is the period of a disease causing
organism between the entrances of the organism till
the commencement of symptoms. Malaria
inoculation period is 10 days, while typhoid has
inoculation period of 7 days only.
3.7.1.1 L AG P HASE (P HASE OF A DJUSTMENT )
Immediately after inoculation of the cells into fresh
medium, the population remains temporarily
unchanged. Although there is no apparent cell
division occurring, the cells may be growing in
volume or mass, synthesizing enzymes, proteins,
RNA, etc., and increasing in metabolic activity.
The length of the lag phase is apparently dependent
on a wide variety of factors including the size of the
inoculum; time necessary to recover from physical
damage or shock in the transfer; time required for
synthesis of essential coenzymes or division factors;
and time required for synthesis of new (inducible)
enzymes that are necessary to metabolize the
substrates present in the medium.

3.7 GROWTH CURVE 3.7.1.2 A CCELERATED G 1 P HASE

A graphic representation of growth (population
changes) of bacteria in culture medium is known as
growth curve. When a fresh medium is inoculated
with a given number of cells, and the population
growth is monitored over a period of time, plotting
the data will yield a typical bacterial growth curve.
The graph is plotted between logarithm of number
of microbes (y – axis) and time (x- axis).
The cell division just start at this point.
3.7.1.3 E XPONENTIAL ( LOG ) P HASE
The exponential phase of growth is a pattern of
balanced growth wherein all the cells are dividing
regularly by binary fission, and are growing by
geometric progression. Cell obtains the maximum
size just before reproduction i.e. in log phase.
Immediate after reproduction the cell show the
smallest size. The cells divide at a constant rate
depending upon the composition of the growth
medium and the conditions of incubation. The rate
of exponential growth of a bacterial culture is
expressed as generation time, also the doubling
time of the bacterial population. Generation time (G)
is defined as the time (t) per generation (n = number
of generations). Hence, G=t/n is the equation from
which calculations of generation time (below)
derived.

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3.7.1.4 S TATIONARY P HASE
Exponential growth cannot be continued forever in a
batch culture (e.g. a closed system such as a test
tube or flask). Population growth is limited by one of
three factors: 1. exhaustion of available nutrients; 2.
accumulation of inhibitory metabolites or end
products; 3. exhaustion of space, in this case called a
lack of "biological space".
During the stationary phase, if viable cells are being
counted, it cannot be determined whether some
cells are dying and an equal number of cells are
dividing, or the population of cells has simply
stopped growing and dividing. The stationary phase,
like the lag phase, is not necessarily a period of
quiescence. Bacteria that produce secondary
metabolites, such as antibiotics, do so during the
stationary phase of the growth cycle (Secondary
metabolites are defined as metabolites produced
after the active stage of growth). It is during the
stationary phase that spore-forming bacteria have to
induce or unmask the activity of dozens of genes
that may be involved in sporulation process.
3.7.1.5 D EATH P HASE
If incubation continues after the population reaches
stationary phase, a death phase follows, in which the
viable cell population declines. (Note, if counting by
microscopic counts, the death phase cannot be
observed.). During the death phase, the number of
viable cells decreases geometrically (exponentially),
essentially the reverse of growth during the log
phase.
3.8 DILUTION
 Blank: It is refer to a sterile media with no
microbe
Dilution are of two types (parallel and
serial)
 Simple sterile water, simple buffer, sterile
slime water can be used to make the
bacterial dilution suspension that help in
easy count of the bacterial cells.
 Normally 1 ml Sample is added in 9ml of
sterile water to form a diluted suspension in
case of serial dilution, and it may be
repeated to get the require dilution.
Reproduction and Growth 26
 In parallel dilution equal ratio of bacterial
sample and dilatant is mixed gentally.
3.9 SYNCHRONOUS GROWTH
Studying the growth of bacterial populations in
batch or continuous cultures does not permit any
conclusions about the growth behavior of individual
cells, because the distribution of cell size (and hence
cell age) among the members of the population is
completely random. Information about the growth
behavior of individual bacteria can, however, be
obtained by the study of synchronous cultures.
Synchronized cultures must be composed of cells
which are all at the same stage of the bacterial cell
cycle. Measurements made on synchronized cultures
are equivalent to measurements made on individual
cells.
A number of clever techniques have been devised to
obtain bacterial populations at the same stage in the
cell cycle. Some techniques involve manipulation of
environmental parameters which induces the
population to start or stop growth at the same point
in the cell cycle, while others are physical methods
for selection of cells that have just completed the
process of binary fission. Theoretically, the smallest
cells in a bacterial population are those that have
just completed the process of cell
division. Synchronous cultures rapidly lose
synchrony because not all cells in the population
divide at exactly the same size, age or time.
In factories we desire to keep one of the phases of
bacterial growth prolong so we may provide extra
food and remove extra waste, which keep the phase
prolong for some time and in this way the microbes
are synchronized and the phase is called
synchronous phase.
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Blank: It is refer to a sterile media with no
microbe
Dilution are of two types (parallel and
serial)
Simple sterile water, simple buffer, sterile
slight buffer can be used to make the
bacterial dilution suspension that help in
easy count of the bacterial cells.
Normally 1 ml Sample is added in 9ml of
sterile water to form a diluted suspension
in case of serial dilution.
3.10 CONTINUOUS CULTURE
In booth experimental research and in industrial
processes, it is often desirable to maintain a
bacterial population growing at a particular rate in
Reproduction and Growth 27
the exponential or log phase. This condition is known
as steady-state growth. The culture volume and the
cell concentration are both kept constant by
allowing sterile medium to enter the culture and
vessel at the same rate that ‘spent’ medium
containing cells is removed from the growing
culture. In this way the rate at which the new cells
are produced in the culture vessel is exactly
balanced by the rate at which cells are being lost
through the overflow from the culture vessel.
Since the nutrients are not renewed, exponential
growth is limited to a few generations. Bacterial
cultures can be maintained in a state of exponential
growth over long periods of time using a system
of continuous culture designed to relieve the
conditions that stop exponential growth in batch
cultures. Continuous culture, in a device called
a chemostat, can be used to maintain a bacterial
population at a constant density, a situation that is,
in many ways, more similar to bacterial growth in
natural environments.
In a chemostat, the growth chamber is connected to
a reservoir of sterile medium. Once growth is
initiated, fresh medium is continuously supplied
from the reservoir. The volume of fluid in the growth
chamber is maintained at a constant level by some
sort of overflow drain. Fresh medium is allowed to
enter into the growth chamber at a rate that limits
the growth of the bacteria. The bacteria grow (cells
are formed) at the same rate that bacterial cells (and
spent medium) are removed by the overflow. The
rate of addition of the fresh medium determines the
rate of growth because the fresh medium always
contains a limiting amount of an essential nutrient.
Thus, the chemostat relieves the insufficiency of
nutrients, the accumulation of toxic substances, and
the accumulation of excess cells in the culture, which
are the parameters that initiate the stationary phase
of the growth cycle. The bacterial culture can be
grown and maintained at relatively constant
conditions, depending on the flow rate of the
nutrients.
In continuous culture growth no breaking is required
as food is provided before need.
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 Reproduction and Growth 29

3.11.1.6 M EDIA FOR E NUMERATION OF B ACTERIA

3.11 MEDIA Specific kinds of media are used for determining the
bacterial content such materials as milk and water.
In order to grow the microorganism in laboratory a
typical media is provided having proper nutrition
3.11.1.7 M EDIA FOR C HARACTERIZATION OF
values, so the desired organism could grow properly.
B ACTERIA
The beef extract is a complex substance containing
A wide variety of media conventionally used to
almost all important required substance which is
determine the type of growth produced by bacteria
necessary for the growth such as carbohydrate,
as well as to determine their ability to produce
protein, amino acid, some vitamins and certain
certain chemical changes.
lipids. Peptone is a source of protein, and helpful in
proper growth, reproduction and easy division.
Sodium Chloride is a source which can be used for 3.11.1.8 M AINTENANCE M EDIA
the growth of particularly marine microbes. Agar is Satisfactory maintenance of the viability and
added as solidifying agent because no bacteria can physiological characteristic of culture over time may
use agar as substrate till now. The concentration of require a medium different from that which is
agar (15-20g) varies from season to season. optimum for growth.
3.11.1 TYPES OF MEDIA
3.11.1.9 S OLID M EDIA AND S EMI -S OLID M EIDA
Media can be in form of Broth (liquid form) or Solid.
In solid media i.e. 2% Agar , no penetration occur but
Fungi require Sabouraud’s Dextrose Agar, while
in semi-solid media, i.e. 0.5% agar, only flagellated
anaerobes require thioglycollate broth medium.
bacteria can penetrate.
Other includes Enriched media, blood riched media,
3.11.1.1 S YNTHETIC MEDIA
chocolate agar, MacConkey agar (esp. for pin-
The chemically defined media, containing all known
pointed pink colony forming bacteria).
ingredients.
Nutrient Broth Nutrient Agar
3.11.1.2 C OMPLEX M EDIA Beef 5g Beef 5g
The media or a complex mass showing various types Extract Extract
of ingredients in different concentration is labeled as Peptone 10g Peptone 10g
complex mass or complex media. Sodium 5g Sodium 5g
Chloride Chloride
3.11.1.3 S ELECTIVE M EDIA Distilled 1000ml Agar 20g
These media provide nutrients that enhance the Water
growth and predominance of a particular type of Distilled 1000ml
bacterium and do not enhance (and may even
Water
inhibit) other types of organisms that may be
present. E.g. Eosin methylene blue agar (EMB agar)
3.11.2 MIXED CULTURE
3.11.1.4 D IFFERENTIAL M EDIA The culture conitaing two or more than two types of
Certain reagents or supplements, when incorporated known microbes.
into culture media, may allow differentiation of 3.11.3 CONTAMINATED CULTURE
various kinds of bacteria. For example, if a mixture of The culture containing a few unknown microbes or
bacteria is inoculated onto blood-containing agar the entrance of unknown microbe/s in the pure or
medium (blood agar), some of the bacteria may mixed culture.
hemolysis (destroy) the red blood cells; other do not.
This one can distinguish between hemolytic and non- 3.12 SELECTION
hemolytic bacteria on the same medium.
To study the characteristics of one species that
species must be separated from all the other
3.11.1.5 A SSAY M EDIA
species, i.e. it must be isolated in pure culture.
Media of prescribed composition are used for the
However before attempting isolation, it is often
assay of vitamins, amino acids, and antibiotics.
helpful to use a selective method first.
3.12.1 CHEMICAL METHOD OF SELECTION

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 Use of Special Carbon or Nitrogen Source
 Use of Dilute Media
 Use of Inhibitory or Toxic Chemicals
3.12.2 PHYSICAL METHOD OF SELECTION
 Heat Treatment – To select endospore
forming bacteria, a mixed culture can be
heated to 80’C for 10 mins before use to
inoculate culture media.
 Incubation Temperature
 pH of Medium
 Cell Size and Motility
3.12.3 BIOLOGICAL METHOD OF SELECTION
 A disease- producing species occurring in a
mixed culture can often be selected by
taking advantage of its pathogenic
properties.
3.13 PURE CULTURE
Pure culture is the culture or growth of the organism
of only one specific type of microbe.
If the bacterial species being sought comprises a
suitable high proportion of the mixed population, it
can be isolated in pure culture. A strain is usually
made up of succession of cultures and is often
derived from single colony; however, the number of
bacteria which gave rise to the original colony is
usually unknown. If a strain is derived from a single
parent cell, it is termed as clone. Each strain is
designated by an identifying number and its history
is recorded.
3.14 METHOD OF ISOLATING PURE
CULTURE
3.14.1 SPREAD PLATE METHOD (MILE AND
MISRA COUNT)
This method allows determination of the number of
cells that will multiply under certain defined
condition. Molten media (not very hot and not
coagulated i.e. about heated about 45’C) is solidified
in a petri dish and sterilized. A measured amount of
the bacterial suspension(0.1ml) is introduced into a
petri dish containing solidified media and spread
throughout the surface through L-shape glass rod.
The petri dish is incubated. One organism give rise to
one colony, hence a colony count performed on the
plate reveals the viable microbial population of the
Reproduction and Growth 30
inoculum. The number of colonies formed are
counted and multiplied by the dilution factor.
Usually less conc. bacteria are counted through this
method. The original sample is usually diluted so
that the number of colonies developing on the plate
will fall in the range of 30 -300. Within this range the
count can be accurate and the possibility of
interference of the growth of one organism with that
of another is minimized. Colonies are usually
counted by illuminating them from below (dark field
illumination) so that they are easily visible and a
large magnifying lens is often use.
3.14.1.1 D ISADVANTAGES
 Concentrated (need dilution)
 Overlapping of colony
3.14.2 STANDARD PLATE METHOD (POUR PLATE
METHOD )
Known amount of (serial or parallel diluted) bacterial
sample (1ml) is first poured in a petri dish. Molten
media is added in it and the petri dish is slight jerked
to distribute the sample over the petri dish. Media is
then made solidified and incubated. Each separate
colony will be formed in such a way that the quality
(type) and quantity of bacteria can easily be noted.
Colonies may be visible as:
 Imbedded colony – in between media
 Surface colony
 Bottom colony
So the colony is counted from bottom as well.
Any colony can be picked and transferred into the
fresh media to obtain pure culture.
3.14.3 STREAK PLATE METHOD
Streak Plate Method is also used to separate
different type of microbes (mixed culture and
contaminated culture to pure culture). Molten
media (i.e. at 45’C) is added in petri dish, and
solidified. It is then incubated so that there is no
media present on it. Near the burner, sterile wire
loop is used to pick up some culture from
contaminated or mixed culture. Then that wire loop
is used to make streak on the solidified media
present on petri dish.
Wire loop is sterilized again and more streaks are
made by stretching the already present streak at
different angle. This process is repeated twice and
thrice. In this way, sample is thin out on the agar
surface so that some individual bacteria separate
from each other.
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When incubated, some visible isolated colonies of
bacterial cells is formed. Indirectly the number of
bacterial cells can also be counted by this technique.
3.14.4 MICROMANIPULATOR
A device called micromanipulator can be used in
conjunction with a microscope to pick a single
bacterial cell from a mixed culture. The
micromanipulator permits the operator to control
the movements of a micropipette or microprobe (a
fine needle) so that a single cell can be isolated. This
technique requires skilled operator and is reserved
for studies in which a clone must be obtained clearly.
3.15 MAINTENANCE AND PRESERVATION
OF PURE CULTURE
Most microbiologist laboratories maintain a large
collection of strains, frequently referred to as a
stock-culture collection. These organisms are
needed for laboratory classes and research works as
test agent or as reference strains for taxonomic
studies.
Every practical require fresh culture. 18 hours old
culture is best for practical. Preservation of culture
media is required to prevent dehydration and keep
the microbe alive.
3.15.1 METHOD OF MAINTENANCE AND
PRESERVATION OF PURE CULTURE
3.15.1.1 P ERIODIC T RANSFER TO F RESH M EDIA
Periodically transfer the culture after
certain period of time into fresh media.
3.15.1.2 P RESERVATION BY O VERLAYING
C ULTURES WITH M INERAL O IL
Sterile cooking oil (heated over 200’C for 1
hour and cooled) is added to the test tube
containing slant culture. Cooking oil prevent
dehydration keeps the microbe alive. It is
best for this purpose as it does not freeze
and boil easily.
3.15.1.3 P RESERVATION BY E MBEDDING W AX
Microtome is used for micro cutting of a
growth which is introduced into melted wax
Reproduction and Growth 31
and solidified. A block is cut out of the wax
and preserved. Each time we need to use
the growth we have to melt it.
3.15.1.4 P RESERVATION BY L YPHOLIZATION
(F REEZE D RYING )
Lypholization or freeze drying preserves the
growth for many years. Pure culture is
prepared in flash and thick culture is
obtained by continuous growth. Freezer
with special lining of N2 gas and alcohol is
used to reach to temperature -78’C.
Vacuum pump is also used to decrease the
external pressure and the culture is
preserve in form of powder or fine
granules. Instead of flask, injection vials are
used to store these granules, plastic
container may also be employed. The
lyophilized culture is preserved in normal
freezer/ fridge (not at room temperature).
Media containing glycerol prevent cell
damage by crystal formation (Crystal can
damage almost all bacterial species).
3.16 QUANTITATIVE MEASUREMENT /
ESTIMATION OF BACTERIAL GROWTH
There are two types of quantitative measurement:
 Total count (death cell + living cells)
 Viable count (only living cells – more
important)
Quantitative measurement can be performed
directly and indirectly and there are three basic
method:
 Cell Count
 Cell Mass
 Cell Activity
3.16.1.1 S UMMARY :
 Total Count by Direct Method
By microscope, by Petroff- Hausser
Bacterial Counter Chamber, Electronic
Enumeration of Cell Numbers
 Total Count by Indirect Method
 Count by Cell Mass, and Turbidimetric
Method
 Viable Count by Indirect Method
 Plate Count Methods, By Cell Activity
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3.17 CELL COUNT
Cell can be counted directly(by microscopy and
electronic particle channel) as well as indirectly (by
colony count).
3.17.1 CELL COUNT BY MICROSCOPE
Bacterial sample is taken in the form of suspension
(broth). Suspension may be diluted as per need. 0.01
ml of suspension is required to prepare smear on a
microscopic slide. Smear should be evenly,
2 the bacteria are passed through a tiny orifice 10 to
homogenously distributed in an area of 1cm in one
direction and at one attempt.
The smear is stained and observed under
microscope. Average cells present in one
microscopic field is calculated by counting the cells
present on the 4 corner and one center of smear
(total 5 microscopic field) and dividing it by 5.
The diameter of one microscopic field is calculated
by the help of stage microscope and by the help of
the diameter, area of one microscopic field is found
out.
Then using the following equation we can find out
the number of microscopic fields
3.17.2 CELL COUNT BY PETROFF- HAUSSER
BACTERIAL COUNTER CHAMBER
Bacteria can be counted easily and accurately with
the Petroff- Hausser counting chamber. this is a
special slide accurately ruled into squares of
 Depth = 0.2mm or 1/50 mm
 Length= 1/20 mm
 Width= 1/20 mm
 Volume = 1/50 x 1/20 x 1/20 = 1/20,000
3 3
mm = 1/20,000,000 cm = 1/20,000,000 ml
A suspension of unstained bacteria can be counted
in the chamber, using phase contrast microscope.
Bacterial cells are counted 4 corners and 1 center
small square and average number of cells (x) present
in one small square is calculated.
Direct microscope counts can be made rapidly and
simply with a minimum of equipment; moreover the
morphology of bacteria can be observed as they are
Reproduction and Growth 32
counted. Very dense suspension can be counted if
they are diluted appropriately; however, suspension
having low numbers of bacteria, e.g. at the
beginning of a growth curve, cannot be counted
accurately.
3.17.3 ELECTRONIC ENUMERATION OF CELL
NUMBERS
In this method, the bacterial suspension is placed
inside an electronic particle counter, within which
30 um in diameter. The orifice connects the two
compartments of the counter which contain an
electrically conductive solution. as each bacterium
passes through the orifice, the electrical resistance
between the two compartments increases
momentarily. This generates an electrical signal
which is automatically counted. Although this
method is rapid, it requires sophisticated electronic
equipment; moreover, the orifice tends to become
clogged.
3.18 INDIRECT CELL COUNT
3.18.1 PLATE COUNT METHOD
Streak Plate method, Spread Plate Method, Standard
Plate Method (as discussed above) can also be
utilized for cell count, it give viable indirect count by
colony forming unit.
3.18.2 FILTRATION
If sample (broth) is very large in quantity but the
microbial growth is very less, filtration may be
adopted. Millipore filters are used for microbial cells
retension. Millipore filters retains all microbes
except virus and the size of pore is known ( usually
0.22um or 0.45um are used). Vacuum pump is
required which provides vacuum as well apply
pressure to enhance the process of filtration. All the
bacterial cells are retained on the filter paper which
can be inverted on a petri dish containing solidified
media and incubated. The colonies form on petri
dish can be counted which indicated the Colony
forming unit i.e. the viable count of bacterial cell
present in the culture.
3.18.3 TURBIDIMETRIC METHOD
Turbidimetric method is standard for research.
Spectrophotometer is use in this method. The zero
error is checked and the optical density is found out
by the help of spectrophotometer. Bacteria in a
suspension absorb and scatter light passing through
them which is noted by spectrophotometer as
optical density of the culture. In this method instead
of handling microbes, chemicals are used such as
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barium sulphate ppt. (Suspension) (Soln. of chloride
+ sulphuric acid = barium sulphate ppt.). It also has
some demerits as the pigmented microbes cannot
be used in this method.
Graphical representation can be form for different
parameters which saves the time.
3.19 BY CELL MASS
3.19.1 QUANTITY OF NITROGEN (N2) –
MICROKJELDHAL METHOD
The major constituent of cell material is protein and
since nitrogen is a characteristic part of proteins,
one can measure a bacterial population In terms of
bacterial nitrogen. Bacteria average approximately
14-16 % nitrogen on a dry-weight basis. To measure
growth by this technique whole mass of bacterial
sample is digested with sulphuric acid. A catalyst
Copper Sulphate-Dipotassium Sulphate- Sillinium is
used which release the ammonia. The ammonia
which is released is trapped immediately in 2% Boric
acid solution and titrated to calculate the amount of
nitrogen, which can indirectly indicated the total
count of bacterial cell in a concentrated medium.
3.19.2 BY THE WEIGHT OF CELL WALL
Bacterial cell can also be counted by the help of
weight of cell wall. Thick mass of the sample is
required for this test. The thick mass of bacterial
sample is freeze dried. Weight of flask (W1) is noted
Reproduction and Growth 33
and the sample is placed in it. Flask and sample is
weight again (W2) to calculate the actual weight of
sample (W= W1- W2). The sample is then heated for
1 hour at 120’C. The assembly is cooled down in a
desiccator containing phosphorous pentaoxide
(P2O5) which prevents the absorption of moisture.
The Sample is heated and cooled down 3 times and
then weigh. This weigh will be the weight of cell wall.
Cell count can be calculated indirectly by the help of
total cell weight and total cell wall weight.
3.20 BY CELL ACTIVITY – FERMENTATION
Fermentation is anaerobic conversion of substrate.
In this method the specific broth is used e.g. lactose
broth (contain lactose) is used. The bacterial cell
which uses lactose as substrate will convert lactose
into lactic acid. More is the formation of lactic acid in
the media; more will be the count of cell.
Fermentation can be visible by evolved gas or
change in colors which also show the intensity of
fermentation. If no gas is visible Durham’s tube (type
of fission tube) is placed in the flask containing
media and bacterial culture and jerked. If the gas is
evolved the Durham’s tube will be lifted upward.
3.21 GROWTH CHARACTERISTICS
Characteristics of colony observed by naked eyes in
petri dish when we obtain growth are known as
growth characteristic.
 Obesity
o Transparent – light can pass
through
o Opaque - no light can pass through
o Translucent – some light can pass
through
 Color
 Size
 Big, small, pin-pointed
 Glaziness
 Smooth colony (S) or Rough Colony (R)
Escherichia coli in certain media shows pin pointed
pink (PPP) colony.
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3.22 COLONY CHARACTERISTICS
A bacterial colony is defined as a visible cluster
of bacteria growing on the surface of or within a
solid medium, presumably cultured from a single
cell.
3.22.1 SIZE
Reproduction and Growth 34
Colonies range in size from extremely small
(pinpoint), to large colonies measuring 5-10 mm in
diameter.
3.22.2 MARGIN OR EDGE
The periphery of bacterial colonies may take one of
several different patterns, depending on the species.
3.22.3 SURFACE TEXTURE
Depending on the species, the colony surface may
be smooth (shiny, glistening); rough (dull, granular
or matte); or mucoid (slimy or gummy). Certain
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 Reproduction and Growth 35

species have colonies possessing a highly wrinkled Depending on species colonies may be thin to thick
surface. and the surface may be flat or it may exhibit varying
3.22.4 ELEVATION degree of convexity.

Black Bacteroides
3.22.5 CONSISTENCY melaninogenicus
This can be determined by touching a transfer Pink Flectobacillus major
needle to the colony. Some bacterial species form Pink Staphylococcus roseus
colonies having a butyrous or butterlike consistency. Red Serratia marcescens
Others may form colonies that viscous, stringy, or Golden Yellow Staphylococcus aureus
rubbery. Citrus Yellow Staphylococcus citreus
3.22.6 OPTICAL FEATURES White Staphylococcus albus
Bacterial colony may be Yellow Micrococcus luteus (Sarcina
 Transparent – light can pass through lutea)
Brown Derxia gummosa
 Opaque - no light can pass through
 Translucent – some light can pass through
3.22.8 CHARACTERISTIC OF BROTH CULTURE
 Amount of growth: Scanty, moderate or
3.22.7 PIGMENTATION OR CHROMOGENESIS
abundant
Colour Typical Example
 Distribution and type of growth
Violet color Chromobacterium violaceum
Green (ppt) Pseudomonas chlororaphis
Blue (Soluble in Pseudomonas aeruginosa
water) (Phycocyanim)
Bright Green Pseudomonas fluorescens
(Fluorescent)

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 Reproduction and Growth 36

o Osmosis  Halophiles
Do You Know? o Hydrophiles  Barophiles
Instead of 1 wire loop, a plastic bead may o Light (energy)
also be used. Plastic of known sizes are o pH
available in market. Plastic beads or glass is  Acidophiles
added to broth culture to provide solid  Alkalophiles
surface for better growth  Neutrophiles

3.23 FACTOR AFFECTING GROWTH
 Nutritional requirement
 Chemical requirement
o Media
 Physical requirement
o Temperature
o Gaseous requirement- Oxygen,
carbon dioxide
3.24 ENERGY
Energy is mainly obtained from sunlight the primary
source
 Phototroph  require light
 Chemotrophes  require chemicals
 chemolethotroph  need inorganic matter
 chemoorganotroph  need organic matter

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 Reproduction and Growth 37

3.25 ENVIRONMENTAL FACTORS AFFECTING

Do You Know? GROWTH
Less water content make the 3.25.1 NUTRITION FACTORS
microbe more stable Chemical substances are necessary for growth and
function. Biosynthetic capacitis of microbes depends
on their nutriental requirements.
3.25.2 CARBON
Category Energy Source Carbon Source Representive Microbes
Photoautotroph Light CO2 Cyanobacteria/ Algae
Photosynthetic bacteria
Heterotroph Light Organic compound Cyanobacteria/ Algae
Photosynthetic bacteria
Chemoautotrophs Inorganic compounds CO2 S,Fe, NH3, Oxidizing
bacteria, methane
producing bacteria
Chemoheterotroph Organic compound Organic Compound Protozoa, fungi, bacteria
3.25.3 ESSENTIAL ELEMENTS  Traces elements (Zn++, Cu++, Mn++, Ni, B3,
 Nitrogen (amino acid etc) CO2, K+, Ca++, Mg++)
 Oxygens (H2O or O2 etc)  Vitamins and their linked compouds
 Sulphur (Amino acids: Cysteine,  Water (heat labile/sensitive/heat resistant/
methionine) Radiation resistant)
 Phosphorus ( PO4  Teichoic acid, nucleic  Media
acid, phospholipids)  Growth Factor

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 Reproduction and Growth 38


o
A growth factor is a vital requirement Mesophiles Grow Best Between 20 c And
without which there is no growth, these o
40 c (Mosty Cause Food Spoilage)
factors cannot be synthesized by the  Thermophiles grow best at temperatures
microbes themselves during the entire o
above 40 C. (Pseudomonoas sp.)
process of their life; thus these factors must
be supplied from outside into the substrate
Thermoduric organisms can survive high
or media. This is the principal of
temperatures but don't grow well at such
microbiological assay.
temperatures. Organisms which form endospores
The growth is entirely dependent or directly
would be considered thermoduric.
related to quantity of growth factor. If we
Some organisms have exotic temperature
increase the growth factor to double the
requirements. Thermus aquaticus is a bright orange
growth will also be double.
gram negative rod isolated from hot water and
o E.g. Vitamins Amino Acids
steam vents at Yellowstone Park. This organism
o
grows best at temperatures between 70-75 C. Some
Vitamin K Bacteroides
of its unique enzymes are in demand for molecular
melaninogenicus
biological and industrial applications.
Thiamine (B1) Bacillus anthracis Neisseria sp. are called cold-sensitives
Riboflavin Clostridium tetani Pseudomonas are normal
Niacin (Nicotinic acid) Brucella abortus Slow down exyme activity, induce protective
Pyridoxine (B6) Lactobacillus species dormancy (3-4’C to inactivate most mesophiles
Cobalamin (B12) Lactobacillus species pathogen)
Biotin Leuconostoc Fever about 105’C can cause permanent brain
mesenteroides damage.
Folic acid Leuconostoc dextranicum
Pantothenic acid Morganella morganii
3.26.2 OXYGEN
Microbes display a great diversity in their ability to
use and to tolerate oxygen. In part this is because of
3.26 PHYSICAL REQUIREMENTS the paradoxical nature of oxygen which can be both
3.26.1 TEMPERATURE toxic and essential to life.
Obligate aerobes rely on aerobic respiration for ATP
Generally, an increase in temperature will increase
and they therefore use oxygen as the terminal
enzyme activity. But if temperatures get too high,
electron acceptor in the electron transport chain.
enzyme activity will diminish and the protein (the
Pseudomonas is an example of this group of
enzyme) will denature.
organisms.
On the other hand, lowering temperature will
Microaerophiles require O2 for growth but they are
decrease enzyme activity. At freezing temperatures
damaged by normal atmospheric levels of oxygen
enzyme activity can stop. Repeated cycles of freezing
and they don't have efficient ways to neutralize the
(thawing) and thawing can denature proteins. In
toxic forms of oxygen such as superoxide (O 2-) and
addition, freezing causes water to expand and also
hydrogen peroxide (H2O2). The Streptococci are
forms ice crystals, hence cells begin to rupture.
examples of this group.
Every bacterial species has specific growth
Obligate anaerobes will die in the presence of
temperature requirements which is largely
oxygen because they lack enzymes like superoxide
determined by the temperature requirements of its
dismutase and catalase.
enzymes. Each organism will have:
 A minimum growth temperature, 3.26.3 IONIC STRENGTH AND OSMOTIC
 An optimum growth temperature, PRESSURE .
 And a maximum growth temperature.
LIGHT
Organisms can be classified according to their Optimum condition for growth is darkness.
optimum growth temperature. 3.26.4 PH OF THE MEDIUM
 Psychrophiles Grow Best Between -5 c And
o
Most pathogenic bacteria grow best in pH 7.2-7.4.
o
20 c (Bacillus Stearothermophilus) Vibno cholerae can grow in pH 8.2-9.0

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Chapter 4 VIRUSES
A virus is a small infectious agent that replicates only
inside the living cells of other organisms. Viruses can
infect all types of life forms, from animals and plants
to bacteria and archaea.
Viruses are intracellular obligate parasites which
require a host cell to perform every biological
function necessary for the survival of virus. It lacks
cellular machinery. They are incapable of performing
any independent metabolic function. Many viruses
can be purified in form of crystal with an ability to
infect any host and thus can propagation (commonly
replication is the process of multiplication).
Viruses are surrounded by protective coat commonly
known as capsid, containing a few kind of protein,
having limited genetically information, e.g. Tobacco
mosaic virus (largest virus) and polio virus (smallest
virus). The Tobacco mosaic virus (TMV) has 2100
copies of single protein.
These repeating structural units in capsid are known
as protomers. If it bounds to each other by a knob
like structure than it is called capsomers. If it has
additional layer it is called envelope. Some envelope
has an additional layer it is called envelope. Some
envelopes have protenious projection known as
spikes.
Virion is the subunit of virus. Virion is the entire
infective virus which alters to recognize the
susceptible host to attack. The enveloped virus if
dissolved into non-enveloped then the remaining
nuclear capsid cannot cause the disease or infection.
Viruses 39
It is smallest microbe and can pass through filter
(Millipore filter (0.22um) can retain all other
microbes other than microbes). So also known as
filter passing microbes.
A coccus can contain 10,000 viral particles. Viruses
are either RNA or DNA and the power of RNA/DNA is
dominant than host RNA/DNA.
They are non-cellular
Can adopt according to the host so shape and
structure is variable.
Viruses are obligate parasites – require living host –
Best tissue to cultivate virus in lab is chicken embryo.
Icosahedral shape with cubic symmetry may show
sphere like structure with twenty triangular sides,
while the helical shaped virus appear as long rods
with protomers. Head is analogus to capsid.
However still some virus show complex morphologic
e.g. bacteriophage with cubical symmetry and the
head is attached with tail: a hollow tube covered
with sheath of contractile protein, which terminate
to a base plate having fibbers which are used to
recognize and attack to a specific host. If any
structure is removed or damage, the virus will no
longer attack or inject.
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 Viruses 40

Interferon is naturally present in our body as anti-

viral.
Plague formation is due to colony of virus.
Bacteriophage (virus) is present in fecal method of
birds and animals if grows in bacteria either
dominate (lysogenesis) or causes the lysis of
bacteria.

BACTERIOPHAGE

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 Viruses 41

Sterile blade is used to remove the shell without

4.1.1 CULTIVATION OF VIRUS IN LABORATORY
Chicken egg with embryo is usually used to cultivate
virus in laboratory.
4.1.1.1 G ENERAL M ETHOD
damaging the membrane. Place the shell on petri
dish and use needle to inject virus at particular place
on embryo. Place the shell back by the help of wax.
Incubate it and filter it.

4.2.1.1 DNA
4.2 CLASSIFICATION OF VIRUSES

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 Viruses 42

Example
th
Parvovirus Single Stranded Icosahedral Without Enveloped 5 Disease
Papovirus Double Stranded Icosahedral Without Enveloped Warts
Adenovirus Double Stranded Icosahedral Without Enveloped Respiratory
Infections
Herpes Virus Double Stranded Icosahedral Enveloped Chicken pox
Cold Sore
Poxvirus Double Stranded Complex Enveloped Small pox

4.2.1.2 RNA
Reovirus Double Stranded Icosahedral Without Enveloped Diarrhoea
Picornavirus Single Stranded Icosahedral Without Enveloped Polio
Common Cold
Togavirus Single Stranded Icosahedral Enveloped Yellow fever
Encephalitis
Orthomyoxcirus Segmented Single Helical Enveloped Influenza (8
Stranded Segments)
Paramyxovirus Single Stranded Helical Enveloped Measles
Mumps
Rhabdovirus Single Stranded Helical Enveloped Rabies
Retrovirus Single Stranded Helical Enveloped AIDS
Tumor Induction
Corona virus Single Stranded Helical Enveloped Cold Virus

4.3 VACCINE
A vaccine is a biological preparation that improves
immunity to a particular disease. A vaccine typically
contains an agent that resembles a disease-causing
microorganism. Vaccines are material derived from
living organism or they are directed or use as whole
material. Attenuated vaccines- a vaccine created by
reducing the virulence of a pathogen, but still
keeping it viable (or "live") e.g. Cow Pox vaccine.
Sometimes death material is used such as in case of
typhoid-paratyphoid A and B vaccine (TAB Vaccine).
 No proper treatment is present for viral
disease but prevention from complication
of secondary disease (cause usually by
bacteria) is important.
 Guarneri’s bodies are used to identify
viruses
Do You Know?
Virus = Viridae (former name use in
classification)
Making of antibodies start after at least 1
week of incubation
4.4 POXVIRUS / POXVIRIDAE
 Small pox is representative of this class.
 Incubation period is 9- 15 days
 Variola Major (Mortality rate 10-30%)
 Variola Minor (Mortality rate 0.1- 0.3%)
 Gaurinier’s bodies are used to identify
viruses
 It is brick shape, double stranded DNA
without envelope.
 Attenuated vaccine is used for cow pox

4.4.1.1 H ISTORY
 It is known to show it symptoms from 1122
B.C. In 1500 first known epidemic was
appeared and was carried by Cortez’s Troop
resulting in 3.5 million death, Collaps of
Inca; Azetec Civilization.

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 It causes 50,000 deaths in Indo-Pak. In 1950
2 lacs deaths were reported by an epidemic.
4.4.1.2 S YMPTOMS
A symptom appears after 8-18 days as headache,
backache, high fever, and vomiting then appearance
of red spot on third day of the start of symptoms.
They spread to the trunk and then to extremities.
They will appear in the form of macules (deep spots),
then pimple, then pustule (puss is added it is
converted to pink colors) then to blister (boil) and
vesicle scabs are removed leaving behind permanent
scars.
4.4.1.3 D IAGNOSIS
 Direct Microscopic Examination
 Giemsa Staining to Observer Guarneri’s
bodies
 Can be cultivated in chicken embryo and
proceed
 Paul’s Test
Rabbit eyes are injected with this virus
 Complement fixation test
4.4.1.4 V ACCINE
Vaccine cow pox virus us attenuated and modified
into avirulent and use in the dilution of 1:1000.
4.5 INFLUENZA
 Each virus contains 500 copies of H-antigen
and 100 copies of N-antigen.
 Influenza is a Latin word meaning influence
of flue.
 Influenza has3 types A, B, C
 ‘A’ causes more severe illness as compare
to B. C rarely causes disease.
Viruses 43
4.5.1.1 H ISTORY
Major pandemic usually occur after 11 years
(periodically). Most dramatic pandemic occur in
1918 when 20 million people died throughout the
world.
4.5.1.2 S YMPTOMS
Due to viral destruction of cell lining of upper
respiratory tract, trachea, and bronchi.
The symptom begins suddenly which include fever,
chill, headache, muscle ache, weariness, sore-throat,
non-productive dry cough, and nasal obstruction.
Mostly deaths occur by disease complications which
are associated with bacterial pneumonia.
Person suffering from heart or kidney disease
diabetic persons, or having severe anemia, impaired
immunological deafness are highly susceptible to the
flue. So annual vaccination is recommended and it is
90% effective in reducing death rate.
4.5.1.3 T REATMENT
Amidative (medicine) prevent the flu however it is
ineffective if once the symptoms appears.
4.6 ACQUIRED IMMUNE DEFICIENCY
SYNDROME (AIDS)
 Causative agent is known as HIV – Human
immunodeficiency virus
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4.6.1.1 H ISTORY
 In 1950, first reported in Central Africa. In
1980, a deficiency in immune system was
reported in young homosexual patient who
may also be extended to heterosexual
person.
 The disease is developed as opportunistic
way. In some person it appears as skin
cancer in form of “Kaposi- Sarcoma”. A
group of French scientist at Pasteur
institute in 1993, the scientist Montagnier
studied it in detailed. It was named
lymphadenopathy associated virus (LAV) by
him.
 American group named it lymphoric virus III
and also Persistent- Generalized
Lymphadenopathy (PGV). International
Commission Finally recommended it HIV.
HIV may effect nervous system;
megakaryotes, monocytes, macrophages
(reservoir of HIV virus). It is diagnosed after
6-8 weeks by ELISA Test (Enzyme-linked
Immunosorbent Assay), PCR (polymerase
chain reaction). It may be sympathetic or
asympathetics.
4.6.1.2 S YMPTOMS
 HIV- A
Fever, weight loss, unexpected diarrhea and
ARC (AIDs related Complex Symptoms)
Viruses 44
 HIV-B
Neurological disease, myelopathy and
secondary infection
 HIV-C
Similar to Herpes zoster multidermatomal,
leukoplakia, candidiasis
 HIV-D
Secondary cancer (Kaposi’s sarcoma)
 HIV- E
Abnormal neurological difficulties
4.6.1.3 T REATMENT
 The disease may be life long, so the
sufficient time may be there to provide the
treatment. The life cycle of virus can be
interrupted in different steps:
o Decreasing the binding capacity of
HIV to susceptible T-Lymphocyte
with medication like soluble CD4
o Synthesis of pro-viral nuclear
material could be inhibited by
reversed transcriptase i.e.
Zidovudine
(azidothymidine AZT), it is
similar to an important building
block loosed in the synthesis
ofviral DNA
 Other anti-HIV agent include dideoxyinosine
(ddE) and dideoxycytidine (ddc), both can
be used with minimum sided effect.
 Other can also be used as anti-HIV i.e.
foscarnet and ampligen. A vaccine for HIV
infection that protect human has not been
developed yet inactivated whole virus may
protect susceptible animals
4.7 HEPATITIS
 Hepatitis is inflammation of liver. Hepatitis
is of two types
 Small spherical particles mostly of hepatitis
B
 Tubular or Filamentous root
 An enveloped nucleocapsid structure also
known as Dane’s particle
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 Incubation period varies from 4 weeks to 6
months and therefore also known as long
incubation hepatitis
 Severe hepatitis may appear suddenly by
showing dark urine and clay coloured stool
before proper jaundice
4.7.1.1 H EPATITIS A
No specific treatment except injection alpha-
interferon
Intron A® can be used as prophylactic measurement
4.7.1.2 H EPATITIS -B
Genetically engineered yeast cells is used to produce
Recombinax – I ®
4.7.1.3 H EPATITIS -C AND D
It is nor-A nor-B also called NANB, the disease is less
severe. In 1984, it is discovered as retrovirus RNA;
using the enzyme reverses transcriptase to form
DNA from the bacterial code in RNA. Thus its
replication process is reversed.
Hepatitis C is about 80nm in diameter having lipid
coat, single standard RNA causing yellow fever and
dengue fever. 40% cases are infected and lead to
chronic viral disease.
Hepatitis D was discovered in 1977 having
antigenetic RNA fragment. It causes infection only if
Hepatitis B is present. In 1984 it was reported with
evidence that Hepatitis B assist in replication of
Hepatitis D. Hepatitis Delta (hepatitis-D) is also
known as piggyback virus.
4.8 POLIO
 Causative agent is Poliomyelitis
 Polio vaccine is a typical vaccine that can be
prepared by three immunological types of
vaccine. it is one of the smallest virus. 10-
100 thousand virus of polio can be place in
a cooci
 Trivalent vaccine is used for
o Dumb Polio (avirulent, asymptotic)
o Abortive Polio
o Paralytics Polio (cause disease and
also known as bulbo-spinal nerve
paralysis)
 Paralytic
Viruses 45
 Non-paralytic (can be
recovered)
 May causes convulsion, stiffness of neck
and abdomen. Polio virus multiplies in
nerve cell of man and monkey. Therefore
the kidney of affected person, most
commonly animal (monkey) is removed and
2
grounded into fraction of 1cm .
4.8.1.1 O RAL V ACCINE OR S ABIN ’ S V IRUS
V ACCINE
The vaccine normally pours into the mouth,
consisting of virus from monkey kidney tissue; which
are culture and then checked for neurotropic
symptoms in the animal.
The oral vaccine is prepared as syrup, in hard gelatin
capsule containing a dilution of stock virus.
Sabin’s gave the idea of stock culture virus retain in
laboratory for storage, do not multiply.
4.8.1.2 S ALK ’ S V IRUS V ACCINE
Virus multiply in vertical column and develop
immunity in monkey tissue virus is obtained from
animals and killed by formaldehyde to prevent
paralysis or any paralytic disease.
4.9 RABIES VIRUS
The most important member of the rhabdovirus
family is the rabies virus, a parasite of domestic and
wild mammals. Only one immunological type of this
virus is known to exist. Transmission to humans
occurs through the bite of an infected animal. Dogs,
cats, bats and skunks are most frequent source of
the virus infecting humans.
4.9.1.1 H ISTORY
 In 1885, Pasteur produced an effective
rabies vaccine by using brain tissue from
rabbits in which rabies virus had been
propagated. By drying the infected nerve
tissue for increasing periods of time, the
infectivity of the virus could be
progressively diminished to point where the
preparation could be used to immunize
person who had been bitten by rabid
animals. In 1919, Semple modified the
Pasteur vaccine by adding phenol to
completely inactivate the virus.
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 Viruses 46

 1949, “The Flury Strain: vaccine was develop symptoms of rabies, the human victim must
developed by using duck embryonated to not be vaccinated.
give attenuated strain egg for rabies.
 In 1980, an inactivated vaccine is prepare
from virus propagated in culture, if diploid
human cell, devoid of nerve tissues aging
was licensed to use. It appears safe as well
as highly immunogenic.

Rabies is essentially an overwhelming

encephalomyelitis. In humans the incubation period
from time of infection caries from 6 days to 1 year
but is usually about 3 to 8 weeks. The development
of symptoms and the length of incubation period (in
untreated cases) depend largely on the severity and
location of the bite. It has been estimated the only 5-
15 percent of all persons bitten by a rabid animal
contract rabies. Symptoms in humans include severe
headache and high fever, with alternating stages of
excitement and depression. Patients have difficulty
in swallowing and slight stimuli incite muscular
spasms in the throat and chest. Death usually
follows paralysis or convulsive seizures. The
mortality rate from rabies is nearly 100 percent.
If a person has been bitten by a rabid animal, the
long incubation period for rabies allows time for
measures to be taken to prevent the virus from
reaching central nervous system. these measure
include a combination of passive immunization (by
administration of immune human or horse globulin,
which provides an immediate source of antibodies
against the virus but which last for only about 14
days) and active immunization (adminsitrations of a
rabies vaccine, to stimulate a longer-lasting
production of antibodies by the patient).
Laboratory confirmation of rabies in the animal
which has bitten the patient is done by any of
several means
 Detection of rabies virus antigen in clinical
specimens by use of fluorescent antibodies
 Isolation of virus from saliva, urine, spinal
fluid, or tissues by inoculation into the
brains of mice
 Demonstration of inclusion bodies (Negri
bodies) in the nerve cells of the brain

In all these test negative results do not exclude

rabies and for this reason the animal should be
destroyed prematurely by should be kept under
observation for at least 2 weeks. If the animals

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Chapter 5 PROTOZOA
Protozoa (singular, protozoan) are non-
photosynthetic unicellular organism with a
eukaryotic cell structure. They are further
characterized by the absence of cell walls, which
distinguishes them from the fungi and non-
photosynthetic algae. Some protozoa possess a
pellicle, a protein layer that increases the rigidity of
their cell membrane, it also provide some protection
from osmotic stress and is responsible for the
characteristic shape of protozoa.
Protozoa ranges in length from about 2um (the
smallest, Leishmania parasite) to 20,000um (2cm or
4/5 inch). Thus the smallest protozoa are similar in
size to many bacteria and the largest are large
enough to be seen without the aid of microscope.
Most protozoa are polymorphic; they undergo
morphological changes during different phases of
their life cycle. Some of these protozoa alternate
between two forms, the actively feeding trophozite
and the other one are the cyst. Trophozites feed and
reproduce as long as environmental conditions are
tolerable. The onset of adverse condition would kill
the trophozite may triggers its transformation into a
cyst, thereby increasing the chances of species
survival. The cyst is dehydrated, dried, protective
form of organism. Cysts survive in dried and aquatic
environment. Some cysts are reproductive.
Trophozite forms of the same organism survive
poorly outside the body and if ingested are quickly
destroyed by stomach acid. Without cyst the
protozoan could not establish an infection in the
gastrointestinal tract. The cyst is therefore the only
infectious stage of intestinal protozoa; trophozite
play no role in transmission of intestinal illness. Once
in the intestine, however the fragile trophozite
emerge from the cyst and reproduction resumes in
the new host.
Not all protozoa forms cyst. Protective cysts are
unnecessary if the organisms are transmitted
directly between living hosts with exposure to
hostile environment. Trichomonas, for example, a
genital pathogen that is transmitted from one
person to another by direct sexual contact, forms no
cyst. Similarly no cysts are formed by protozoa that
are transmitted by vector, living organism (usually a
biting arthropod) that transfers the protozoa from
one vertebrate host to another. Vectors protects the
parasite from extreme changes in temperature,
moisture, nutrient availability, and other factors in
Protozoa 47
the external environments. Malaria, for example, is
prevalent in humans only in areas populated by its
vectors, the Anopheles mosquitoes, and human
sleeping sickness only where there are tsetse flies.
Control of these diseases depends on eliminating
their vectors from the regions.
Some protozoa are pleomorphic. This means that
they have several different trophozite forms often
depending on their environment. The term
pleomorphic should not be confused with
polymorphic, which denotes the capability of
producing both trophozite and cysts forms.
Organisms with multiple hosts are often
pleomorphic with different trophozite morphologies
in the different species. For example, an organism
may bear flagella for swimming though body fluids
but lose the appendages when it invades heart
tissue.
5.1 NUTRITION AND METABOLISM
5.1.1 NON-PHOTOSYNTHETIC CHEMO -
HETEROTROPHS
They require an external source of food for energy
and material for biosynthesis. They obtain their
nutrients on one of two ways
5.1.1.1 O SMOTROPHS
Absorbing dissolved nutrients directly through their
cell membrane. For example, parasitic protozoa that
absorb their nutrients from the blood and tissue of
their host are osmotrophos.
5.1.1.2 P HAGOTROPHS
Feed by engulfing soluble organic material or solid
food particles, forming intra-cytoplasmic vesicles
called food vacuoles. Amoebas, for example can take
up particulate matter anywhere along their surface
by phagocytosis.
Ciliated organism such as the paramecia creates
currents to sweep food particles into a specialized
mouth like structure on the membrane called
cytostome, where they trapped in food vacuoles.
Enzymes stored in lysosomes are discharged into the
vacuole; where the ingested particle is digested. The
end products of digestion are absorbed into the
cytoplasm, where they are either metabolized by cell
for energy or used to synthesize structural
components.
Any waste products remaining, in the food vacuoles
are usually released from the cell by exocytosis, a
process in which the vacuole membrane fuses with
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plasma membrane, spilling the vacuole contents out 5.2 REPRODUCTION

of the cells.
Contractile vacuoles accumulates the water, when  Binary fission (most common)
they are full the vacuoles contract and their contents  Longitudinal fission
from the cell, bail out the excess water.  Transverse fission
Free-living protozoa are usually aerobic. Some  Multiple fission
parasitic protozoa, however, live in environment
 Conjugation
where oxygen may be limited; indeed some
intestinal protozoa require low oxygen  Fertilization
concentration, even anaerobic conditions.

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5.3 CLASSIFICATION
Group Characteristics Representative Diseases
Mastigophora  Mastigo=whip; phora=bearer (whip bearer)  African Sleeping sickness
(flagellated)  Move by flagella *(or by undulating (Trypanosoma gambiense
membrane) and Trypanosoma
 Reproduce asexually by longitudinal binary rhodesiense)
fission  Chagas’s Disease
 Sexual (trypanosoma cruzi)
reproduction absent  Giardiasis lamblia (intestinal
infection)
 Trichomonas vaginalis
(genital infection)

Ciliophora  Move and/or feed by cilia*  Balantidiasis coli (causes

(ciliated)  Genetic exchange by conjugation diarrhea) (spread through
 Reproduce asexually by transverse binary fecal material)
fission
Sarcodina  Move and feed by pseudophods*  Amebic dysentery
(amoeboid)  Reproduce asexually by binary fission (Entamoeba histolytica)
 Foraminifera and radiolarian are amoebas
that have rigid internal or external skeletal
structures made of calcium or silica.
Sporozoa  No apparent trophozoite motility* (non-  Malaria (Plasmodium)
(Apicomplexa) motile)  Toxoplasmosis
 Interacellular parasites  Pneumocystis (interstitial

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 Alternate asexual and sexual cycles cell)

 Many require alternate hosts for completion  Pneumonia
of life cycle
* This property that distinguishes protozoa
from the members of the other three
subdivisions

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5.4 DISEASE CAUSED BY PROTOZOA
Generally pathogenic protozoa initiate infection in
human at four primary sites
 Intestine
 Genital Tract
 Tissues and blood stream
 Central nervous system
Intestinal Infection are commonly caused by
 Giardia lamblia (Flagellated)
 Blantidium coli (Ciliated)
 Entamoeba histolytica (amoeba)
All three organisms have similar life cycles
Since 1972 Giardia lamblia has caused more
outbreaks of waterborne disease in US than any
other pathogen. The outbreak have usually been
associated with deficiencies in water treatment
system.
Entamoeba histolytica causes the most serious
disease, amebic dysentery (bloody diarrhea). The
pathogen release enzymes that break down
intestinal tissues (histolytica =tissue-lytic). The
pathogen can spread and cause fatal abscesses of
the liver, lungs, heart and brain. About 10 percent of
the World’s population may be carriers of this
protozoan, each infected person capable of shedding
3 million cysts per day.
5.4.1.1 T RICHOMONIASIS
Protozoa 53
The flagellated protozoan Tichomonas vaginalis is
one of the world’s most common causes of genital
infections. In some areas of US, 50% of the women
examined shows microscopic evidence of
Trichomonas infection. It is a sexually transmitted
disease. Fomites (inanimate objects) such as towels
and clothing have also been implicated in
transmission. The disease trichomoniasis is localized
usually causing vaginitis. Males are also infection
with the organism but usually show no symptoms.
Tichomonas vaginalis is a pear shaped protozoa with
two pairs of anterior flagella and one posterior
flagellum. it thrives in the slightly acidic environment
of the human vagina. Establishment may be
encouraged physical or chemical trauma, including
poor hygiene, drug therapy, diabetes or mechanical
contraceptive devices such as intrauterine device
(IUD). The organism has no cyst stage.
In females, trichomoniasis is accompanied by intense
itching (pruritis), and burning pain during urination.
Usually, a creamy white, frothy discharge is also
present. The symptoms are frequently worse during
menstruation and erosion of the cervix may occur.
In males the disease occurs primarily in the urethra,
with pain on urination and thin, mucoid discharge.
The disease can occur concurrently with gonorrhea.
5.4.2 BLOOD INFECTION
Human blood parasites are nearly always acquired
by the bite of an infected arthropod vector, and are
also transmitted by blood transfusion. The blood is
primary site of infection but subsequent invasion of
the brain, viscera, and other secondary sites may
occur. The most common protozoan blood infections
are trypanosomiasis and malaria.
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American trypanosomiasis, also known Chagas’s
disease is caused by trypanosome cruzi. The vector
are triatomine bugs, blood sucking insections.The
insects attraction to the lips account the nickname
“the kissing bug”. Another common site of attack is
eyelid. The kissing bug may deposit feces
contaminated with trypanosomes. From the
bloodstream, the organism localize in heart and
central nervous system.
Chagas’s disease is the major cause of heart disease
for those under 40 year old. the damage they cause
is often fatal.
More than a million people die of malaria every year.
Virtually all malaria deaths occur in areas in habited
by the female Anopheles mosquito. Four members
of the genus Plasmodium causes malaria in humans
 Plasmodium malariae
5.4.3 INFECTION OF CENTRAL NERVOUS SYSTEM
Several free-living amoebas are capable of causing
meningoencephalitis (an inflammation of the brain
and the meninges) the two most common causes of
amebic meningoencephalitis are Naegleria fowleri
Protozoa 54
 P. vivax
 P.ovale
 P. falciparum
The disease occurs when any of the four species is
present in the saliva of an anopheles mosquito and is
injected into the human bloodstream by insect’s
bite. The parasite infects the liver and ultimately
release progeny back into the blood stream, where
red blood cells become infected. The red blood cells
lyses, showering the bloodstream with more
parasites, which then infect additional RBCs. Rupture
of the RBCs, occurs in synchronized manner,
resulting in the periodic episodes of chills and fever
that typical of malaria.
and several species of Acanthamoeba. Naegleria
infection is contracted while swimming in water
containing the parasites which enter through the
nasal cavity and occasionally invade the brain.
Acanthamoeba probably enters through broken skin
or the lungs. Factors such unfortunate person for
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 Protozoa 55

amebic meningoencephalitis include diminished

immune response. Acantheamoeba infections of the
ey have been among wearer of contact lens who
failed to properly disinfect their lenses. Severe cases
required removal of the eyeball.

5.5 OPPORTUNISTIC INFECTION

Some protozoa primarily cause disease in person
with severely reduced defenses. Three protozoan
diseases
 Toxoplasmosis
 Cryptosporidiosis
 Interstitial cell pneumonia

are becoming important opportunistic pathogens

Taxoplasma gondii, infects one-third of world’s
adults. These sexual forms are produced only in cats
and are shed in the cats’ feces. The disease is also
severe when transmitted across infected mother’s
placenta, often leading to miscarriage or serious
defect in her fetus.
Cryptosporidiosis has long been known to cause
intestinal infections in animals. These sporozoans
usually cause no more than a self-limited diarrhea
unless person is immunosuppressed. the disease in
person suffer in from AIDS, for example, may lead to
dehydration and death.
Another opportunistic pathogen commonly
associated with AIDS is Pneumocystis carinii. This
organism causes intestine cell pneumonia, a
frequently fatal pulmonary infection that strike
immunosuppressed or premature infants. This
disease is most frequent cause of death among
children with acute lymphoblastic leukemia.

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Chapter 6 FUNGI
Fungi is placed in an independent group described
by Whittaker. They are basically decomposers. They
require higher humidity and carbohydrate (greater
concentration). About 500,000 species of fungi in
which only 100 are pathogens and mostly fungi are
beneficial, used in various industries. The fungal
spores are already coloured and showing different
characteristic in shape. The fungal spores are either
exospore or endospore. The spore bearing hyphae
are known as fruiting bodies.
The fungal spores are also reproductive i.e. before
germination they show reproduction.
Fungi are basic decomposer.
Example of spores with hyphae (exospres):
 Aspergillus
 Penicillum
Endospore with hyphae
 Mucor
 Rhizopus
6.1 COMMON YEAST (SACCHAROMYCES
CEREVISIAE )
The fungi are heterotrophic microbes ranging from
unicellular to multicellular macroscopic organism.
The most important species of the fungi include
yeast, a unicellular having a number of
chromosomes varying from 8-16.
At the time of reproduction the number of
chromosomes would be double. The way of
reproduction also varies from asexual to sexual.
Budding and conjugation are frequently observed.
The fungi or yeast must be studied from top as well
as bottom of petri dish without inverting the petri
dish.
6.1.1 IMPORTANCE AND CLASSIFICATION OF
YEAST
The yeast can be classified as following:
6.1.1.1 T RUE Y EAST
Most important and mostly use in daily life for
preparation of baking products. It is also a source of
vitamin particularly Vit B. Sometime it is also used as
digestive. Their commercial preparations are
available in market. The yeast can also be
Fungi 56
incorporated in the media use for the growth of
fungi.
The most common media used for the growth of
fungi is known as Sabouraud’s Dextrose Agar (SDA)
media. When the yeast is also incorporated in this
media it is named as SDA+Y.
6.1.1.2 F ALSE Y EAST
The yeast that are pathogenic are commonly term as
false yeast. E.g. Candida albicans
6.1.1.3 B OTTOM Y EAST
Used particularly for preparation of low grade wine
e.g. beer
6.1.1.4 T OP Y EAST
Used for preparation of high grade wine e.g. Ales.
6.2 FUNGAL DISEASES
Any organ of the body can be affected. Throughout
the world about 20% of the person are suffering
from fungi disease due to hyphae.
Do You Know?
Infection: When any microbes establish and causes
damage to host
Infestation: a way of carrying microbes without any
harm/symptoms
Inflammation: A localized protective reaction of
tissue to irritation, injury, or infection,
characterized by pain, redness, swelling, and
sometimes loss of function.
6.2.1 SYSTEMIC MYCOSES (DEEP)
6.2.1.1 A CTINOMYCOSIS
Actinomycosis israelii and actinomycosis bovis
These are gram positive bacillary and branching
forms called higher bacteria. They occur as obligate
parasite in man and animals. In tissues they form
hard sulfur granules. They require blood for growth
on media.
Infection in man begins in throat, abdomen or lung
cavity. The organism may penetrate the skin
develop draining sinuses, It occurs around the world
and most common in rural males 20-40 years old. A.
bovis causes abscesses on face and head in cattle is
called “lumpy jaws”
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6.2.1.2 N OCARDOSIS
Norcardia asteroids
Gram-positive filamentous higher bacteria.
Fragments of hyphae appear as bacilli or cocci. Many
strains are acid-fast. They are often found in soil;
grow well on Sabouraud’s glucose agar and other
ordinary media.
One type of this infection produce chronic,
granulomatous lesions the lungs and intestines and
sembies tuberculosis. Sulfur granules are not
formed. It mainly causes abscess and sinuses of feet,
resulting in a deformlity. This is called mycetoma
pedis.
6.2.1.3 C RYPTOCOCCOSIS
Cryptococcus neoformans
These are yeast like capsule producing organisms
that reproduce by building. No hyphae or spres are
formed. They grow well on ordinary culture media.
This organism may infect any part of the body but
usually start from the lungs and spreads through the
blood stream. Infection in the brain and meninges
usually causes death.
6.2.1.4 M ONILIASIS
Candida albicans
C.albicans are yeast like cells with pseudohyphae.
They produce large thick walled spherical
chlamydospores. On ordinary culture media, pasty,
smooth colonies having a yeast odor develop.
Monilia may infect any body area and is found in the
mucous membrane of the intestinal tract of many
health persons. Infections, with C. albicans in the
mouth are called thrush.
6.2.1.5 N ORTH A MERICAN BLASTOMYCOSIS
Blastomyces dermatitidis
These are large roung cells (5 to 15 u) with a single
bud. Intercalary and terminal chlamydospores
appear in old cultures. The optimum temperature
for growth is 37’C. On infusion blood agar the
colonies resemble M.tuberculosis.
Infections with Blastomyces dermatitidis resembles
pulmonary tuberculosis involvement of the lungs
pleura. It is characterized chroni, granulomatous,
superlative lesions of any body tissues.
6.2.1.6 S OUTH A MERICAN BLASTOMYCOSIS
Blastomyces brasiliensis
These are yeast like cells that are larger than B.
dermatitidis. Parent cells give rise to many buds.
Fungi 57
Smooth, waxy, yeast like cells appear on blood or
meat medium after several days’ incubation at 37’C.
South American blastomycosis is clinically similar to
the North American blastomycosis. Lesions are most
commonly found in the moth and gastro intestinal
tract and in the lymph nodes of neck.
6.2.1.7 H ISTOPLASMOSIS
Histoplasma capsulatum
These are small, oval cells found intercelluarly in
tissues. Colonies on blood agar resemble
Staphylococcus albus. Cells have single buds. At
room temperature on Sabouraud’s glucose agar,
delicate branching, septate hyphae appear with
chlamydospores present in old cultures.
This may occur as an acute or chronic, localized or
disseminated infection of the recticuloendothelial
system. Clincally it may be confused with carcinoma
of the nose, tongue or pharynx tuberculosis;
Hodgkin’s disease; and aplastic anemia. Most
infection regress spontaneously but fulminating
cases are usually fatal.
6.2.1.8 C OCCIDIOIDOMYCOSIS
Coccidioides immitis
In culture of Sabouraud’s glucose agar these
organism develop as typical white to buff coloured
mold colonies that sporulate by arthospore
formation.
This disease goes by many other names such as
valley fever, San Joaquin fever and desert
rheumatism. The fungus is highly infectious and
widely disturbed in the soil. Most cases are mild and
transitory but few terminate fatally.
6.2.1.9 S POROTRICHOSIS
Sporotrichum schenckii
Organisms are rarely found in tissues but in
experimentally infected rats the organism are gram-
positive, resembling fusiform bacilli. On Sabouraud’s
glucose agar incubated at room temperature
delicate, branching septate hyphae with spherical or
pyriform micro-conidia in clusters on lateral
branches are found.
This is chronic infection that usually begins as a
subcutaneous nodule at the site of an injury. Initial
lesions resembles warts, boils. In rare cases involving
spread of the organism thoroughout the body the
patient may die.
6.2.2 CERTAIN IMPORTANT SUPERFICIAL
DISEASE OF SKIN
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6.2.2.1 C OSMETIC DISEASE  e.g. Amphotericin- B

Mostly hair and skin
6.4.1.2 S TEROIDS
6.2.2.2 S UBCUTANEOUS DISEASE  Temporary treatment can be done by
Present on subcutaneous tissue but can penetrate steroids as well
into deeper tissue. Infection can lead to bone and
lumphatic vessel esp. if there is a punchure or
6.4.1.3 A ZOLE
wound. E.g. Sporotrichosis
 ketoconazole
6.2.2.3 C UTANEOUS DISEASE ( COMMONLY  imidazole
DERMATOPHYTES )
It includes fungal growth in nail and skin. E.g. 6.4.1.4 P OLYOXIN
whitlow fungal disease and ring worm  Anti-metabolic and anti-mycotic group for
opportunisitic fungi
6.2.2.4 O PPORTUNISTIC FUNGI I NFECTION  E.g. Flucytosine ( Mode of action: 5-
Establish due to weak defensive system fluorocytosine is converted to 5-
fluorouracil that inhibit the microbe)
6.3 TOXIN
Mycotoxin is toxin produce by fungi. alpha-toxin
(1mg can kill the whole population of Lahore) is
Do You Know?
Fruiting body (full fungi body) is a sporangium with
produces by Aspergillus flavus.
hyphae
Aspergillus niger and Rhizopus attack carbohydrate
Stolen: The hyphae connecting two fruiting bodies is
and wheat
known as stolen E.g. Rhizopus
Toxin from spore is alarming

6.3.1.1 P OISONOUS / T OXIC MUSHROOM

Almost all beautiful looking mushrooms are toxic.
E.g. Death cap. 6.5 CLASSIFICATION OF FUNGI
Fungi basically grow on sub-terrain (superficial layer)
6.4 CONTROL of soil as has the presence of air and moisture both.
Mostly (4 classes) are terrestrial, only one class is
Fungi disease can primary be treated by KMnO4.
aquatic.
6.4.1 GROUPS OF MEDICATION Distinguishing Characteristic of four terrestrial and
one aquatic groups of fungi
6.4.1.1 P OLYENES (G ROUP OF ANTIBIOTICS )
Group Hyphae Sexual Spores Commonly Some Medically
Observed Asexual Important Genera
Spores
Terrestrial
Zyhomycetes Nonseptate Zygospores Sporangiospores Mucor
Rhizopus
Ascomycetes Septate Ascospores Conidia Aspergillus
(largest group) Arthrospores Histoplasma
Blastospores Trichophyton
Penicillium
Claviceps (Ergot)
Basidomycetes Septate Basidiospores Characteristically Crytococcu
none Amanita (“death
angel” mushroom)
Agaricus
Deuteromycetes Septate None Conidia Candida

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Arthrospores Sporothrix
Blastospores Coccidioides
Chlamydospores
Aquatic
Oomycetes (Aquatic Asexual zoospore, flagellated spore after growth they become non- Phytophthora
molds) flagellated, non-motile infestans, causes
Vegetative Spore potato blight
Looks like without cell wall or without chitin Controlled by
Contain N-acetyl glucosamine Bordeaux mixture
(CuSO4 and lime
water mixture)

FIGURE 1: ASPERGILLUS SPECIES

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FIGURE 2: PENICILLIUM

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 Normal Flora 65

 Capsulated streptococci are a transient and

are present in buccal cavity. If enter into
Chapter 7 NORMAL blood can cause disease.
FLORA
 Trillion of microbes all over the skin 7.1.4 SKIN
 The microbes which are present on the  Staphylococci (normally coagulase –ve)
surface of our body in harmonious to us  Lactobacilli
(with agreement), we called them normal  Diphtheroids or Corynebacterium diphtheria
flora. Harmless flora present over our body  pathogen if lamda phage is present
is called normal flora. Mostly residing in our  Micrococci acnes (causes acne)
colon.  and also some fungi and viruses
 If normal flora is removed, pathogen flora
may find space for its growth. It causes 7.1.5 ORAL CAVITY
 Treponema pallidum
infestation.
 Nisseria (pathogen if in genital)
7.1.1 RELATIONSHIP OF MICROBES PRESENT AS  Leptotrichia
NORMAL FLORA  Bacteroides
7.1.2 SYMBIOSIS
The relationship between two different kinds of 7.1.6 UPPER RESPIRATORY TRACT
living things that live together and depend on each  Pneumococci non-capsulated(capsulated
other. causes pneumonia )
 Haemophilus
7.1.2.1 C OMMUNALISM  Mycoplasma
Only one get benefit and sometime it may act as
 Phyranx
parasite
 Streptococci
7.1.2.2 M UTUALISM  Bordettela
Both get benefit, e.g. lactobacilli from lactic acid and
get nutrition 7.1.7 STOMACH
Escherichia coli do not harm if an appropriate  Helicobacter pylori (opportunistic, can
number but if number increase, it may become cause ulcer)
harmful Number of incoming E.coli should be equal  Bacteroides
to outgoing. E.coli never produces pili normally but
 Enterobacter (Enteric Fever Typhoid)
sometime does and sticks to body increasing in
number and becomes parasites
Physiology and anatomical may show some effect 7.1.8 SMALL AND LARGE INTESTINE
susceptibility, body may become susceptible.  Enterobacter
7.1.3 MICROBIAL FLORA OF VARIOUS PART OF  Escherichia coli
BODY  Mycobacteria
 Permanent flora  Klebsiella
 Transient (Temporary)  Pseudomonas
 Proteus
7.1.3.1 E XAMPLES
 Colon, Intestine  Escherichia coli 7.1.9 SEXUAL ORGAN
(permanent)  Staphylococci (normally coagulase –ve)
 Skin  lactobacilli (permanent) form  Streptococci
lactic and kill some pathogenic  Fusobacterium
 Mycobacterium

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 Lactobacilli
 Diphtheroids

7.1.10 EAR
 Pseudomonas
 Enterobacter
 Diphtheroids
 Staphylococci (normally coagulase –ve)

7.1.11 EYES (CONJUNCTIVITIS FLORA)

 Haemophilus
 Staphylococci (normally coagulase –ve)
 Pseudomonas
 Streptococci
(Lysozymes kill mostly pathogenic microbes)
7.1.12 NOSE
 Staphylococci (normally coagulase –ve)
 Streptococcus viridans
 Diphtheroids

7.1.13 NAIL
 Fungi
o Aspergillus
o Penicillium
o Mucor
o Cladosporium

There are certain parts which are supposed to be

free from normal flora e.g. Brain, blood, urine and
cornea.
Gastrointestinal infection may be appeared when
normal flora is altered. Sometime only 10 microbes
can cause infection or disease because there would
be competition between normal flora and pathogen.
Axilla and premaxillary.
Person using antibiotic or chemotherapeutic agent,
they loss their Normal Flora and thus become
susceptible to other pathogen which are normally
present in the atmosphere. Thus unnecessary use of
antibiotic, chemotherapeutic agent on any other
antimicrobial should be avoided.

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Chapter 8 MICROBES OF AIR

We inhale air about 500,000 liter daily, which
contain numerous microbe laden on dust particle.
They travel through dust particle. The transport of
air microbes is dust particle.
Dust particle are not a substrate/food. Entrance of
dust particle in a sterile container or media, render
it, contaminate it, thus the dust particle are source
of contamination. The presence of microbes in a
sterile container indicates the entrance of dust
particle. Throughout the world the indicator
microbes of air is Bacillus subtilis. Most common 8.1.2 FILTRATION
contaminant is Bacillus subtilis (it is indicator Micro filter can be used and cultivated to in a petri
organism of air). dish.
8.1.3 OTHER DEVICES
8.1 AIR IMPINGEMENT TECHNIQUE OR  Invert the funnel
 Put a source in funnel tube
AIR SETTLING PLATE METHOD
 Take a known volume of balloon blown by
 15 Sterile solidified media containing petri pump (not by mouth), use it as source
dish  Inject known quantity of air into the petri
 Place 3 petri dish on each corner and 3 in dish containing media through funnel and
the center of room. excess is removed through pump
 Open one petri dish in each position after 5
minute, other after 10 mins and then the
last one after 15 minutes
 Calculate count per mins (count divided y
time for kept opened)
 A controlled experiment should be done on
unopened petri dish place at all place, the
count (if present) should be subtracted
from total count.
 The growth, colony characteristic can be
checked, and if need streaking and staining
may be employed

8.1.1 TRAPPING
Trap the microbial flora present in air in the water by
the help of glass beads leaving behind the air
without microbes. Beads facilitate capturing and
8.2 FACTORS AFFECT THE MICROBIAL
retaining of microbes. FLORA OF AIR
 Environment factors
 Physical Condition
o Temperature
o Season
o pH
o Humidity etc.

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8.3 AIR BORNE DISEASE  Factories use fumes of formaldehyde (40%

formalin) and after using it, KMnO4 fumes
8.3.1 BY BACTERIA should be used to neutralize
 Bacteroides
 Mycobacteri
um tuberculosis Do You Know?
 Streptococci (Streptococci pneumoniae) Bacillus subtilis is present normally in
 Haemophilus air, Escherichia coli in water and
 Scarlet fever causing bacteria Mycobacterium tuberculosis in milk
 Whooping cough causing bacteria

8.3.2 BY VIRUS
 Small Pox
 Influenza
 Polio

The air borne infection usually affects the respiratory

tract and may cause epidemic. Microorganisms
occur in secretion from nose and throat transmitted
to healthy individual by aerosol.

8.4 CONTROL OF AIR MICROBES

Area of practical work should prevent people from
coughing, talking or sneezing there. During
Centrifuge, the speed should be increase gradually.
Simply removing a container lid up can also cause
microbial contamination.
8.4.1 PHYSICAL METHOD
 Burner (near burner) or any other heat
source
 Ultraviolet tube
UV inhibitory to bacteria to fungus
 Use of goose neck (Pasteur experiment)
 Petri dish
The assembly of petri dish is not air tight;
air is allowed to enter without dust particle
 Air Filter
Cotton is the best filter for dust particle
 Maintaining air pressure
Special technique may be employed to
retain pressure of the room in such a way
that the external air containing dust particle
cannot enter
8.4.2 CHEMICALS
 Glycol lactic acid
 0.1% soln. of Mercuric chloride

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 Microbiology of Water 69

If the presumptive test is positive, to confirm the

presence of E.oli we may proceed further and if the
presumptive test is negative then there is no need
Chapter 9 MICROBIOLOGY OF for any further tests.
WATER 9.1.2 CONFIRMATORY TEST
 We need to provide water in every media We may use following media, to check the
characters of colony formed
for growth
 EMB agar (Eosin methylene blue agar) ,
 The water which is fit for drinking is
E.coli shows pin pointed pink (PPP) colony
tasteless, colorless, and must be free from
in this media.
all pathogenic microbes. The water which is
 MacConkey’s Agar
fit for drinking is potable water; it is
 Lauryl SO4 broth with phenol red as
microbiologically and chemically free for
indicator and also contains lactose shows
harmful substances.
yellow colony
 Escherichia coli (indicator microbe),
indicates other pathoen may be present. We use pour plate method i.e. molten media is used
E.coli is present in normally in fecal matter. and then the sample is added. It is solidified and
Presence of E.coli indicated contamination then incubated.
of water with sewerage water. 9.1.3 COMPLETE TEST
 Normal water is not sterile. Presence of one Brilliant Green Bile Lactose Broth is used for the
E.coli can reject the whole sample of water. complete test.
 Water is also chemically checked for toxic,
potassium, iron and other matter. 9.2 IMVIC TEST
IMViC test includes
9.1 PRESUMPTIVE TEST  Indole test
 Methyl red test
 Presumptive test is performed to check the
 Voges and Proskauer test (VP test) (Kovac’
presence of Escherichia coli, although the
Reagent)
test is also positive if Enterobacter
 Citrus Test (Simmon’s Citrate Test)
aerogenes is present in the water sample.
 E.coli is gram –ve small thin, non-spor
Indole Methyl VP Citrus
forming, non-capsulated, peritrichous
test red test Test
bacilli.
test
E.Coli +ve +ve -ve -ve
9.1.1 PRESUMPTIVE TEST
Enterobacter -ve -ve +ve +ve
9.1.1.1 U SE OF L ACTOSE B ROTH
Lactose broth contains no other microbes except
lactose. E.coli is a lactose fermenter, it always use 9.3 NATURAL WATER
lactose as substrate and convert it into lactic acid. The Earth’s moisture is in continuous circulation, a
Fermentation is anaerobic oxidation. In this test a process known as the water cycle.
test tube containing lactose broth is used. The test
9.3.1 ATMOSPHERIC WATER
tube also contains Durham’s tube. if the media
The moisture contaminated in clouds and
changes it colour to pink or red, it indicates the
precipitated as snow, sleet, hail and rain consititute
formation of lactic acid. Depending on the degree of
atmospheric water
colour “+” signs are used to indicate that the
presumptive is positive. If gas is evolved is used
9.3.2 SURFACE WATER
Bodies of water such as lakes, streams, river, and
to indicate it.
oceans represent surface water
9.3.3 GROUND WATER

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Ground water is subterranean water that occurs 9.5 FACTORS AFFECTING MICROBES OF
where all pores in the soil or rock-containing
material are saturated. Bacteria as well as WATER
suspended particles are removed by filtration, in  Temperature
varying degree, depending of the permeability  Nutrition
characteristic of soil and the depth to which the
 Dissolved Oxygen
water penetrates.
 Source of Energy (availability of sunlight)
9.4 MICROBES PRESENT IN WATER  Salinity
 Hydrostatic pressure
(WATER BORNE DISEASES)
 Turbidity
 Thyphoid  pH
 Cholera  Inorganic and Organic Constituents
 Amoebiasis  Plankton (Phytoplankton, Zooplankton)
 Streptococcus faecalis
 Clostridium perfringens
9.6 PURIFICATION OF WATER
 C. welchii
Boil water at 80’C at reduced pressure

dismiss chlorine affects. It is better to store water for

9.6.1 STERILE WATER sometimes and then use it, which help in escape of
Water can be sterile by the addition of chlorine. chlorine, as chlorine is also damaging for our body
1.8% aqueous solution of sodium thiosulphate to and act as slow poison.

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9.7 SANITY ASPECT OF MICROBIOLOGY

 Sanitary Inspector
o Potability test
o IMViC Test
 Supply to Colony
 Sewerage System

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Chapter 10 MICROBIOLOGY
OF SOIL
 Geology: Science of soil
 Most bacteria are in soil. Minimum 10
9 10.3.1 DIRECT MICROSCOPIC EVALUATION
bacteria are present per gram of soil.
Clostridium tetani is present in soil.
 Soil is a region where geological systems
and biological system meets together.
All waste materials including dead bodies are buried
into the soil; by microbial action all the material is
disintegrated into black colour stable mass which is
known as humus. Thus all these material also
constituents the soil
10.1 CONSTITUENTS OF SOIL
 Solid Particles
o Large
o Small
o Sands
 Water
 Minerals – Clay, fine particles
 Gases – O2, H2S, NH4, CH4, CO2, N2, SO2
 Organic residue – plant, humus (last stage
of decomposition), dead bodies etc.
 Ores
 Biological System (dead bodies as well as
living microbes huge in number)
 These constituents affect the soil nature,
natural nutrient, texture, water holding
content and buffering capacity.
 Upper layer of soil different because of O2.
 Soil also shows different types of microbial
interactions.
10.2 MICROORGANISM OF SOIL
Maximum microbes are present in root sphere.
Microbes are present in huge number in soil which
includes:
 Actinomycetes (cause of smell after rain)
 Certain Algae, Protozoa, Viruses
 Aspergillus
Microbiology of Soil 72
 Penicillium
 Cysts of Protozoa
 Bacteriophage
10.3 EXAMINATION OF MICROBES OF SOIL
Soil is observed under microscope directly.
10.3.2 SERIAL DILUTION METHOD
Serial dilution is best method for microbial
examination.
 Soil sample is collected and check for
microbes.
 Soil sample should not be dried or muddy.
The simplest way to check that is the
sample is not dried is to take the sample on
a piece of paper, and check if it is blown by
air or not, and it also confirm if the sample
is muddy or not, if the sample is muddy it
will stick to paper.
 About 50g of sample is taken. It is stirred
properly and 1g is dissolved in 10 ml of
water, to make dilution (1:10). Serial
dilution is made, using this first diluted
sample and dissolving it in 9ml (1:100). This
method is continuing for 7 times. Last tube
7
is 10 times diluted. Last three test tubes
are used while the rest are discarded.
Incubated by pour plate method, and
counted. The number of count x dilatant
7
(10 ) = count/g of soil.
10.3.3 BURIED SLIDE TECHNIQUE (CONTACT
SLIDE METHOD)
 Dig the soil by the help of spatula. The soil
should not be dried nor muddy, and if need
water may be sprinkle.
 Two slides are joined together and buried in
the soil keeping only a certain part of the
slide out of soil.
 Slides are kept in soil for 9-10 days.
 One Slide is simple stained with Erthrocyine
stain( Simple stain for fungi), while the
other slide is gram stained.
10.3.4 CELLULOSE ENRICHMENT TECHNIQUE
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Soil mostly contains dead plant leaves which
comprises mostly of cellulose, so the soil growing
microbes are usually celluloselytic microbes (use
cellulose as substrate).
Cellulose broth is prepared, which contain no other
carbohydrate except for cellulose. 1 of soil sample is
placed in this broth and incubated. Microbes growth
is replaced in fresh cellulose broth for several times
again and again to obtain celluloselytic microbes
only.
10.4 BIOCHEMICAL ACTIVITIES
10.4.2 CARBON CYCLE
Microbiology of Soil 73
Complex organic materials deposited on or into the
Earth’s surface are degraded by microbes and end
up with humus and its related products etc. The
source of nutrients alter the texture of soil, help to
produce water holding capacity and increasing of
buffering nature and availability of the nutrients. All
these microbial activities going on in the soil bring
various chemical changes, and show different cycles,
such as:
10.4.1 NITROGEN CYCLE
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10.4.3 PHOSPHOROUS CYCLE

10.4.4 SULPHUR CYCLE

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Chapter 11 IMMUNOLOGY
11.1 IMMUNOLOGY
It is the science which deals at one aspect with the
disease caused by pathogens having virulent power
and on the other aspect deals with the resistant of
the host.
It is simply illustrated as
 Pathogenicity, virulence and invasiveness by
microbes to cause infection etc.
 Defense system i.e. Resistance of the host
11.2 PATHOGENICITY
This is a qualitative trait, referring to the inherent,
genetic capacity of a micro-organism to cause a
disease, mediated by specific virulence factor and is
the result of a specific host-pathogen interaction
11.2.1 VIRULENCE
 Degree / Power of Pathogenicity
 This is a quantitative trait; representing the
extent of the pathology caused by micro-
organism. It is therefore a trait expressing
Immunology 75
the interaction between a pathogen and its
host.
11.2.2 INFECTION
 Establishment of microbes in the host after
defeating it
 Multiplication of a pathogenic micro-
organism in a bodily part or tissue which
may produce subsequent tissue injury and
progress to obvious disease through a
variety of cellular or toxic mechanism.
11.2.3 INVASIVENESS
 Capability of microbes to select and react at
particular site and cause disease.
11.3 DEFENSE SYSTEM
The main function of defensive system of the body is
to prevent or limit infection by micro-organism such
as bacteria, viruses, fungi, parasites, protozoa and
worms.
It is classified as:
 1 Line  Mechanical, chemical and
st
microbiological defense
 2 line Phagocytes (lymphocytes)
nd
 3 Line  Immunity
rd
11.4 1ST LINE OF DEFENSE
11.4.1 MECHANICAL, CHEMICAL AND
MICROBIOLOGICAL
Our body is constantly exposed to microbes present
in our environment, including infectious agents that
have been shed from other infected individuals.
Contact with this micro-organism may occur through
external or internal epithelial surface. For a microbe
to invade the body or to cause a disease it has to
bind or cross the epithelium layer.
Epithelia comprise the skin and the lining of the
body’s tubular structures – the gastrointestinal
respiratory and urogenital tracts. Epithelial cells are
held together by tight junctions which effectively
forms a seal against the external environment.
11.4.1.1 S KIN
Skin is the important physical barrier between the
external and internal world. In the case of wounded
skin, microbes can enter the body and cause disease.
11.4.1.2 SWEAT GLANDS:
 Sweat glands are also present in the skin
which secretes anti-microbial substances
present in the sweating material. It also
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contains an enzyme called lysozyme which
kill microbe.
 Sweating is a way of removing of waste
products.
 Dry and over perspired skin is susceptible to
microbial attack.
11.4.1.3 MICROBIOLOGICAL BARRIER:
 Mostly healthy epithelial surfaces are also
associated with a large population of
normally non-pathogenic bacteria known as
“Commensal bacteria” or “micro biota”
 These “Commensal bacteria” or “normal
flora” complete with pathogenic microbes
for nutrients and for attachment site on
epithelial cell.
 The normal flora also produces anti-
microbial substances such as lactic acid
produced by lactobacillus species.
11.4.1.4 GASTROINTESTINAL TRACT:
11.4.1.4.1 MECHANICAL:
 Peristaltic movement (one way) of stomach
is a part of defensive system of a body and
is responsible for keeping the food and f
infectious agent moving through the body.
 Failure of peristalsis is typically
accompanied by the overgrowth of
pathogenic bacteria within the lumen of the
gut.
11.4.1.4.2 CHEMICAL:
The low pH of stomach is due to the secretion of HCl
from the parietal cells of gastric mucosa. It is
essential for proper digestion and it is anti-microbial
and indicates good defensive system.
11.4.1.4.3 MICROBIOLOGICAL:
 Normally Escherichia coli are essential for
digestion but its number increases then it
may become parasite.
 The excessive use of antibiotics kills the
normal flora like E.coli and pathogenic
bacteria frequently replace them and cause
disease.
11.4.1.5 RESPIRATORY SYSTEM:
11.4.1.5.1 MECHANICAL:
Immunology 76
 Hiccups are the sign of defensive system
and these are due to the reflex action of
epiglottis.
 Hair of the nostrils entrapped dust particles.
 The internal epithelia are known as mucosal
epithelia because they secrete viscous fluid
called mucus which contains glycoproteins
called mucins. Microbes coated in mucus
may be prevented from adhering to the
epithelium and in the respiratory tract;
microbes can be expelled in the outward
flow of mucus driven by the beating of cilia
on the mucosal epithelium.
11.4.1.6 EYES/NOSE/ORAL CAVITY:
 Lysozyme is present in
1) Tears
2) Saliva
3) Sweat material
Lysozyme is anti-microbial in nature and
preventing the microbe to invade the body
by killing it.
 Nasal cilia are present which play important
role in the removal of micro-organism.
11.4.1.7 INTERLEUKINS (FEVER INDUCER):
Fever producing substances i.e. interleukins are
naturally present in body. When microbes enter the
body which causes the pyrexia the interleukins
activated and cause the further increase in
temperature which become inhibitory for the
microbes and cause death. However, a high
temperature exceeds 103 F, it is alarming and that
can bring near to 101 F.
11.4.1.8 TRANSFERRIN:
Transferrin is iron-binding blood plasma glycoprotein
that controls the level of free iron in biological fluids.
It plays a key role where erythropoiesis and active
cell division occur. It is also associated with immune
system. It is found in mucosa and bind iron. Thus,
creating an environment low in free iron that
impedes bacterial survival in a process called Iron
withholding. The level of transferrin decreases in
inflammation.
11.4.1.9 INTERFERONE:
It belong to the large class of glycoproteins knows as
cytokines and are released by host cells in response
to the presence of pathogens such as virus or tumor
cells. Interferon is named after their ability to
“interfere” with viral replication within host cells.
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 Immunology 77

They also activate immune cells such as natural killer immune system destroy the virus or bacteria by
cells and macrophages. producing inflammatory cytokines like
phospholipase A2 which also lead to fever 1/3 of
11.4.1.10 INFLAMATION: antibodies are contributed by it.
“A localized physical condition in which part of the Tonsils prevent the entry of microbes into the body
st
body becomes reddened, swollen, hot and often which is 1 line of defense and produce antibodies in
rd
painful and in some cases immobilization especially the case of infection which is 3 line of defense.
as a reaction to injury or infection is termed as
inflammation.”
TONSILLITUS:
Inflammation is part of the body’s immune response.  It is the inflammation of tonsils most
Initially it is beneficial when, e.g. your knee sustains commonly caused by viral or bacterial
a blow and tissue needs care and protection. infection. Enlarged tonsils reduce the size
However, sometimes inflammation can cause further of the airway making snoring or sleep
inflammation it can become self-perpetuating. More apnea.
inflammation is created in response to the existing  Majority of people recover completely
inflammation as it will cause to the secretion of with or without medicaments.
mediators like histamine, leukotriene etc.  Symptoms may include sore throat and
Normally inflammation indicates the presence of fever when caused by bacterium
infection which leads to disease. belonging to the group A Streptococcus;
it is typically referred to as strep throat.
11.4.1.11 TONSILS:  In 40% symptoms will resolve in three
Tonsils are police guard. It is the body’s first line of days and within one week in 85% of
defense as a part of the immune system. These people, regardless of whether
occur in pair and are the collection of lymphoid streptococcal infection is present or not.
tissue facing into the aero digestive tract.  Saucuna lappa is the best medicine to
Under normal circumstances, as virus and bacteria cure tonsillitis.
enters the body the nose and mouth, they are
filtered in the tonsils within the tonsils WBC’s of the
Sequelae: a condition that is the consequence of a previous disease or injury
A person suffering from a disease would be resistance to any outbreak of a typical microbial infection
but as soon as the patient recovers from the previous disease they would become susceptible to that
infection.
Person suffering from infection of Streptococcus pyogene become susceptible to kidney disease after
recovery. Similarly person suffering from Rheumatic infection become susceptible to heart disease.
Herpes zoster (viral disease occurs to patient who was suffering from chicken pox.
Patient suffering from viral infection after recovery become susceptible to cancer.

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 Immunology 78

st
Summary of 1 line defense:
Its involve skin which provides mechanical barrier in which sweat gland, Sebaceous gland are also present.
These secretions are chemically antimicrobial
That is why a dry skin is the susceptible, but over secretion by skin can also cause problem
50,000 l air is inhaled by us in a single day and hair of nostril mechanically stops the microbes from entering
the body. Hairs of nostrils are natural filters.
Tonsils are natural police guards.
Lysozymes are also antimicrobial present in saliva, eyes etc.
Flushing of urine and the excretory system also comes under chemical defense.
Removal of waste products.
st
Peristaltic movement is also 1 line mechanical defense.
Mucin layer(epithelium) present in GIT and other organs systems
HCl present in the stomach is also kills the microbes.
Reflex action of epiglottis i.e. coughing is a sign of defense.
Coughing, sneezing and fever is also defensive system.
If temperatures rises 102’F, it will kill microbes. Interlukin are proteins involve in fever production.
Iron binding substances like protein transferin help in defense system

were instead aggregated around the foreign bodies

as if to drive them out.
A cell that specializes in this process is known as
11.5 SECOND LINE OF DEFENSE: phagocytosis.
Phagocytes engulf a solid particle to form an internal
vesicle known as phagosomes.
11.6 PHAGOCYTOSIS: 11.6.1 MECHANISM OF PHAGOCYTOSIS:
“It is the process used by cells to engulf and Phagocytosis occurs in three steps:
subsequently ingest particles of nutrients or 1. Unbound phagocyte surface
bacteria. This process is very important part of cell receptors do not trigger
function, allowing cells to grab vital nutrients and phagocytosis.
allowing the body to protect itself from harmful 2. Binding of receptor causes
bacteria.” them to cluster.
In 1882, Metchnikoff worked on phagocytes. He 3. Phagocytosis is triggered and
inserted small thorns from garden plant into the star the particle is taken up by the
fish larvae. He noticed that the cell within the larvae phagocyte.
were no longer moving around the aimlessly, but

11.7 ROLE IN IMMUNE SYSTEM

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In the immune system, phagocytosis is a major Maturation of T-lymphocytes occurs in thymus

mechanism used to remove pathogens and cell gland.
debris. T cells are involved in the cell mediated response.
Phagocytes are large white cells that can swallow White B cells are involved in the humoral response.
and digest microbes and other foreign particles
Monocytes are phagocytes that circulate in blood. 11.11 B-LYMPHOCYTES
When monocyte migrate into tissue, they develop
into macrophages. Specialized type of macrophages
11.11.1.1 (H UMORAL I MMUNE R ESPONSE )
can be found in organ, including lungs, kidney, brain,
It is the interaction b/w lymphocyte and phagocytes
and liver
B- Lymphocytes originate in bone marrow, tonsils
Dendritic cells are found in the parts of lymphoid
and gut where they continue to differential. Each B-
organs where T-cell also exists. Like macrophages
cell is genetically programmed to encode a unique
dendritic cell in lymphoid tissue display antigen to T
antigen binding receptor on its membrane. 1 B-cell
cell and help stimulate T cells during an immune 8
has capacity to capture 10 antigenic sites is able to
response. They are called dendritic cells because
make 1000 antibodies.
they have branch like extension that can be interlace
to form a network
Milk has a substance which is analogues to
phagocytes. Fresh healthy milk has no microbes.
Milk suitable for drink is known as patable milk. Milk
has ability of phagocytosis.

11.8 OPSONINS
Antibodies produced by the involvement of
phagocytosis are called opsonins.

11.9 ADAPTIVE IMMUNITY (THIRD LINE OF

DEFENSE)
The third line of defense against pathogenic invasion
is the innate immunity response and it is called
specific immunity because it can be differentiated
into specific micro organism
Adaptive immunity is activated only against invading
foreign material and never against its own molecules
(except in auto-immune disease). Thus it has ability
to distinquish between self and non self.
Adaptive and inmate immunity co-operate to
produce a more effective and have vast defense
mechanism against infectious agent.
There are two types of adaptive immune response
 Humoral Immunity (mediated by antibody
produced by B-lymphocyte)
 Cell mediated immunity (mediated by T-
Lymphocytes)
11.10 LYMPHOCYTES
The third line of defense depends on lymphocytes.
There are two basic type of lymphocyte and both are
produced in bone marrow.
When B-cells come in contact with an antigen it
multiplies and differential into two types of cells.
 Plasma cells (that are for immediate use)
 Memory Cells (That are for future use)
11.11.2 PLASMA CELLS
Plasma cells produce large number of antibodies.
The antibody molecule is large poly-functional
glycoprotein found in the blood and other tissue
fluid. They travel in the blood stream, distributing
throughout the lymph nodes, spleen and tonsils.
Once these B-cells reach their destination, they
remain inactive until they encounter a foreign cell
with an antigen that matches their particular
receptor site.
11.11.3 MEMORY CELLS
Unlike plasma cells which last for a few days,
memory cells have a longer life. Memory cells
express some membrane bound antibodies
molecules.

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Memory cells are functionally inactive unless they
are stimulated by the same antigen again.
11.12 ANTIGEN –ANTIBODY COMPLEX
It includes:
 The pathogen clumping together makes
them more vulnerable to phagocytes.
 The antibody “tags” the bacteria when it
sinks to it, making it more easily
recognizable to phagocytes.
Any antigen acting as toxin in your body are
neutralized when antibody stick to it i.e. antibodies
can act as antitoxins. In a similar way, if a virus has
an antibody attach to it, it will no longer be able to
attach or enter a host cell.
11.13 T – LYMPHOCYTES
11.13.1.1 C ELL M EDIATED R ESPONSE
T – Lymphocytes are also produced in bone marrow
but they move to thymus for maturation. In the
blood, t –cell make up 60-70% of peripheral
lymphocytes. These are also found in lymph nodes
and spleen.
11.13.2 FUNCTIONS:
 T-cells are cytotoxic cells and are toxic to
invader (foreign particle).
 They directly kill the antigen by producing a
product called interleukin
 They will release the chemical to dissolve
the capsule which helps the phagocytosis of
capsulated microbes
 It only targets the specific and invader and
does not harm the other cells
 They also kill tumor cells. These cell
confiner or sealed such clusters and
dissolve such cells.
 It can also eliminate our own body cell that
acquired antigenic determinants
 It can also cause autoimmune disorders.
11.14 FACTOR AFFECTING OUR IMMUNITY
 Smoking is the factors which affects and
disturb our immunity.
 Radiations are also harmful and weaken the
defensive system
 Cyclosporine promotes the survival of
microbes and excessive use of steroid also
weaken the defensive system
Immunology 80
 The excessive dose of drug weakens the
defensive system and promote microbial
product which ultimately lead to death.
11.15 GROUPS OF T-CELLS
These are as follow
11.15.1.1 K ILLER T C ELLS
The combine with the antigen on the surface of any
invading cell and release a powerful group of
chemical called lymphokines.
11.15.1.2 L YMPHOKINES
They attack with macrophages, thereby increase the
phagocytic activity.
 Migration inhibition factor (MIF). There will
be increase in phagocytosis by creating a
special type of macrophages known as
angry macrophages.
 Migration activator factor (MAF) cause large
lysosomal granules which have increase
capacity of killing
 Specific macrophages arming factor (SMAF)
they have increased killing capacity.
11.15.1.3 L YMPHOTOXIN
 These are tumor necrosis factor. It can even
destroy the potential pathogen
 They don’t need the previous exposure of
antigen.
 They are present in the blood stream and
protect the body specially rom viral
infection and tumor formation
 They can also detect the abnormal cells and
lyzed them by inserting a pure fermentation
protein “perforins” into the membrane of
microbe (puncturing the microbe).
11.15.1.4 H ELPER T C ELL
Regulatory system of immune response by means of
Helper T cells.
Helper cells has two approaches
 Increase the anitbodies production
 Regulates the production i.e. prevent over
reaction that causes allergic reactions.
11.15.1.5 S UPPRESSOR T-C ELLS
It keep the immune system in check so that once the
antigens have been dealt with the system is switch
off.
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 Immunology 81

11.15.1.6 M EMORY T-C ELLS These cells are naturally present in the body. They
Remain after the pathogens have been killed to stop can also increase the quantity of interference.
re-infection.
11.16 IMMUNITY
11.15.1.7 N ULL C ELLS
It is the state of having sufficient biological defenses
There are certain cells which are neither A nor B
to neutralize infection, disease or other unwanted
such are called lymphocytes.
microbial invasion. It is the capacity of the body to
resist the harmful microbes from entering it.
11.15.1.8 N ATURAL K ILLER CELL (NKC)

This is granted immunity to some race contrary to it.

11.17 COMPONENTS OF IMMUNITY
 Innate immunity (non-specific resistance)
 Adaptive Immunity (specific resistance)
11.17.1 INNATE IMMUNITY
This mechanism is active right from the time a child
is born. Specificity of innate immunity is relatively
low as it lacks the ability to distinguish one microbe
from the other. It provides early lines of defense
against microbes.
It is supposed or assumed to be absolute natural or
racial. Example:
 Algerian sheep are 100% resistant to the
disease anthrax
 Negros is 100% resistant to yellow fever.
They don’t have specific antibody but y
nature they are resistant to this disease.
They are certain races which are sensitive to some
particular diseases. Examples:
 Swiss mice are 100% sensitive /susceptible
to yellow fever.
 European people are sensitive to
tuberculosis (TB).
11.17.2 ACQUIRED IMMUNITY
 It is called specific immunity or third line of
defense.
 Adaptive immunity is activated only against
invading foreign material and never its own
molecules (except in the auto-immune
disease). Thus it has the ability to
distinguish between self and non self.
 The production of antibodies depends upon
the entrance of micro-organism.
 One week is required to produce antibody
for a specific disease

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11.18 NATURAL ACQUIRED IMMUNITY
11.18.1 ACTIVE IMMUNITY
 Immunity is resistance to attack by micro-
organisms.
 Immunity depends on the presence in the
serum of the immune individual of modified
gamma-globulins known as antibodies
 Natural immunity is the usual consequence
of infection. Either overt or in apparent. The
presence of invading microbes within the
tissue of the host stimulates the production
of antibody which not only eliminates the
infection but provides subsequent
immunity to a further infection by some
species of microbes.
 Since the immunity which follows infection
is a response of the host to the invading
microbes it is known as active immunity.
 Example: Typhoid and Malaria etc.
11.18.2 PASSIVE IMMUNITY
 Antibodies produces as a consequence of
infection are transferred during pregnancy
across the placenta from mother to fetus so
that a newborn infant is immune to that
infection to which the mother is immune.
However, since there is no production of
antibody by the infant this form of natural
immunity is known as passive immunity
 Colostrum also contain antibody. Mother
feed milk to the baby.
11.19 ARTIFICIAL ACQUIRED IMMUNITY
11.19.1 ACTIVE IMMUNITY
 Artificial immunity like natural immunity
may be either active or passive
 Active artificial immunity is the immunity
which develops as a consequence of
vaccination following successful
vaccination, the presence of vaccine in the
tissue of the host stimulates the production
of antibody in much the same way as
natural infection.
 Toxoids are also administered which are
treated or weaken antigen. That increases
the production of antibody.
 They are prepared by heating toxins at 56’C
for about 1 hour.
 E.g. Vaccine and toxoids
11.19.2 PASSIVE IMMUNITY
Immunology 82
 When body is failed to produce antibodies
then artificial passive immunity is given. The
injection of antibody-containing serum
obtained from animal induces a state of
artificial passive immunity and it is on this
technique that serum prophylaxis and
therapy are based.
 E.g. Anti-toxins
11.20 REGULATION OF IMMUNITY
A basic principle of immunology is that lymphocytes
respond to foreign antigens but tolerate self-tissues.
For developing T-cells, the ability to distinguish self
from non-self is acquired in the thymus, where the
majority of self-reactive cells are eliminated.
Recently, however, it has become apparent that
some self-reactive T-cell avoids being destroyed and
instead differentiates into specialized regulatory
cells. This appear to be beneficial.
The activated T-cells include regulatory T cells by
setting off the antibody production when antigen
has been eliminated. This is also done by other cells
Helper T cells i.e. they regulate the immune
response. They initiate the humoral immunity.
Hence, the excessive reaction between antigen and
antibody can be dangerous.
11.21 PREPARATION OF ANTIBODIES
Inject the toxic material to the neck of horse in
jugular vein. This process is specially for the
diphtheria caused by the corynebacterium
diphtheriae.
5ml of toxin/toxiod inject into the horse. Then after
several days inject 25, 50, 125, 250, 500ml. There
would be accumulation and maximum quantity of
antibodies.
rd
Take blood from horse add 1/3 of saturated
solution of the ammonium sulphate and then
become precipitate of euglobin which is false
antibodies filtered and wasted. Again repeated the
same procedure with 0.5% saturated solution. Then
washed, suspended washed with chlorinated water
and used
11.22 THEORIES OF ANTIBODY FORMATION
11.23 SIDE CHAIN THEORY
Paul Ehlrich believed that toxin or antigenic material
is composed of two parts
 Heptohoric portion
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 Toxophoric portion
Elrich in 1900 postulated that each cell has on its
surface a series of side chains or receptors that
function by attaching to certain food molecules
while each side chains interact with other molecules
such as disease causing toxins (antigens) produces
by infectious agent. The toxin attaches with receptor
with their antigenic portion known as heptophoses
and this interaction is irreversible and blocks
subsequent binding and uptake of nutrients. The
body then tries to overwhelm the obstruction by
producing a great number of replacement side
chains so that they cannot fit on the surface of the
cell and instead are secreted into the circulation.
According tot him these circulating side chains are
the antibodies which are able to neutralize the
disease causing toxin.
Antigenic material can be administered into the
body to increase the antibody production by
removing toxophoric portion.
11.24 TEMPLATE THEORY / INSTRUCTIVE
THEORY
Linus Pauling spearheaded instructional theories
which proposed that antigen forming a
complementary molecule which would neutralize
similar antigen molecules in the future.
This theory explain specificity and diversity
It did not explains how the body recognized self from
non-self as a blank template would be blind;
memory. Since subsequent responses to particular
antigens are exponentially higher and faster than in
the initial encounter.
11.25 NATURAL SELECTION THEORY
Natural Selection Theory given by Niels K. Jerne in
1955 is stated as follow:
The role of antigen is neither that of a template not
that of an enzyme modifier. The antigen is solely a
selective carrier of spontaneously circulating
antibody to a system of cells which can reproduce
this antibody. Globulins molecules are continuously
being synthesized in an enormous variety of
different configuration.
Among the population of circulating globulin
molecule there will spontaneously be fraction
possessing affinity towards any antigen tow which
the animal can respond. These are so called natural
antibodies.
The introduction of an antigen into the lymph leads
to the selective attachment to the antigen surface of
Immunology 83
those globulin molecules which happen to have
complementary configuration. The antigen carrying
the molecules may be engulfed by phagocytic cell.
When the globulin molecule thus brought into a cell
have been dissociated from the surface of the
antigen the antigen has accomplished its role and
can be eliminated.
11.26 CLONAL SELECTION THEORY
Antibodies are also produce by special identical cells
called clone i.e. aggregation of antibodies
Antibodies are highly specific and used to treat only
specific disease
This is given by Burnet
The main features are as follow:
 The phenomena of immunity is based on
the presence in the body of clones of
immunologically competent cells which can
react directly with the corresponding
antigen and can give rise to cells which can
synthesize and liberate antibody
 During embryonic life, those clones which
can reaction with antigenic determinants in
the body are eliminated. In this way, the
information needed to differentiate self
from non-self is corporate into the body.
11.27 UNITARIAN CONCEPT OF ANTIBODY
According to this concept in immunology, a single
pure antigen will produce only one variety of
antibodies which when brought into contact with
the antigen in appropriate form can react in various
ways i.e.
 Agglutination
 Precipitation
 Opsonization
 Complement fixation
Detection of the disease or the presence of antibody
by different methods this is called Unitarian concept
of antibodies.
11.27.1 AGGLUTINATION
When antibody combine with antigen it forms
clumps, these thread like structures are called
agglutinin and the process is called agglutination
11.27.2 PRECIPITATION
Instead of microbes, its antigenic portion is used,
which this antigenic portion combine with antibody
it form precipitate the antibody is called precipitin
and the process is called precipitation
11.27.3 OPSONIZATION
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Antibodies coat microbes and promote their
ingestion by phagocytes. The process of coating
particles for subsequent phagocytosis is called
opsonization
11.27.3.1 O PSONIN
The antibody production during phagocytosis is
called opsonins
11.27.4 COMPLEMENT FIXATION
An immune response in which an antigen-antibody
combination inactivates a complement so it is
unable to participate in second antigen-antibody
combination.
Absence of compliment in patient serum indicates
the presence of disease because in diseased person
11.28.1 ANTIGEN BINDING
Participation in effector function depending upon
the physical properties of antibody.
There are two antigen binding fragment and single
crystallizable fragment on antibody which possess
the physical properties of the molecules. Only
crystallizable fragment has been shown to fix
complement
Biochemical analysis demonstrated that every
monomeric antibody molecule consists of:
 Four protein chains, two heavy and two
lights linked to each other by disulphide
bond
 Heavy chains identify the class of antibody
(G, A, M, E, or D)
 Each chain has constant region (C) and
variable (V) region
 Antigen reacts to variable region
 The variable region can change in response
to the antigen
Immunology 84
compliment is already used. The antibody in this
case is called complement fixing antibody and the
test is called complement fixing test.
It is the basis for various diagnostic tests to detect
the presence of a specific antigen or antibody.
11.28 STRUCTURE OF ANTIBODY
Antibodies are glycoprotein bivalent molecules also
known as immunoglobulin (Ig) is a large Y shape
protein produced by plasma cell that is used by the
immune system to identify and neutralize foreign
objects such as bacteria and viruses. All antibodies
have two basic functions
 The macrophage introduces antigen to the
variable region
11.29 TYPES OF ANTIBODY
Immunoglobulin are further classified into five
classes/ types
11.29.1.1 IGM
 First immunoglobulin to appear after
exposure to antigen
 Indicates new infection
 Activates complement
 Big, cannot cross placenta
 Naturally occurring IGM, against A and B
blood antigens
11.29.1.2 I G G (G AMMAGLOBULIN )
 These are most common
 Small in molecular weight
 Only produced in large amount during
secondary immune response
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 Crosses the placenta  Usually found in tissue attached to mast

 Act as an opsonins (coats the bacteria so, it cells
is easily phagocytized)  Mediates allergy reactions
 Important in fighting parasites
11.29.1.3 I G A ( ALPHA )  Systemic release can cause anaphylactic
 Found in secretion (mucous, milk eyes, shock
sweat etc.)
 Offers primary protection 11.29.1.5 I G D ( DELTA )
 Is protected from digestive enzymes  Found on B cells
 Is proceeded by IgM  Helps the B cells become antigenically
active
11.29.1.4 I G E (E PSILON )
 Only a small amount serum

Agglutinins
They cause clumping of bacterial cells for which they
11.30 FUNCTIONAL NAMES OF ANTIBODIES are specific

Antibodies are all produces as a result of antigenic 11.30.1.2 P RECIPITINS

stimuli. They are humoral antibodies an can be They causes the precipitation of extracts of bacterial
differentiated as: cells or other antigens

11.30.1.1 A NTITOXIN 11.30.1.3 L YSINS

They neutralize toxins

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They cause the lysis of bacterial or other cells that
are specifically sensitive to their actions
11.30.1.4 C OMPLEMENT F IXING
A NTIBODIES
These antibodies participate in complement fixation
reactions
11.30.1.5 O PSONINS
They render micro-organism more susceptible to
ingestion by phagocytes
11.31 ANTIGEN-ANTIBODY REACTION
The reaction between antigen and antibody occur in
three stages
 In first stage the reaction involves the
formation of antigen-antibody complex
 The second stage lead to visible event like
precipitation, agglutination etc.
 The thirds stage includes destruction of
antigen or its neutralization
Ag+ AB  Ag –Abs complex
11.32 GRIFFITH EXPERIMENT:
He gave the concept of transformation. Initial
experiment was performed by Griffith in England.
1. He injected the culture of rough colony into
the mice, mice became immunized.
2. He injected the culture of smooth colony
into another mice result the mice is died.
3. He injected the filtrate(heated culture) of
rd
smooth colony in immunized and 3 mice
immunized mice is died because non
capsulated bacteria converted into
rd
capsulated bacteria and 3 remain healthy
because culture did not contain any cellular
particle.
Transformation occurs naturally among few
genera of bacteria, including Bacillus,
Haemophilus and certain strains of
Streptococcus and Staphylococcus.
11.33 AGGLUTINATION TEST
A blood test used to identify unknown antigens;
blood with the unknown antigen is mixed with
known antibody, agglutination may or may not occur
and help to identify the antigen used in tissue
matching blood grouping and diagnosis of infection
Immunology 86
It is relatively easy to perform simple to interrupt
and is serological method of choice when suitable
cellular antigen can be prepared:
Agglutination test are classified as
11.34 SCREENING TEST
11.34.1.1 M ACROSCOPIC A GGLUTINATION
T EST
A drop of bacterial suspension one drop of NaCl and
1-3 drops of patient serum are taken in test tube and
Streptococcus pyogenes act as normal
transient flora but if enters blood causes
disease. Its virulent power is capsule.
Capsulated bacteria can be converted to
non-capsulated and vice versa. This
concept was named as transformation
by Griffth. Gram +ve bacteria can be
converted to Gram –ve but gram –ve
cannot be transformed to gram +ve.
mixed t. Within 3-5 minutes the microbial cells starts
clumping. Such type of test are called macroscopic
agglutination test.
11.34.1.2 M ICROSCOPIC AGGLUTINATION
T EST
A drop of bacterial suspension is placed on drop of
NaCl and 1-3 drops of patient serum are placed on
cavity slide for 1 hour. The reaction occurs and
clumps formed then observe it under microscope.
This is the rapid method to determine the presence
of agglutinating antibodies.
Measuring the agglutination of particular antigen by
its specific antibody is the simplest way to estimate
the quantity of that antibody in serum
Agglutination test is a diagnostic value for typhoid
fever, salmonellosis, brucellosis, thypus fever and
Rocky Mountain spotted fever.
11.35 WIDAL’S TEST
A test of blood serum that uses an agglutination
reaction to diagnose typhoid fever and other
salmonella infections.
This test is particularly used in the diagnosis of
typhoid fever by agglutinating typhoid bacilli with
the patient’s serum but the term is loosely applied to
other agglutination test using heat or killed cultures
of organism other than salmonella typhi.
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 Immunology 87

The extent of agglutination indicated the extent of 11.38 PRECIPITATION REACTIONS

disease
In this reactions only metabolites/wastes are taken
which are soluble and in solution form.
11.36 WEIL FELIX REACTION It is also used for blood grouping but it is very
It is used as diagnostic test for rickettsia diseases. important in order to detect very small quantity as
Fortunately, the bacteria Proteus vulgaris is highly some time it is observed for medico legal
antigenically similar to rickettsia which very requirements. It is used for the diagnosis of
dangerous. These proteus species are not very Syphilis..it is also used for the typing of hemolytic
dangerous and not cause any disease in human and strains of streptococci. It is also used for the
are rather beneficial. So serum from patient with detection of food adulteration.
rickettsia infections agglutinates suspensions of 11.38.1 RING TEST
proteus organisms This consists of layer of antigen solution over a
The most commonly used strains of proteus are OX- column of antiserum in a narrow tube. it is the
19, OX-2 and OX-K simplest type of precipitation test. A precipitation
This test is differential for certain rickettsia diseases forms at junction of two liquids.
because of selective agglutination of different strains
of Proteus species. 11.38.1.1 C LINICAL A PPLICATIONS
OX19 OX2 OXK Ascoli’s test(suspension of Bacillus anthraces is
prepared and injected in healthy rabbits. 1 or 2g
+++ + - Typhus spleen of deed organism is removed and boiled for
Fever 5-10min in saline water)
++ ++ - Rocky The grouping of streptococci by Lancefield
technique.
mountain
spotted
fever
- - +++ Tsu Tsu
Gamushi
Fever
- - - Rickettsial
pox Fever
The agglutination reactions are also used to
determine the blood groups and to diagnose syphilis.
11.38.2 SINGLE DIFFUSION IN ONE
11.37 TUBE AGGLUTINATION TEST DIMENSION (OUDIN’ S TEST )
Antibody is incorporated in agar gel in test tube.
This is a standard method for quantitative
Antigen solution is then layered over it. The antigen
estimation of antibodies. The serum containing
is diffuse downward through agar gel and whatever
antibody is diluted serially with saline in several
it reaches in optimum concentration with antibody,
small test tube to which the constant volume of
a line of precipitation is formed. The line of
antigen suspension is added.
precipitation moves downward as more antigens
Maximum agglutination occurs in those test tubes
diffuse. The number of bands indicates the number
that contain equal number of antigens and
of different types of antigens present.
antibodies.
A control tube is kept which has no antiserum. The 11.38.3 DOUBLE DIFFUSION IN ONE
tubes are incubated until visible agglutination in DIMENSION (OAKLEY AND FULTHROPE
diluted serum of antibodies shows the severity of MODIFICATION)
disease and activeness of microbe.

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 Immunology 88

The antibody is incorporated in gel in a test tube, Some related organism such as those of genus
above which is placed a column of plain agar and rickettsia, Salmonella, or Shigella may contain
antigen is layered on surface of this. Antibody and common antigens that cause several species or
antigen diffuse towards each other and form a band strains to agglutinate with anti-serum prepared from
of precipitate where they meet at optimum a specific species or variety of group. Hence they can
proportion be differentiated.
For Example
Antigen 1 Antigen
2
ABCDE EFGHI
Abs: abcde Abs:
efghi
“e” is the correspondence antibody , which is
common in both.

11.40 COMPLEMENT FIXATION TEST

Complement fixation test are based on the presence
of complement-fixing antibodies in serum. These
antibodies are produced in an animal body
stimulated by bacterial antigens.
Complement fixation is classic method for
demonstrating the presence of antibody in patient
serum. In the actual test, two systems are involved
 Bacteriolytic/Cytolytic/ Amboceptor/
Complement Fixation System
 Hemolytic/ Indicator System

11.38.3.1 C LINICAL USES 11.40.1.1 B ACTERIOLYTIC C OMPLEMENT

To quantitate serum immunoglobulin, complement
FIXING S YSTEM
proteins this method is commonly used.
In this system serum bacterial suspension and
For screening sera for antibodies to influenza
complement are united/ mixed/ if the antigen and
viruses.
antibody in the serum are capable of union, the
11.38.4 ELICK’S MODIFICATION complement is said to be fixed.
Dip the fiter paper in suspension and a place it on an
agar medium. 11.40.1.2 H EMMOLYTIC / I NDICATOR
S YSTEM
 The complement fixation test consist of two
components. The first component is an
indicator system that uses combination of
sheep red blood cells, complement fixing
antibody such as immunoglobulin G
produced against the sheep RBCs in the
rabbit and an exogenous source of
complement. When these elemnts are
mixed in optimum condition, the anti-sheep
antibody bind on the surface of RBC’s cell.
 Complement subsequently bind to this
antigen antibody complex formed and will
cause the RBCs to lyse and this indicate no
11.39 AGGLUTINATION ABSORPTION disease is present. Lysis of the indicator
TECHNIQUE sheep RBC’s is cell signified both lack of

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antibody in patient serum and negative
complement fixation test.
11.40.1.2.1 N EGATIVE F IXATION TEST :-
Ag +? + C’→ serum (anti sheep RBCs prepared by
inject the RBCs into rabbit)→blood(sheep
RBCs)→haemolysis→ disease –ve
 The second component is known as antigen
and patient serum added to a suspension of
sheep RBC’s in addition to complement.
THses two components of the complement
fixation method are tested in sequence.
Patient serum is first added to the known
antigen and complement is added to the
solution. If the serum contains antibody to
the antigen, the resulting antigen-antibody
complex will bind all of the complement.
Sheep RBC’s and the anti-sheep antibody
are then added. If complement has not
been bound by an antigen-antibody
complex formed from the patient serum
and known antigens, it is avaialbel to bind
to the indicator system of sheep cells and
antisheep antibody.
 If the patient’s serum does contain a
complement fixing antibody, a positive
result will be indicated by the lack of RBC’s
lysis.
11.40.1.2.2 P OSITIVE C OMPLEMENT FIXATION
T EST
Ag +Abs + C’→ serum (anti sheep RBCs prepared by
inject the RBCs into rabbit)→blood(sheep RBCs)→no
haemolysis → disease +ve.
11.41 WASSERMANN’S TEST FOR SYPHILIS
Immunology 89
Wassermann test for syphilis is one of the best
applications of component fixation test. In this
instead of using the causative microorganism i.e.
Treponema pallidum, artificial antigenic material is
used which heptophorical resemble the causative
agent. The artificial antigenic material is made by
extracting beef heart powder with ether and alcohol.
This extract contains a complex phospholipid called
cardiolipin. Cholesterol and Lecithin; they are added
as an electrolyte.
Since the material is not capable of inducing
antibody formation in an animal it is not a true
antigen, but because it combines with syphilitic
antibodies in the complement fixationtest, it is
reffered to as “non-specific antigen”
The antibody formed in response to cardiolipin is
called “regain”
11.42 KAHN TEST
He modified Wassermaann Test which is cheap,
simpler and faster. Instead of using of Beaf heart he
used alcoholic extract of beef eart muscle in which
cholesterol is added. The serum is made inactivated
by heating it at 56’C for about 30 minutes and is
mixed with known amount of antigen.
Incubation period for Wassermann test is 2 days
while for Kahn’s test is just 30 minutes.
11.43 KLEIN TEST
Klein further modified the technique instead of using
petri dish he used slide. The formation of
microscopic precipitate in the mixture of patient’s
serum and an antigen on the slide give faster result
and indicate the syphilic condition
Ag + Abs + C’  Serum (antisheep RBCs)  Blood
(sheep RBCs)
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 Immunology 90

Sometimes our own tissue even organ of the body

becomes foreigner and the disease will not identified
11.44 AUTO IMMUNITY by using biopsy technique. Microbe is taken and
auto vaccine is made. Sample of patient is used
The resistance produce as a result of auto vaccine is instead of pure culture. This is time consuming.
called auto immunity

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Sample(patient) -Heat Attenuated Microbe 
Auto Vaccine
Vaccine made from the patient serum is called auto
vaccine.
11.45 ELISA TEST (ENZYME LINKED
IMMUNE ABSORBENT TEST)
Elisa test was first described Enguall and Permann in
1971. Applied for microbiological diagnosis by Voller
and his co-worker for AIDs, rubella virus and certain
condition resulted by drug.
The enzyme linked immunosorbent assay (ELISA) is a
common serological test for the presence of
particular antigen or antibody.
Enzyme activity in it is the measure of quantity of
antigen or antibody present in test sample
Antigen and antibodies are detachable at levels of
-10
about 10 g/ml or 1 part in 10 billion for immune
assays.
This test is used for the detection of HIV and
Hepatitis with great success.
There are two method of this assay
11.45.1 DOUBLE ANTIBODY SANDWICH
METHOD (DIRECT METHOD)
 In this method, specific monoclonal
antibody is first attached on to the walls of
microtiter plate.
 A suspension of serum or other fluid is
added to the well to test for the presence of
a complementary antigen.
 If the antigen is present in the specimen it
will bind to the antibodies that are attached
to the walls of well.
 Binding of the antigen to antibody is strong
enough to withstand washing that removes
the test fluid and unbound antigen.
 After washing to remove unbound antigen,
another aliquot of the monoclonal antibody
is added to the well. IN this antibodies are
modified so that they carry enzyme
conjugate i.e. peroxidase. It is designed to
produce color change when the enzyme
reacts with its substrate.
 The sample is again washed. If the antigen
is present, a complex is formed that
Immunology 91
includes the antibody bound to the well,
the antigen and the enzyme conjugated
antibody.
 If antigen is not present the enzyme bound
antibody will have been washed away. The
enzyme substrate i.e. H2O2 is now added.
 A color change reveals the presence of
enzyme labeled antibody as well as it bound
antigen
 No color change indicates that antigen was
not present in the test fluid- a negative test
result.
11.45.2 INDIRECT METHOD
 This indirect ELISA test is one that
determines whether a specific antibody
(e.g. HIV antibody) is present in a sample
such as serum. In this case the appropriate
antigen is first attached to the wall of a
microtiter plate.
 Serum that must contain antibody against
the antigen is added to the well.
 If the specific antibodies are present in the
sample they will bind to the antigen
attached to the well
 Washing removes any antibodies that do
not specifically attach to the antigen
absorbed to the well.
 If antibodies do attach to the antigen they
can be detected by the addition of an
antibody-enzyme conjugate. As the serum
contains human antibodies due to this
enzyme conjugated anti human IgG
antibodies are used,
 If the antibody is present in the serum it will
bind to the attached antigen and enzyme
conjugated antibody will bind to this
complex. Washing is again done to remove
any unbound antibodies
 The substrate for the enzyme is added
 A colour change indicates that serum
contains antibodies that react against the
original antigen which indicates the positive
result.
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11.46 SOME OTHER IMPORTANT
SEROLOGICAL TEST
11.47 FLUORESCENT ANTIBODY
TECHNIQUE
11.47.1.1 (I NDIRECT IMMUNOCHEMICAL
T ECHNIQUE )
 Fluorescent antibody technique is used to
identify specific microorganism (antigen).
 In this technique known antibody is
tagged with fluorochrome dye
(fluorescein – isothicocynate) that will
combine with its homologues antigen
(corresponding). This mixture is placed on
a slide and observed by dark field
microscopy. If the organism is present it
will appear bright,
 However if this preparation is observed
with the fluorescence microscopy by
using UV light the antigen coated with
antibody will be visible.
11.48 NEUFLED QUELLUNG REACTION
 Quellung is the German word meaning
“Swelling”
 It is the biochemical reaction in which ant
capsular antibodies binds to the bacterial
capsule which causes the capsule to swell
and become larger. This reaction is
performed with only those species of
bacteria that forms capsule like
Streptococcus pneumoniae, Escherichia
coli and salmonella etc.
 The antibody reaction allows these
species to be visualized under
microscope.
 A loop full of organism is teased on the
slide along with specific antiserum and
methylene blue.
 The dye stains the body of the cell and
the surrounding fluid, leaving the capsule
clear. If the reaction is positive the
capsule becomes opaque and appears to
enlarge
 Pneumococci can be differentiated into at
least 75 known capsular serotypes on the
basis of polysaccharides in their capsular
material
11.49 COAGULASE TEST
Immunology 93
 Ability of the organism to clot the plasma
cell of the human body or rabbit from the
patient.
 This test is used for Staphylococcus
aureus. Diluted blood is mixed with a
broth culture of Staphylococcus aureus
and is incubated for 2 hours.
 Clotting indicates the presence of enzyme
i.e. coagulase which makes the bacteria
more virulent.
11.50 HEAMAGGLUTINATION TEST
 The clumping or clustering of RBC’s
caused by certain viruses’ antibodies or
other substances.
 Certain viruses have the ability to
agglutinate RBC’s from certain species of
animal like chicken guinea pig or human
type O red blood cells.
 Serial dilution of the virus antigen are
made then RBCs suspension is added in
each tube and the tubes are incubated for
1 hour
 Positive test is indicated when
agglutination occurs in bottom of tube. In
negative test, agglutination does not take
place.
11.51 HEMAGLLUTINATION INHIBITION
TEST
In the case of influenza or other viruses which
cause hemagglutination an increase in the
antibody which inhibits the agglutination, indicates
the presence of virus of the type of antigen
employed.
11.52 INTRA-CUTANEOUS TEST
11.52.1 DICK’S TEST
This is the method of determining the
susceptibility of scarlet fever by injection into the
3
skin of 0.1 cm of scarlet fever toxin. Reddening of
skin within 24 hours indicates lack of immunity to
the disease.
11.52.2 SHICK’S TEST
This method of determining susceptibility to
diphtheria by injection of small amount (0.1 ml) of
diluted diphtheria toxin intradermal into the arm
of person. The reddening of skin indicates lack of
immunity to the disease.
11.52.3 TUBERCULIN TEST
Tuberculin is an extract of Mycobacterium
tuberculosis that is used in skin testing in animals
and human to identify a tuberculosis infection
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 Immunology 94

Several types of tuberculin have been used for

which are as follow:

 P URIFIED P ROTEIN D ERIVATIVE (PPD)

It is the most important. It is a complex
mixture of antigens. Test based upon PPD
are relatively unspecific since many of its
proteins are found in different
mycobacterial species
Dose of PPD is 0.002 mg/ml

 O LD T UBERCULIN T EST
The virulence of microbe is decrease by
making them old or diluted thorough
serial dilution. It is diluted in the ratio of
1:10,000. It is checked after 48 hours.

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 Sterilization 95

12.5 INFECTION
Chapter 12 STERILIZATION Once bacteria establish, multiply and occupy a
part, it does not allow other microbe to grow there
12.1 STERILIZATION and use your body metabolites causing damage
and is called infection
“A process through which a substrate or an article
is made free from all life, by removal of all types of
microbes.”
12.6
OR “Blood get septic” e.g. Streptococcus enters blood
“Removal of all microbes from a material/ due to any cut inbuccal cavity and form capsule.
substrate/ article”
12.7 INFESTATION
12.2 PROCESS OF STERILIZATION Presence of microbes on or inside the body
“Any attempt towards sterilization, is considered Trillion of microbes is carried by an individual.
13
as sterilization” Body has 10 cells and bacterial/ microbes cell it
14
carries is about 10 .
12.3 STERILE
“A condition in which no microbial life is retained.”
12.8 INFLAMMATION
OR Redness, hotness, swelling, pain, immobilization at
“A condition, area or system which is free from the site of infection as a sign of infection.
microbes”
12.9 ASEPTIC
12.4 SEPTIC “Free from contamination caused by harmful
Enterance of microbe on a material/ substrate or bacteria, viruses, or other microorganisms;
article” surgically sterile or sterilized.”
Septicemia

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12.10 METHODS
12.11 PHYSICAL METHODS:
12.12 PASTEURIZATION:
“Pasteurization is a process of heating food, which
is usually a liquid, to a specific temperature for a
predefined length of time and then immediately
cooling it after it is removedfrom the heat.”
(Yeah, it’s from Wikipedia)
It is named after microbiologist Louis Pasteur for
his work on his pasteurization process in 1864.
This process slows spoilage caused by microbial
growth in the food.
It can be done in the following methods:
 Low temperature holding method: Heat
o
at 62 C for 15-30 minutes
 High temperature holding method: Heat
o
at 72 C for 15 seconds
 Heated at Ultra-high temperature of
o
149 C for 2 seconds
The key to the process of pasteurization is that
each and every particle is exposed to the desired
temperature to enable proper sterilization. This is
why the process is known as holding.
Advantages of High Temp Method (High
temperature short time method)
 Mycobacterium  Killed
 Spore forming  Killed
 Phospitases (enzyme)  destroyed
Presence of phospitase indicates improper
pasteurization
Disadvantages: Less shelf life of 1 day at room
temperature and 2-3 days in freezer.
12.13 PASTEURIZATION OF MILK
Milk is sterilized by pasteurization, it is tested with
the presence of mycobacterium tuberculosis which
is the indicator bacteria of milk.
To ensure proper heating, the milk should be
passed in a thin opening.
The enzyme Phosphatasecan also work as an
indicator for the confirmation of the process of
pasteurization.
12.14 HISTORIC SITUATION: “THE MILK
MENACE”
Although pasteurization was a good process to
sterilize milk, the problem was still there for the
shelf life of milk. The scientists wanted milk to
have its nutritional value even after sterilization.
Pasteur’s death was an obstacle. These were dark
times in the milk business. Scientists worked on
Sterilization 96
devising methods to sterilize milk and keep its
nutritional integrity.
Holland tried it,but the results were not what the
world needed.
Nowadays, a method was devised to heat milk at
o
149 C for 2 seconds, and then immediate cooling.
It maintain the nutrition value even the
temperature is above 100’C but the time 2
seconds is too less for the milk materials to get
denatured.
12.15 TYNDALLIZATION
This method was devised by the scientist named
“Tyndal”.
This method can also be done in two ways:
 Heat 80 C for 5 consecutive days
o
(repeatedly) for 1 Hour
 Heat 100 C for 3 consecutive days
o
 The process itself takes about 30min-1hr,
however it is repeated again and again up
to duration of 5 days. It is incubated so
that the spores formed are germinated.
This is because when these spore forms
are converted into vegetative form, they
become thermo-labile, and are removed
when heated again.
 Repetition of the process ensures the
removal of all spore forms.
 This method is not feasible for substances
which become denatured above 100 C,
o
e.g. milk.
 If water is heated for 100’C, all the
microbes are kill but some salts are also
destroyed.
 Water should also be store in earthen
pot.
 Boiling water efficacy can be improved by
2% Na bicarbonate. For substrate Na
Bicarbonate is used while for apparatus is
Na carbonate.
12.16 BOILING
Boil at 100C in autoclave. 2% sodium carbonate is
added in article. Sodium bicarbonate may be
added. It removes the gas and increase the
permeability of penetration of water into semi
permeable membrane of microbe and kills it.
12.17 SCREENING OF MILK
 Milk does not contain preservative, and is
excellent media for microbial growth so it
must be free from pathogenic microbes.
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 Sterilization 97

 Add methylene blue (EMB agar) in milk minutes

which inhibit all gram positive bacteria
15 lb 121’C 15
and convert in methylene white.
 When methylene blue is added to milk, it minutes
turns blue. (What a shocker). Methylene 20 lb 126’C 10
blue gets converted into methylene minutes
white, the higher the amount of
microbes, the faster this conversion Another table: Temperature of
takes, and amount of microbes can be Steam under Pressure
estimated. 2 o
Steam Pressure (Lb/in ) Temperature ( C)

12.18 PHOSPHATASE TEST 0 100.0

 Raw milk + Saturated solution of 5 109.0
phenolphthalein DiPO4. The enzyme in 10 115.0
milk converts the solution into 15 121.5
Phenolphthalein and DiNa 20 126.5
paranitrophenylPO4. The steam pressure of 5 is used in pressure
 The free phenolphthalein is mixed with cookers.
NaOH/NH3 indicator, it gives blue colour.
The intensity of this colour indicates 12.22 PRECAUTIONS FOR AUTOCLAVING
amount of enzyme in milk.
 The air must be removed to remove
 Ideally, enzyme should not be there due
pressure
to pasteurization. So this is a test to see
 Autoclave should be used when
the degree of success of the process of o
temperature above 100 C is needed
pasteurization.
 In order to maintain time and pressure
relationship air must be removed from
12.19 METHYLENE BLUE REDUCTION autoclave.
METHOD
 Milk requires O/R (oxidation-reduction) 12.23 MISCELLANEOUS
potential of +300 millivolt.  Death of microbes in moist heat occurs by
 Milk + Aq. solution of Methylene blue coagulation
converts the colour of milk into blue.  Virion particles take two hours in the
 When the O/R potential decreases, the autoclave to be killed
colour changes from blue to white. It  All carbohydrates are to be sterilized
means that milk attains its original colour separately and the aseptically mixed
after a given period of time.  Carbohydrates get disintegrated at high
 Less time requires for colour to change temp so avoid high temp by maintaining
means high bacterial count. 10 lb pressure
 Bacillus stereothermophillus is a heat
12.20 GRADING OF MILK resistant bacteria and is controlled by
autoclaving
Grade Raw milk Pasteurized milk
5 4
A 2x10 3x10 12.24 DRY HEAT
6 4
B 1x10 5x10 The meaning of dry heat, is the exposure of the
C ∞ ∞ article directly to a flame.
This process is called incineration. (Burning) when
using wire loop in this method, spattering should
12.21 AUTOCLAVE be avoided.
In the oven, this method can be done according to
The autoclaving method can be employed at the
the following temperature and time combinations:
following corresponding temperature and time
combinations: Temperature Time
Pressure Temperature Time 160C 2 Hr
10 lb 115’C 20 180C 1.5 Hr

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 Sterilization 98

200C 1Hr 5 10 90=90% 999,990

6 1 9=90% 999,999
 No water containing media can be placed
o
in an oven (since it boils at 100 C). So we
use a liquid which has a higher boiling The table shows that after every minute, 90% of
point. Eg: oil. the microbes are killed and 10% survive. and in 6
minutes, only one microbe is left alive, and the
 Microbes are killed by oxidation in oven.
rest of the 999,999 are dead. The last one commits

o
The starting temperature of oven is 120 C
suicide because it gets lonely. Just kidding.Let’s
have a moment of silence for all the lost lives of
12.25 DECIMAL DEATH RATE the microbes.
Death rate of microbes should be a minimum of
90% at every phase. The survival will be 10%. This 12.27 THERMAL DEATH TIME
is the criteria for heating (every minute).
“The shortest period of time to kill a suspension of
bacteria (or spores) at a prescribed temperature
12.26 DECIMAL REDUCTION TIME and under specific conditions.”
(Simple definition): “The time required (in
minutes) to reduce the population by 90%” 12.28 THERMAL DEATH POINT
(Fancy definition): “The time required for
“The lowest temperature at which microbes are
thermal death curve to pass through one log cycle”
killed in a given time.”
The following table can provide an example for
It is mostly 10 mins. As mentioned in some books.
decimal reduction time:
Time Survivors Deaths per Total
unit time deaths 12.29 REDUCTION TIME FOR MOIST
0 1,000,000 0 0 HEATING
1 1,00,000 900,000=90% 900,000 The table shows reduction time for some bacterial
species interrelated with different temperatures.
2 10,000 90,000=90% 990,000
12.29.1
3 1000 9000=90% 999,000
DESTRUCTION TIMES (MINUTES)
4 100 900=90% 999,900
o o o o o o o o
Organism 100 C 105 C 110 C 115 C 120 C 125 C 130 C 134 C

Bacillus Anthracis 2-15 5-10

Bacillus subtilus Many 40

hours
Clostridium tetani 5-90 5-25

Clostridium welchii 5-45 5-27 10-15 4 1

Clostridium botulinum 300-530 40-120 32-90 10-40 4-20

Soil bacteria Many 420 120 15 6-30 4 1.5-10

hours
Clostridium sporogenes 150 45 12

12.30 REDUCTION TIME FOR DRY

HEATING
o o o o o o o
Organism 120 C 130 C 140 C 150 C 160 C 170 C 180 C

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Bacillus anthracis
Clostridium botulinum 120
Clostridium welchii 50 15-35
Clostridium tetani 20-40
From these tables it can be concluded that the
temperature and reduction time are indirectly
related.
A graph between time and temperature.
Thermal death time curve for spores of a
bacterium encountered in food spoilage.
Temperature and time have indirect relation.
12.31 FACTORS AFFECTING
STERILIZATION
1. Susceptibility of microbes
The susceptibility can play a role in the process of
sterilization. Some microbes are resistant and
some are more susceptible then others.
Eg: SarcinaLuteais highly susceptible and because
of this it is used to test products.
2. Counts/no of microbial cells
The amount of microbes can alter the time and
process of sterilization. Its only logical that higher
the number of microbes, the longer the process
would take.
Initial inoculum: It is the amount of microbe
initially inoculated. It works as a standard.
3. Time of exposure
Self-explanatory…more temperature means less
reduction time…less temperature means more
reduction time
4. Dose of the agents
Sterilization 99
Upto 180 60-120 9-90
60 15-60 25 20-25 10-15 5-10
5
5-15 30 12 5 1
5.Environmental conditions
example:air,temperature etc.
The last point says “environmental conditions”, so
what is the environment basically…
Well…
Environment: “Everything except you and me is
environment”
Yes, that is the definition of environment. Just a bit
of philosophy in it.
An easily understandable definition would be “The
surroundings of an individual including all the
biotic and abiotic factors are called the
environment”.
12.32 STERILIZATION BY RADIATION
 U.V radiation
 Ionizing radiation
 Energy transmitted through space in a
variety of forms is known as radiation
 Radiation refers to any form of radiant
emissions or divergence, as of energy in
all directions from radioactive elements
12.32.1.1 N ON -I ONIZING RADIATION
 Non-ionizing radiation possesses a
distinct wavelength much longer than
that of ionizing radiation
 UV light has anti-microbial, anti-bacterial
but less anti-fungal actions
 UV light, radiation from germicidal lamp is
useful only for surface and transparent
objects
 UV light can cause permanent damage to
DNA of exposed cell
 Vaccines and anti-biotic can be sterilized
by UV light or radiation
 Dust particles absorb radiation
12.32.1.2 I ONIZING R ADIATIONS
 The ionizing radiations normally possess a
wavelength shorter in comparison to non-
ionizing radiation (<1nm) eg: ¥rays, X-rays
etc. the energy of these rays is more than
about 10eV and they are called ionizing
radiations.
 Sterilization by radiation is called Cold
sterilization, because there is no heat
involved.
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 Sterilization 100

60
 Cobalt is mostly used for radiation The bacteria which were considered or deemed
sterilization. It has a half-life of 5 years. dead previously have recovered and are in a live
 Every anti-body has a half-life. state again. Hence, photo-reactivation
 Gamma rays are generated by cobalt OMG Zombie bacteria!!
rods, which are immersed in water within
a protected region of the radiation 12.33 CONTROL OF MICROORGANISMS
facility. Materials to be sterilized are
loaded onto a monorail conveyor system Organism Median
and automatically transported past the lethal dose
radiation source. (rads)
 Cesium-137 is used in some facilities, Viruses Tobacco mosaic 200,000
radiation is effective if it can generate Rabbit papilloma 100,000
peroxidase.
 Radappertization is a form of food Bacteria Escherichia coli 5000
irradiation which applies a dose of
Bacillus 130,000
ionizing radiation sufficient to reduce the
mesentricus
number and activity of viable
Algae Mesotenium 8500
microorganisms.The dose is 4.5-5.6
megarods.Radappertization is derived Pandorina 4000
from the combination of radiation
Protozoa Colpidium 330,000
andNicholas Appert, the name of the
French scientist and engineer who Paramecium 300,000
invented sterilized food for the troops of
Vertebrates Goldfish 750
Napoleon.
 Gamma rays are important source for Mouse 450
preservation of food. Eg: potato
 Bacillus thuringiensis, also called Rabbit 800
crystalofarusbacterio, is a spore and Rat 600
crystal forming bacteria. However, it is
not harmful to humans. The relevant Monkey 450
thing to the topic here is that it can humans 400
resist150 Krads. And In case you may
have forgotten, it’s a biological
insecticide. A “rad” (radiation absorbed dose) is the dose
 Serratiamarcescens, which is a non-spore which delivers 100 ergs/g of irradiated material. It
13
forming bacteria, can resist 30 Krads. is equal to 6x10 eV.
 soil also has radioactive properties A “rad” is also abbreviated as “rd”, wow I was able
 half-life of measles is very long to save so much time by removing the “a” part in
 our body also emits radiations, people of rad and making it just rd.
Gilgit emits more radiations because their
food contains radioactive elements 12.34 LETHAL DOSES OF DIFFERENT
 Mutants are present amongst us (just RADIATIONS
something I wrote on my register coz I
was bored) Type of Cathode Gamma X-rays
12.32.2 PHOTOREACTIVATION organism rays rays From
The word “photoreactivation” means repairing of From Van From cobalt 3-MEV
a DNA. In this case, it’s the repairment of the DNA de Graff 60 source
of bacteria. The procedure is following: accelerators
 Expose to U.V light Vegetative
 Immediately count the amount of Non- 0.1-0.25
microbe who survived pathogenic
 Expose them to visible light right after Pathogenic 0.45-0.55 0.15-0.25 0.03-
exposure to UV light 0.5
 Then finally count Bacterial 0.5-2.1 1.5(approx.) 0.5-
spores 2.0
Molds 0.25-1.15 0.2-0.3 0.25-

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 Sterilization 101

1.0 It is made of powder. Eg:

Borosilicate glass
yeasts 0.5-1.0 0.3 0.25-
 Micro-porous plastic or membrane filter:
1.5
Made of cellulose ester, or
the acetate or the nitrate
12.35 MECHANICAL STERILIZATION  Berkefield:
Has great pore density and
12.36 FILTRATION is more permeable then milipore, hence
they are recommended for aqueous
(Don’t need to write the definition here….right?) it preparation.
is the typical example of mechanical sterilization. Various types of filters use two methods:
The standard pore size for the filter is 0.45µm. But,
 Pressure
it is not viable for some sneaky little microbes, so
 Vacuum
for them we have 0.22 µm pore size.
Milk cannot be sterilized by this method, its
actually because of its protiens.
12.37 TYPES OF FILTER Filterization cannot be used to sterilize viruses
 Ceramic filter from a substance, because of their small sizes
It is porous porcelain
 Fibrous filter 12.38 SONIC WAVES
It is made of Sonic waves are also another way of
asbestos/cellulose sterilization
 Sintered glass filter  Microwaves are not meant for
sterilization.

This is a sort of a summary for all the physical

12.38.1 CONTROL OF methods. If you get the question to write abt the
MICROORGANISMS physical methods of sterilization, this would be a
smart way to attempt the question.
Method Recommended drugs Limitations

Moist heat

Sterilizing instruments, Ineffective against organisms in

Autoclave lines,utensils,treatment trays,
media and other liquids
Free flowing steam or boiling Destruction of non-spore forming
water pathogens, sanitizes clothing and
materials impervious to steam
Cannot be used for
heat-sensitive articles
Cannot guarantee to produce
sterilization on one exposure

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 Sterilization 102

dishes
Dry heat

Sterilizing materials impermeable Destructive to materials which

Hot-air oven to or damaged by moisture, e.g. cannot withstand high
oils ,glass ,metals temperatures for long periods
Disposal of contaminated objects Size of incinerator must be
Incineration that cannot be reused adequate to burn largest load
promptly and completely
Potential of air pollution
Radiation

Control of airborne infection, Must be absorbed to be effective

disinfection of surfaces (does not pass though opaque
Ultraviolet light objects), irritating to eyes and
skin; low penetrations

X-ray, gamma, and cathode Sterilization of heat-sensitive Expensive and requires special
radiation surgical materials and other facilities for use
medical devices
Filtration/ sedimentation

Membrane filters Sterilization of heat-sensitive Fluid must be relatively free of

biological fluids suspended particulate matter
Fiberglass filters (HEPA) Air disinfection expensive

Effective in decontaminating Not effective alone, but as adjunct

Sonic waves/Ultrasonic delicate cleaning instruments procedure enhances effectiveness
of other materials
Hands, skin and objects Sanitizes, reduces microbial flora
Washing

 Paul Ehrlich introduced the anti-biotic

molecule as the “Magic bullet”,that can
12.39 CHEMICAL METHODS OF destroy the organism in the tissues
without harming the tissue. There are
STERILIZATION also certain dyes having anit-microbial
It consists of chemotherapeutic agents, qualities
antibiotics and sulpha drugs.  There are some chemicals with anit-
microbial properties, they are called
12.40 CHEMOTHERAPEUTIC AGENTS Chemical knife. These medications were
used in surgery and were used very
The chemical substances used within the body for carefully, hence called the chemical
therapeutic purposes and synthesized by the knifes. (people in history also had a talent
chemist which is usually a modification of pre- for naming things)
existing chemicals.  Penicillin was initially introduced by a 21
year old French boy, named Duchesne in
12.41 ANTIBIOTICS 1896. (Interesting). The three important
Anti-biotics are obtained from living things, and scientists who worked for penicillin are
they also act on living things to control them. Fleming, Flori, and Chain. All three of
them received Nobel prize in 1945.
However, Fleming Is widely credited with
12.42 IMPORTANT HISTORY the discovery of penicillin. He was
Time to switch on the time machine, and go into working on Streptococcusaureus when he
the past. discovered penicillin. There were spaces

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 Sterilization 103

in the petri dishes of the culture, these The drug is combined with trimethoprim which
vacant spots were due to the growth of inhibits another step in folic acid synthesis. The
penicillin. That’s how he came to discover name of this drug is Bactrim. It is used for urinary
it. and intestinal infections, and pneumocytic
 SahachiroHata (not a name of microbe) pneumonia.
performed research on chemical control Isoniazide (INH) Isonicotonic acid hydrazide:
of syphilis. (syphilis seems to be a very It is an anti-tuberculosis drug. When resistance
popular disease in history). He found out develops, INH is combined with rifampin and
that there were some compounds such as ethambitol. These drugs are then absorbed into
Ars-phenol. (The ars is for arsenic). They the body and they show their anti-tuberculosis
had anti-microbial action, effect. Normally, it is hard to find such a specific
eg: Ars-phenamine. It was called drug.
compound #606. (they had twitter back Nalidixic acid:
then?). It is a compound from the quinolone group. It
The compound was modified and inhibits and blocks DNA synthesis in gram-ve
converted into a modern therapeutic bacteria, which are responsible for causing urinary
agent and then called Salvarsan. A dye of infection.
red colour was added known as Prontosil. The synthetic nalidixic acid is used in form of fluro
quinolone which is used against shigella,
 A heart touching story “daddys little girl”. salmonella, gonnorhea.
Once upon a time there was a scientist Typical example of quinolone group is
named Domagk. He had a daughter. One Ciprofloxasin.
day she fell gravely ill. The poor soul was Flagyl: it is an amoebic drug
suffering from septicemia. Her father Chloroquin: It eliminates malarial parasite inside
tried everything but nothing worked. At the blood cell
last, in 1935, when Domagk was working Primaquin: It eliminates malarial parasite outside
on prontosil, he injected the dyes in his the blood cell
daughter and in a miracle, she recovered. Para amino Salicylic acid (PAS):
And they lived happily ever after, They act as therapeutic agents, are used in
probably because he received a Nobel combinations.
prize for his work in 1939. Lucky guy he Dapsone:
was. Diamidophenylsulphone, used to prevent leprosy.
 Research started on the dye. The active
principle of prontosil was first isolated by 12.43 CHEMICAL ANTI-
Jacques and Trefouel in the form of
sulphonamide.
SEPTICS/DISINFECTANTS :
 it was first synthesized by “Glemo” in 1. Phenol
1908, and it was extensively used in world 2. Alcohols
war 2. 3. Dyes
 In 1940, Wood and Fildsused certain 4. Iodine
sulphonamide to interfere with the 5. Chlorine
growth of bacteria without tissue 6. Quaternary ammoniated compounds
damage, this was because of competitive 7. Soaps/detergents
inhibition. 8. Heavy metals
This ends the history portion. Back to the future! 9. Others: Fumigants, Formaldehyde
The human body lacks in folic acid, so it must be 10. Gases
consumed in food. In the production of folic acid, High level Germicides: Ethylene oxide,
bacterial enzymes join together 3 important Glutaraldehyde
components. Eg: Para amino benzoic acid (PABA), Intermediate Germicides: 2% Glutaraldehyde: not
which acts similar to sulphonamide. Thus, the kill spores
modern sulphonamides are classified by Low level Germicides: Limited to certain kind of
sulphamethazole which is used basically for bacteria
urinary tract infection. It is caused by gram-
vecocci. (Neisseriameningitits). 12.44 PHENOL
Bactrim:
It was discovered by Joseph Lister. It can be used
as anti-septic, disinfectant,

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germicidal,bacteriocidal and almost all the forms.
It kills vegetative cells. it can damage nervous
system.
It has cresol, and Lysol. Bis-phenol is a
combination of phenol. It is taken as standard
disinfectant.
12.44.1.1 P HENOL CO - EFFICIENT TEST
Phenol co-efficient: Def: “It is a ratio of the
effectiveness of a given or new disinfectant, which
can kill the given microbe in a proper
concentration, within 10 minutes, but not in 5
minutes, compared to the effectiveness of the
phenol against the same microbe under same
conditions.” (Believe me, all of that is part of the
definition)
Phenol is being considered the standard here, and
the 5 minute limit is there for our own safety,
“warna microbe humarekaylagte hen”
Phenol however, is taken as a standard
because it is the nearest to an ideal
disinfectant.
12.45 ALCOHOLS
It is usually ethyl alcohol 70%. It causes death of
microbes by coagulation. It is used to disinfect
thermoemters.
Isopropanol is used to reduce microbes on skin.
12.46 DYES
Methylene Blue is an example
12.47 IODINE
Strong Iodine solution can be an anti-dote to a
snake bite. (I have no idea why this statement is
written in this topic). Iodine solution cannot
replace phenol because Iodine burns skin.
It can decontaminate water and wounds.
A compound, iodophore is analgesic, however,
iodine is not.
12.48 CHLORINE
 Direct quote from the professor
“behtreen germicidal”. They can
decontaminate water and wounds.
 A compound of chlorine called
Hypochlorous acid can kill virion. It used
against E.coli, Salmonella typhi and
Entamoebahistolytica. It is a very
effective oxidative agent.
 The slow poisoning effect of
hypochlorous acid is less than chlorine.
 A group of compounds called
hypochlorides can eradicate HIV.
Sterilization 104
 Chlorohexidine can be used for gram +ve
and –ve organisms, even for fungi, viruses
and bacteria without spores. They do not
however, show any action on spore
formers.
 Chlorohexidine interferes with plasma
membrane permeability. They are used as
anti-septic lotions.
12.49 QUATERNARY AMMONIATED
COMPOUNDS
 CetylPyridinium Chloride: cationic
surfactant +ve, wetting agents to remove
oil
 Zephiran: To control Cyst protozoa
 Phemerol
12.50 ETHYLENE OXIDE TREATMENT
(ETO)
 Most of these products are sterilized by
ETO. Ethylene oxide treatment. Infect,
60% of products are sterilized by ETO.
This treatment is not changeable with
oven or incubator.
 Ethylene Oxide (ETO) sterilization is
mainly used to sterilize medical and
pharmaceutical products that cannot
support conventional high temperature
steam sterilization - such as devices that
incorporate electronic components,
plastic packaging or plastic containers.
o
 The temperature is kept at 60 C, and the
humidity is kept at 30.
 It is lightly toxic and explosive, which is
reduced by mixture with Freon in
cryoxide or 90%CO2 in carboxide.
 Also known as Gas-autoclave. It is used to
sterilize paper, leather,wood,rubber and
petri dishes.
 It is basically used for plastic (heat-labile)
for upto 4 hours.
 Care should be taken in humid conditions,
may cause cold burns.
 It is also used to sterilize
catheters,artificial heart valves, heart-
lung machine components and optical
equipment.
 ETO can however, damage nucleic acid
and proteins, it is microcidal and
spermicidal.
 The NASA uses the gas for sterilization of
interplanetary space capsule.
 A similar closely related but less explosive
gas is β-propionolactone.(BPL)
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 Sterilization 105

12.51 SOAPS AND DETERGENTS 12.54.1.6 H OMOGENIZED

The substance should have a single
Heavy metals: Readily bindedprotiens homogenized phase.

12.52 MERCURIC CHLORIDE 12.54.1.7 S TABLE

 Silver nitrate combined with silver The substance should stay stable. Sometimes,
chloride. AgNO3 eye drops given to new when agents act on substances, they change
born babies. stability.
 Used to prevent eye infection
 Anti-bacterial, Anti-protozoan, Anti 12.54.1.8 C HEAP ( LOW COST )
fungal, Anti-virus It should be cheap…do we really need a
reason for that?
By The Way: No chemical is considered as an
12.53 OTHERS ideal disinfectant.
Formaldehyde and Ethylene oxide are used for
fumigation 12.55 PRESERVATION
When we talk about sterilization, why do we do it?
12.54 IDEAL DISINFECTANT What is the most important application of
An ideal disinfectant is capable of adequate: sterilization? Maintaining stability of drugs and
preserving food.
12.54.1.1 P ENETRATION We want to preserve food, pharmaceutical
It acts on substance. It can enter microbial cell products, cosmetics and all the stuff like that.
in substrate Even the sterilized product may contain some
preservatives. This is because upon first usage, the
12.54.1.2 A VAILABILITY sterility of the product is lost, and the
It should be easily available preservatives help in maintaining the sterility.
The preservatives are used to increase the shelf
12.54.1.3 P OWER OF DETERGENT ( NON - life.
IRRITATING AND NON - STAINING ) 12.55.1 EXAMPLES:
It should be powerful enough to control and  0.1% Benzoic acid
kill the microbes, or at least stop the  Sodium Benzoate
metabolism on the microbe.  Sorbic acid
 Paraben
12.54.1.4 N ON - TOXIC TO HOST  Phenol
It should know where its loyalties lie. It should  Formaldehyde
not cause any harm to the person taking the  Nitrate or nitrite (for meat products)
drug. It should not have any fatality.  Antibiotics: Oxytetracycline (disintegrates
Eg: formaldehyde causes irritation. (according when cooking is done so an acceptable
to the professors observation, it also causes preservative)
weight loss).  Silver nitrate (in ophthalmic soultions)
Iodine causes burn on skin and gives color .
 Just a reminder for the reader…”yehi din
12.54.1.5 T OXIC TO MICROBES hen ketchup banana ke”
It should be selected as such that it acts on a 12.55.2 PRESERVATION BY
specific microbe which needs to be
LYOPHILIZATION
eliminated.

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12.56 FERMENTATION
The word “fermentation” is a Latin word, and it
means boiling.
Although it’s not actually boiling process, it seems
similar to boiling because there is release of CO 2 in
the form of bubbles. Eg: cheese has holes due to
these bubbles
It has four definitions:
Sterilization 106
DEF-1: “Any metabolic reaction caused by
microbes is called fermentation” (in an industry)
DEF-2: “Fermentation is a process of anaerobic
oxidation”
DEF-3: “It Is a condition where final electron
receptor of a catabolic pathway is an organic
compound.”
DEF-4: “If an organic compound is electron donor
as well as final electron acceptor, the metabolic
process is called fermentation”
Eg: pyruvate is an excellent electron receptor
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12.57 HARNESSING MICROBIAL
METABOLISM
 (Ok, before we start this, there is a lot of
stuff under this heading, and to be
honest, I don’t know what all of it means.
It seems to be connected, but not sure if
it really is, however, it is easy so here we
go. )
 In order to dispose waste materials,
biocoil reactor is used. It is also called a
Fermenter.
 0.1N hydrochloric acid is put in the
fermenter, and different cultures of
microbes. Its maintained in this state so
the “sewage” is separated. Sewage gets
converted into protein. These protiens
are made into animal feed.
 Cold water is run around the sides of the
tank to keep it cool.
 Also, some compounds are produced
which generate electricity when they are
burnt out.
 1 million tons of human and animal hair
are used for this every year. It also mostly
contains feathers form poultry. These
substances mostly contain keratin. They
Sterilization 107
are difficult to digest, when placed in bio
digester, so a typical bacterium is used .
This special bacterium is
Bacilluslicheniformis. It acts on feathers
and methane (CH4) is released. The
common name of methane is Green gas.
It is released with other digested masses.
These masses are then made into feed for
livestock.
 Methane is also produced by
fermentation as an end product, in
stomach chambers of cattle.
 Cattle release 100 million ton methane
each year. Under certain conditions, they
cannot use the pathway to produce
methane, or the lack them altogether. So
their cells dissemble glucose by
fermentation to generate ATP. By
glycolysis, 6 carbon sugars split into
molecules of pyruvic acid, accompanied
with energy molecules.
 Saccharomyces cerivaceae (yeast)
converts pyruvic acid to CO2 and free
carbon as the end product for glycolysis.
 Acetaldehyde is reduced to alcohol.
Acetobacter degrades alcohol to vinegar.
 (remember those two dudes? The French
one and the muslim one. one wanted to
make vinegar but kep getting alcohol. The
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 Sterilization 108

other “Pasteur” wanted alcohol but kept  Mannose

getting vinegar. Come to think of it, they Disaccharides:
could have done some very lucrative  Lactose
trading, or just sat together and done  Sucrose
some tequila shots…ok im getting off  Maltose
topic here) Trisaccharides:
 Propionobacterium extract energy from  Raffinose
lactic acid, and convert it into propionic Polysaccharides:
acid, acetate and CO2.  Starch
 Propionic acid contributes the  Glycogen
characteristic flavor of cheese. CO2  Dextrin
produced in the form of bubbles makes Polyhydric alcohols:
holes in cheese. The end product of  Sorbitol
microbial fermentation can produce very  Mannitol
important and economical products like  Glycerol
lactic acid and formic acid. Glycosides:
 Fermentation generates organic end  Salicin
products, some are edible, with more
 Coniferin
desirable flavor/texture, and are easily
 Aesculin
digestible, eg: soya sauce, types of
Some acids:
vinegar.
 Citrate
 Cattle possess microbial flora that can
 Gluconate
help in digestion of plant diet. Bacteria,
 Melonate
protozoa and others reside in anaerobic
chamber where the microbes can break
up simple sugars and ferment their 12.59 INDICATORS TO DETECT PROCESS
products. Eg: acetic acid, propionic acid, OF FERMENTATION
butyric acids.
 The dairy products of milk can be The following are some of the indicators used:
produced by various species of 1. Andrade’s indicator: (0.5%)
lactobacilli, streptococci and It has acid fuchsin. When its added to
thermophillus species, which can also carbohydrate, the colour turns yellow. If the color
produce acetaldehyde and lactic acid. turns pink or red, it means fermentation is taking
place. The intensity of the process is indicated
 Non-dairy products e.g.: soya sauce. It’s
made by Aspergillusoryzai. Soya from pink to red.
2. Bromo-Cresol Purple: (0.2%)
bean+wheat. It is soaked. Sugar is
The colour is purple when carbohydrate is added.
released which is then fermented,
resulting in formation of soya sauce. If fermentation occurs, the colour becomes yellow,
indicating fermentation.
 Lactobacilli is also used in making of soya
3. Bromo-Thymol Blue: (0.2%)
sauce.
The initial colour is blue. The fermentation
indicating colour is yellow.
12.58 APPLICATION OF FERMENTATION 4. Phenol Red: (0.1%) got no idea, probably
The most important application of fermentation is better to just leave it.
to identify bacteria.
Bergey’s manual plays a big role in this process. 12.60 PHARMACEUTICAL PRODUCTS OF
Examples of important carbohydrates used in
fermentation for identification of microbes:
MICROBIAL ORIGIN
Monosaccharides 12.60.1 NOW THE PRODUCTS
(1) Pentoses
 Xylose Product Source Uses/Applicati
 Arabinose s ons
 Rhaminose
(2) Hexoses Calcium
 Glucose Glucon Aspergillusniger Food,
ate cosmetics and
 Fructose
Citric juices etc.

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 Sterilization 109

acid to the concern microbes. Antibiotics must be used

to control the infection and showing inhibition to
the growth of causative agent we can determine
Aspergillusterreus Plastic this effect in-virto by simple lab. Test. Bacterial
Itaconic industry sensitivity to the antibiotics is used for diagnostic
acid Steroids/horm purposes because it is standardized test and
ones therefore, gives most accurate test.
omatostatin In summary, we can determine the most effective
antibiotic to be used in treating the patient, so
Dextra Leuconostocmesenter Blood antibiotics sensitivity test is performed by using a
n oides plasma culture obtain from the patient and study its
absorbent sensitivity against various antibiotics.
Stabilizer Antibiotic kill bacteria in several ways e.g.
Xantha Xanthomonascompest Emulsifier penicillin inhibits the synthesis of cell wall, while
ns ris streptomycin completes with PABA (Para-amino
benzoic acid) as substrate for an enzyme reaction.
Filters The drug enter the reaction in place of PABA
Cellulos Acetobacterxylinum Fibre products thereby blocking the synthesis of essential cell
e component
Growth- Some broad spectrum antibiotics interfere with
Amino- Corynebacteriumgluta factors enzyme synthesis
acids micum Food-
suppliments 12.62 DISC METHOD
additives
The death of bacteria/ microbes is manifested by
175000 tons of organic acid annualy…mostly citric zone of inhibition around the antibiotics in
acid impinged disc. Diameter of disc is about 6 to 8
Vaccine production (tobacco plant). The tobacco mm, so if the zone of inhibition has diameter less
plant can be used to make vaccines. than 10mm it is consider negligible.
As the quantity increase the size of injected
12.61 ANTIBIOTICS antibiotic also increases which are name
Antibiotics are different from disinfectant or accordingly as:
antiseptic as these are biosynthesized which refer  Disc (Disk) Method
to the production of a substance from a living  Ditch Plate Method
organism, these substances are of no use to the o By making sharp end of
microbe that produce them so we can also call Cork/Glass borer
them by product of microbial metabolism.  Well Plate Method
The important genera that produce the largest o Greater in depth than ditch
number of antibiotics and useful for human  Cup Plate Method
 Bacillus A growth factor is a vital requirement without
 Penicillium which there is no growth, these factors cannot be
 Streptomyces synthesized by the microbes themselves during
Antibiotics are also different from disinfectant in a the entire process of their life; thus these factors
way of that these are administered internally and must be supplied from outside into the substrate
travel via blood stream to most part of the body. or media. This is the principal of microbiological
This mean the antibiotics must have less toxicity to assay.
the host cell while it should be toxic or destructive

12.62.1 ANTIBIOTICS Amphotaricin-B Streptomyces noduses

Various anti-biotics from microbes:

Anti-biotic Microbe Bacitracin Bacillus licheniformis

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Most produced antibiotic in the world is penicillin.

Chlorotetracyclin Streptomyces aureofaciens It also has the most number of derivatives.
e
12.63 APPLICATION
Chloromphenicol Streptomyces venezulae
Streptomycin is available in form of powder
weighing 1g. It solution is made by adding
maximum 5ml of water, which make the dose of
Erythromycin Streptomyces erythraeus
Streptomycin 1000mg/5ml or 200mg/ml. The
solution is serially diluted to obtain 20mg/ml. The
diluted solution is then parallel diluted by adding
Kanamycin Streptomyces kenamyceticus 5ml of water in 5ml of the 20mg/ml dose to obtain
10mg/ml. Similarly 5mg, 2.5mg and 1.25 mg
parallel dilution are made.
Neomycin Streptomyces fradiae These dilutions are subjected to Ditch plate, well
or cup plate method, to get the zone of inhibition
at different dose.
Mystatin Streptomyces noursei A graph is plotted which help us indicate which
dose is suitable for the patient.
By the help of graph we can also find out the
Oxytetracycline Streptomyces rimosus unknown dose of any drug. This is known as
microbial assay.

Fumagallin Aspergillus fumigatus

Penicillin griseofulvin(10,000
Griseofulvin fold)
Penicillin urtica
Penicillinni garicus

Penicillin Penicillin chrysogenum

Polymyxin-B Bacillus polymyxa

Dechlorination/hydrogenatio
Tetracycline n of chlorotetracycloneby
direct fermentation of
chlorinated medicines

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Chapter 13 MANUFACTURE
OF ANTIBODIES
13.1 INTRODUCTION
Industrial manufacture of most antibiotics is based
on the large scale production of microorganisms
which convert raw materials into antibiotics. This
process is commonly referred to as fermentation.
Other much longer-established fermentation
processes include wine and beer making. Strictly
speaking, fermentations are biological processes
occurring in the absence of air (oxygen). However,
the term is now commonly applied to any large-
scale cultivation of microorganisms, whether
aerobic (with oxygen) or anaerobic (without
oxygen). Following production by fermentation,
anti-biotic are recovered, concentrated and
purified by a series of downstream processing
stages. For semi- synthetic antibiotics there will
then be chemical conversion stages to produce the
bulk antibiotic or active pharmaceutical ingredient
(API). The bulk antibiotic (API) is formulated into
the required dosage form: tablets, vials for
injection, solutions, ointments and so on. Stages
up to bulk API are commonly termed primary
manufacture; stages from API to formulated
product are termed secondary manufacture.
13.2 BACKGROUND
Benzylpenicillin (penicillin G, originally just
‘penicillin’) is the first antibiotic to have been
manufactured in bulk. It is still universally
prescribed and is also used as input material for
some semisynthetic antibiotics. Developments
associated with the penicillin fermentation process
have been a significant factor in the development
of modern biotechnology and therefore it is an
appropriate example to illustrate key aspects of
manufacture. Despite the ever-increasing use of
complex instrumentation, the application of
feedback control techniques and the use of
computers, the science of antibiotic fermentation
is still imperfectly developed. Processes are
difficult to optimize and no two apparently
‘identical’ batches will ever be entirely the same.
This is because living cell populations change both
quantitatively and qualitatively throughout the
production cycle and small changes in control
parameters, such as a fluctuation in air pressure or
a power dip, can potentially impact a batch and
the effect may vary dependent upon the age of
the batch. Also there tends to be significant batch
Manufacture of Antibodies 113
to batch variation in the complex nutrients
commonly used in the fermentations.
No single product can exemplify all the important
features of antibiotic manufacture. Benzylpenicillin
is a b-lactam. Brief accounts are given of the
manufacture of two other b-lactams, penicillin V
(phenoxymethylpenicillin) and cephalosporin C.
However, important as the b-lactams are, they are
but one of many families of antibiotics.
Furthermore, most industrial microorganisms used
to make b-lactams are fungi; this is atypical of
antibiotics as a whole where bacteria, particularly
Streptomyces spp., predominate. All the examples
are of batched fermentations, i.e. of processes
where sterile medium in a vessel is inoculated, the
broth fermented for a defined period (usually
hours or days), the tank emptied and the broth
extracted by downstream processing to yield the
antibiotic. During the fermentation, nutrients,
antifoam agents and air are supplied, the pH is
con- trolled and exhaust gases are removed. After
emptying, the tank is cleaned and prepared for a
new batch (‘turned round’). In continuous
fermentations, sterile medium is added to the
fermentation with a balancing withdrawal of broth
for product extraction. This has a number of
advantages pro- viding the system can be run
without contamination. One advantage is long
fermentation runs of many weeks, hence greater
productivity per vessel due to fewer turn rounds.
In continuous culture the growth rate can be held
at an optimum value for product fermentation. It
is therefore suitable for products whose synthesis
is proportional to cell density, but is not generally
an economical process for antibiotic production
where synthesis is not associated with growth and
there are additional concerns about strain
degeneration.
13.3 THE PRODUCTION OF
BENZYLPENICILLIN
13.3.1 THE ORGANISM
The original organism for the production of
penicillin, Penicillium notatum, was isolated by
Fleming in 1926 as a chance contaminant. By 1940,
Florey and Chain produced purified penicillin and
its tremendous curative potential became
apparent. However, the liquid surface culture
techniques necessary for the cultivation of this
obligate aerobe were lengthy, labour-intensive
and prone to contamination. The isolation of a
higher-yielding organ- ism, P. chrysogenum, from
an infected Cantaloupe melon obtained in a
market in Peoria, Illinois, USA, was the key
advance. This organism could be grown in deep
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fermentations in sealed tanks under stirred and
aerated conditions, in vessels as large as 250m3,
and thus started the antibiotic manufacturing
industry. From this one ancestral fungus penicillin
manufacturers evolved production strains by what
became classic strain selection procedures. Large
numbers of isolates were screened to identify
higher-yielding variants, generally after some type
of mutagenic treatment. This cycle of mutation
and selection was repeated many times. The
selected variants can produce over 20g/l of culture
com- pared with the 2mg/l produced by the ‘wild’
strain, especially when fermented on media under
particular control conditions developed in parallel
with the strains. However, there comes a time
when sequential improvements in penicillin
productivity obtained by these standard strain
improvement techniques (physical and chemical
mutagenesis in conjunction with a variety of
selection techniques that apply pressure for high-
yielding variants) become subject to rate-limiting
returns. At first, it is easy to double the ‘titre’ with
each campaign; later in the genealogy even a 5%
improvement would be regarded as excellent.
Recent developments may well prove to have
transformed this situation. Tremendous progress
has been made since the mid-1980s both in the
isolation and manipulation of the biosynthetic
genes in this pathway and in the related routes to
the cephalosporins (via the cephalosporin C-
producing fungus Acremonium chrysogenum) and
the cephamycins (via the cephamycin C-producing
bacterium Streptomyces clavuligerus). Antibiotic
manufacturers can now apply recombinant DNA
technology to the industrial strains of filamentous
microorganisms used to produce b-lactams and
there are real prospects of making genetic changes
that will very significantly increase productivity.
These are discussed further, later in this chapter.
There is plenty of scope for improvement, because
the best current industrial strains and processes
convert little more than 10% of all elemental car-
bon into penicillin. Production strains are stored in
a dormant form by an appropriate culture
preservation technique. Thus, a spore suspension
may be mixed with a sterile, finely divided, inert
support and desiccated. Alternatively, spore
suspensions or vegetative cells can be lyophilized
or stored in liquid nitrogen biostats. All laboratory
operations are carried out in laminar flow cabinets
in rooms in which filtered air is maintained at a
slight positive pressure relative to their outer
environment. Operators wear sterilized clothing
and work aseptically. Antibiotic fermentations are,
of strict necessity, pure culture aseptic processes,
without foreign growth, i.e. contamination by
other microorganisms.
Manufacture of Antibodies 114
13.3.2 INOCULUM PREPARATION
The aim is to develop for the production stage
fermenter a pure inoculum in sufficient volume
and in the fast-growing (logarithmic) phase so that
a high population density is quickly established.
The time taken for each seed stage is measured in
days and decreases as the sequence progresses.
The final inoculum to the production stage is
generally 1–10% of the total volume of the
fermenter. If the fermenter is under-inoculated
there may be an extended lag before growth starts
and the fermentation period will be prolonged.
This is both uneconomic and may result in
degenerative growth which affects performance,
quality and hence also cost. The inoculum stage
media are designed to pro- vide the organism with
all the nutrients required for that stage. Adequate
oxygen is provided in the form of sterile air and
the temperature is controlled at the desired level.
Principal criteria for transfer to the next stage in
the progression are freedom from foreign growth
and growth to a pre-determined cell density.
Typical of fungi, the organism grows as branching
filaments (hyphae) and by the time that the
culture has progressed to the production stage it is
very viscous.
13.3.3 THE FERMENTER
A typical fermenter is a closed, vertical, cylindrical,
stainless steel vessel with convexly dished ends
and of 25–250m3 capacity. Its height is usually two
to three times its diameter.
13.3.3.1 O XYGEN SUPPLY
The penicillin fermentation needs large quantities
of dissolved oxygen, which is supplied as filter-
sterilized air from a compressor. Oxygen is critical
to aerobic processes and its supply is a crucial
aspect of fermenter design and batch control. As
oxygen is poorly soluble in water, steps are taken
to assist its passage into the liquid phase and from
aqueous solution into the cell, the latter being
particularly problematic in viscous broths. One
option is to introduce air into the bottom of the
vessel via a ring ‘sparger’ with multiple small holes
rather than through a single large orifice. This
breaks the air flow into smaller bubbles which
have a greater surface area to volume ratio and
hence greater oxygen transfer. These bubbles lose
oxygen as they rise up the tank and, at the same
time, carbon dioxide diffuses into them. The vessel
is kept under a positive head pressure which
promotes the dissolution of oxygen and in addition
reduces the chances of contamination. Impellers
and baffles assist the transfer of oxygen as well as
helping to achieve the correct blend of shear and
of bulk circulation from the power sup- plied, and
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generally promoting mixing of cells and nutrients.
Impellers are mounted on a rotating vertical stirrer
shaft which is mechanically driven by a powerful
electric motor. This is a major expense as very
large amounts of energy are consumed. Baffles
located at the vessel perimeter further increase
13.3.3.2 T EMPERATURE CONTROL
The production of benzylpenicillin is very sensitive
to temperature. A lot of metabolic heat is
generated and the fermentation temperature has
to be reduced by controlled cooling. This heat
transfer is achieved by circulating chilled water
through banks of pipes inside the vessel (which
also serve as baffles) or through external ‘limpet’
coils on the jacket of the vessel. These coils consist
of continuous lengths of pipe welded in a shallow
spiral round the vessel. This cooling water system
is also used to cool batched medium which has
been sterilized in the vessel prior to its inoculation.
13.3.3.3 D EFOAMING AGENTS AND
INSTRUMENTATION
Microbial cultures may foam when they are
subjected to vigorous mechanical stirring and
aeration. If this foaming is not controlled, culture
is lost by entrainment in the exhaust gases and so
there are systems, often automatic, for detecting
incipient foaming, for temporarily applying
backpressure to contain the culture within the
Manufacture of Antibodies 115
turbulence. There has been considerable research
into novel, energetically more efficient methods of
aeration, and the next generation of fermenters
may include some that are radically different in
design.
vessel and for the aseptic addition of defoaming
agents.
Instrumentation is also fitted to provide a
continuous display of important variables such as
temperature and pH, the power used by the
electric motor, airflow, dissolved oxygen and
exhaust gas analysis. Manual or computer
feedback control can be based either directly on
the signals provided by the probes and sensors or
on derived data calculated from those signals, such
as the respiratory coefficient or the rate of change
of pH. Analysis of exhaust gases can provide
valuable physiological information.
13.3.3.4 M EDIA ADDITIONS
Not all the nutrients required during fermentation
are initially provided in the culture medium. Some
are sterilized separately by batch or continuous
sterilization and then added whilst the
fermentation is in progress, usually via automatic
systems that allow a preset programme of
continuous or discrete aseptic additions. This type
of fermentation is called fed batch.
13.3.3.5 T RANSFER AND SAMPLING SYSTEMS
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Aseptic systems are used to transfer the inoculum
to the vessel, to allow the removal of routine
samples during fermentation, for early harvesting
of aliquots when the vessel becomes full as a
consequence of the media additions and to
transfer the final contents to the extraction plant
when fermentation is complete. Asepsis is assured
by engineering design and by steam, which must
reach all parts of the vessels and associated
pipework. Any pockets of air or rough surfaces
that steam does not penetrate could act as
reservoirs for foreign growth. Sampling is essential
to monitor the amount of growth, the running
levels of key nutrients and the penicillin
concentration. It is necessary also to check that
there has been no contamination by un- wanted
microorganisms.
13.3.3.6 C ONTROL OF THE FERMENTATION
If growth in the fermenter proceeds unchecked at
the rate prevailing in the seed stages, the culture
would become very dense and the available
aeration would no longer be sufficient to maintain
penicillin production. Should oxygen availability
fall below a critical level, benzylpenicillin
biosynthesis is greatly reduced although culture
growth continues. Accordingly, conditions are so
adjusted that fast growth is achieved only until the
cell population has reached the maximum density
that the vessel can support. Further net growth is
constrained by deliberately limiting the supply of a
key nutrient (in practice, a sugar). The cells can
then be stimulated to an ‘overproduction’ of
benzylpenicillin while restricting the amount of
growth and a stable, highly productive cell
population can be sustained.
13.3.4 BATCHED MEDIUM
The medium initially batched into the fermenter is
a complex one but designed only to support the
desired amount of early growth. The principal
nitro- gen source is corn steep liquor (CSL), a by-
product of the maize starch-producing industry.
This material was originally found to be specifically
useful for the penicillin fermentation, but it is
recognized as valuable in many fungal antibiotic
media. Apart from its primary purpose in supplying
cheap and readily available nitrogen, CSL also
contains a useful range of carbon compounds,
such as acids and sugars, together with inorganic
ions and growth factors—in short, it is virtually a
complete growth medium in itself. However, like
some of the fed nutrients, CSL is a complex
nutrient, not chemically defined, derived from
natural products and with significant batch-to-
batch variation. It is therefore one of the reasons
why no two fermentations are ever absolutely
Manufacture of Antibodies 116
identical. In recent years a number of
manufacturers have moved from using these
relatively cheap but chemically undefined complex
nutrients to defined media, thus reducing
variability in their fermentation processes. The
batched medium contains subsidiary nitro- gen
sources and additional essential nutrients such as
calcium (added in the form of chalk to counter the
natural acidity of the CSL), magnesium, sulphate,
phosphate, potassium and trace metals. The
medium is sterilized with steam at or above 120°C
either in the fermenter itself or in ancillary plant,
which may be worked continuously.
13.3.5 FED NUTRIENTS
The sterile medium is stirred and aerated and its
pH and temperature are adjusted to the
appropriate values. It is then inoculated and the
growth phase begins. The initial carbon source is
sufficient in quantity to maintain early growth but
not sufficient to provide the energy that penicillin
production and maintenance of the cell population
need during the rest of the fermentation. Carbon
for these subsequent stages is ‘fed’ continuously in
such a way as to limit net growth. Either sucrose or
glucose is used, possibly as cheaper, impure forms,
such as molasses or starch hydrolysate. As the
concentration of residual sugar in the broth is too
low to measure, the rate of feeding has to be
learned by experience and modified on the basis
of systematic observation. An alternative way of
attaining carbon limitation with- out the
complication of a carefully monitored carbon feed
rate is to supply all the carbohydrate at the outset
as lactose. The rate-limiting hydrolysis of lactose to
hexose is then relied upon to give a steady, slow
feed of assimilable carbohydrate. Originally, all
benzylpenicillin was manufactured using lactose in
this way and some manufacturers still prefer this
technique. Calcium, magnesium, phosphate and
trace metals added initially are usually sufficient to
last throughout the fermentation, but the
microorganisms need further supplies of nitrogen
and sulphur to balance the carbon feed. Fed
nitrogen is often supplied as ammonia gas. The
word ‘balance’ is used quite deliberately; the
whole system is a balanced one. Thus, the carbon
and nitrogen feeds not only satisfy the organisms’
requirements for these elements in the correct
molar ratio, they also maintain an adequate
reserve of ammonium ion and contribute to pH
control, the carbon metabolism being acidogenic
and balanced by the alkalinity of the ammonia.
Sulphate is usually sup- plied in combination with
the sugar feed and, by obtaining the correct ratio,
there is a balanced presentation of sulphate with
an adequate pool of intermediates. The growth
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phase passes rapidly into the antibiotic-production
phase. The optimum pH and temperature for
growth are not those for penicillin production and
there may be changes in the control of these
parameters. The only other event that marks the
onset of the production phase is the addition of
phenylacetic acid (PAA) by continuous feed. All
feeds are sterilized before they are metered in to
the fermenter. Contaminants resistant to the
antibiotic rarely find their way into the fermenter,
but when they do their effects are so damaging
that prevention is of paramount importance. Any
foreign growth has the potential to disrupt the
fermentation process with both quality and
financial implications. For example, a resistant b-
lactamase-producing, fast-growing bacterial
contaminant can destroy the penicillin already
made, as well as consuming nutrients intended for
the fungus, causing loss of pH control and
interfering with the subsequent extraction
process. Hence the key requirement in both design
and operation of all stages of the fermentation is
that aseptic operations be maintained with growth
of the process microorganism but no unwanted
foreign growth by contaminating microorganisms.
13.3.5.1 S TIMULATION BY PAA
Phenylacetic acid (PAA) supplies the side-chain of
benzylpenicillin without PAA, the organisms
synthesize only small quantities of this penicillin.
Indeed, it was the chance presence of phenylacetyl
compounds in CSL (formed by phenylalanine in the
grain by the natural bacterial flora during
processing) that caused it to be established in
early experiments as the best of the cheap
complex nitrogen sources and led to the use of
PAA. Not only does PAA stimulate benzyl- penicillin
biosynthesis but it also suppresses the formation
of other (unwanted) penicillins. High levels of PAA
are, however, toxic and PAA is also expensive. The
feed is controlled to supply an adequate standing
level of PAA without approaching the toxic limit;
the feed is reduced just before the end of the
process so that the amount of unused
(irrecoverable) precursor in the final culture is not
excessive.
13.3.5.2 T ERMINATION
When to stop a fermentation is a very complex
decision and several factors have to be taken into
account. Quite often a manufacturer will find it
appropriate to harvest shortly after the first signs
of a faltering in the efficiency of conversion of the
most costly raw material into antibiotic.
13.3.5.3 E XTRACTION
Manufacture of Antibodies 117
13.3.5.3.1 R EMOVAL OF CELLS
At harvest, the benzylpenicillin is in solution extra-
cellularly, together with a range of other
metabolites and medium constituents. The first
step in downstream processing is to separate the
cells from the liquid broth by techniques such as
filtration, ultrafiltration or centrifugation. This
stage is carried out under conditions that avoid
contamination with b-lactamase-producing
microorganisms which could lead to serious or
total loss of product.
13.3.5.3.2 I SOLATION OF BENZYLPENICILLIN
The next stage is to isolate the benzylpenicillin.
Solvent extraction is the generally accepted
process al- though other methods are available,
including ion-exchange chromatography and
precipitation. In aqueous solution at pH 2–2.5,
there is a high partition coefficient in favour of
certain organic sol- vents such as amyl acetate,
butyl acetate and methyl isobutyl ketone. The
extraction has to be carried out quickly, as
benzylpenicillin is very unstable at these low pH
values. The penicillin is then extracted back into an
aqueous buffer at pH 7.5, the partition coefficient
now being strongly in favour of the aqueous
phase. The solvent is recovered by distillation for
re-use. The benzylpenicillin is then crystallized,
blended and dried. The downstream processing
stages are designed not only to recover the
antibiotic but also to ensure that it is of the
appropriated potency and purity and that this is
achieved by cost-effective processes.
13.3.5.3.3 F URTHER PROCESSING
Benzylpenicillin is produced as various salts ac-
cording to its intended use, whether as an input to
semisynthetic b-lactam antibiotics manufacture or
for clinical use in its own right. The treatment of
the crude penicillin extract varies according to the
objective but involves formation of an appropriate
salt, usually followed by treatment to remove
pyro- gens, and by sterilization. This last is usually
achieved by filtration but pure metal salts of
benzylpenicillin can be safely sterilized by dry heat
if desired. For parenteral use, the antibiotic is
packed in sterile vials as a freeze-dried power
(reconstituted before use) or suspension. For oral
use it is prepared in any of the standard
presentations, such as film- coated tablets.
Searching tests are carried out on a significant
number of random samples of the finished
product to ensure that it satisfies the stringent
quality control requirements for potency, purity,
freedom from pyrogens and sterility.
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13.4 THE PRODUCTION OF PENICILLIN V
By the addition of different acyl donors to the
medium, different penicillins can be biologically
synthesized. For example, penicillin V is made by a
similar process to benzylpenicillin, but with
phenoxyacetic acid as the precursor instead of
PAA. In the biosynthetic pathway, the a-
aminoadipyl side-chain of isopenicillin N is
replaced by a phenoxyacetyl group. The
microorganism is again P. chrysogenum. A
manufacturer may use the same mutant strain to
make both products or may have different
mutants for the two penicillins. Parallel situations
of a single organism producing more than one
natural product occur with other types of
antibiotics; for example strains of Streptomyces
aureofaciens are used for both chlortetracycline
and demethylchlortetra- cycline fermentations.
Like benzylpenicillin, penicillin V is still used in its
own right, but can also be used as a starting
material for the manufacture of semisynthetic
penicillins which cannot be made by direct
fermentation.
13.5 THE PRODUCTION OF
CEPHALOSPORIN C
It is possible to convert penicillin V or benzyl-
penicillin to a cephalosporin by chemical ring
expansion. The first-generation cephalosporin
cephalexin, for example, can be made in this way.
Most cephalosporins used in clinical practice,
however, are semi-synthetics produced from the
fermentation product cephalosporin C. The
ancestral strain of Acremonium chrysogenum (at
that time called Cephalosporium acremonium) was
isolated on the Sardinian coast in 1945 following
an observation that the local sewage outlet into
the sea cleared at a quite remark- able rate.
Developments were slow because the activity was
associated with a number of different types of
compound. Cephalosporin C was first isolated in
1952, but it was a further decade before clinically
useful semi-synthetic cephalosporins be- came
available. The biosynthetic route to cephalosporin
C is identical to that of the penicillins as far as
isopeni- cillin N. The further route to
cephalosporin C is shown below. Note the branch
into a third series of b-lactam drugs, the
cephamycins .
Manufacture of Antibodies 118
The similarities in the routes to the three classes of
antibiotics have facilitated progress in the under-
standing of the underlying molecular genetics.
Most of the genes coding for the relevant enzymes
have been isolated. Modern DNA techniques are
being targeted at rate-limiting biosynthetic steps.
Amplification of gene copy numbers, improving
gene expression efficiencies, transferring genes to
bacterial host organisms and manipulation of
pathways of antibiotic synthesis all have potential
in strain development. However, in the production
of antibiotics, economic benefits from the
application of recombinant DNA technology have
thus far been limited. Manufacturing processes for
cephalosporin C and benzylpenicillin are broadly
similar. In common with many other antibiotic
fermentations, no specific precursor feed is
necessary for cephalosporin C. There is sufficient
acetyl group substrate available from the
organism’s metabolic pool for the terminal
acetyltransferase reaction. The product is
extracted from the culture fluid by adsorption
onto carbon or resins rather than by solvent. This
illustrates an important general point that
antibiotic manufacturing processes differ from one
another much more in their product recovery
stages than in their fermentation stages.
13.6 GOOD MANUFACTURING PRACTICE
(GMP)
All stages of antibiotic manufacture from
fermentation through to finished product are
governed by the code of good manufacturing
practice (GMP), of which quality control is one
aspect. GMP requires that ‘there should be a
comprehensive system, so designed, documented,
implemented and con- trolled, and so furnished
with personnel, equipment and other resources as
to provide assurance that products will
consistently be of a quality appropriate to their
intended use’. Quality control is concerned with
testing the quality of the product by a combination
of in- process and final product testing. However,
that a product meets specification does not
necessarily mean that it is suitable for use. GMP is
about ensuring that quality is built in to all stages
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of the manufacturing process. Basic GMP
requirements outlined in the Medicines Control
Agency Rules and Guidance (the “Orange Guide”)
are as follows:
1 All manufacturing processes should be clearly
defined and be capable of consistently producing
material of the required quality and complying
with specification.
2 Critical steps of manufacturing processes and
significant changes to processes should be
validated.
3 All necessary facilities for GMP should be pro-
vided including:
(a) appropriately qualified and trained personnel;
(b) adequate premises and space;
(c) suitable equipment and services;
(d) correct materials, containers and labels;
(e) approved procedures and instructions; and
(f) suitable storage and transport.
4 Instructions and procedures are written in an
instructional form in clear and unambiguous
language, specifically applicable to the facilities
provided.
5 Operators are trained to carry out procedures
correctly.
Manufacture of Antibodies 119
6 Records are to be made during manufacture
which demonstrate that all the steps required by
the defined procedures have been taken and that
the quantity and quality of the product was as
expected. Any significant deviations are to be fully
recorded and investigated.
7 Records of manufacture and distribution, which
enable the complete history of a batch to be
traced, are retained in a comprehensible and
accessible form.
8 The distribution (wholesaling) of the products
minimizes any risk to quality.
9 A system is available to recall any batch of
product, from sale or supply.
10 All complaints are examined, the cause of
quality defects investigated and appropriate
measures taken in respect of the defective
products and to prevent recurrence. Failure to
comply with current GMP may result in a number
of sanctions from the regulatory authorities, up to
and including recall of product from the
marketplace and withdrawal of manufacturing and
marketing licences. Thus appropriate standards for
the manufacture of antibiotics are monitored and
maintained.
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Chapter 14 FACTORY AND
HOSPITAL HYGIENE
14.1 INTRODUCTION
There are many definitions of quality (see Sharp,
2000). For the purpose of pharmaceutical products
the term quality is usually taken to mean fitness for
purpose. Not only must the product have the de-
sired therapeutic properties; it must also be safe for
administration by the route intended. Some
products such as injections must be sterile, while
others such as oral drugs need not be sterile, but
must be free from pathogens that can be contracted
via the oral route (British Pharmacopoeia, 2003,
Appendix XVI D). A great deal more space in the
literature is dedicated to quality of sterile products,
but this reflects the additional quality assurance
required compared with that for non-sterile
products (Sharp, 2000). Manufacturing is carried out
in both industry and hospitals. In the latter, batches
tend to be much smaller, sometimes only one item,
and the products are stored for much less time,
usually less than 24 hours (Beaney, 2001).
The difficulty in demonstrating quality is that the
tests carried out are designed to detect its absence.
For example, the test for sterility (Chapter 20)
involves taking samples at random and testing for
microorganisms. The absence of microorganisms
only allows an estimate of the statistical probability
that the batch is sterile. Therefore it is important
that a product be manufactured in a suitable
environment by a procedure that minimizes the
possibility of contamination occurring. At the end of
this process the tests can be performed as an
additional measure. The UK Orange Guide (Rules and
Guidance for Pharmaceutical Manufacturers and
Distributors, 2002) emphasizes the fundamental
point that the manufacture of sterile products: must
strictly follow carefully established and validated
methods of preparation and procedure. Sole reliance
for sterility or other quality aspects must not be
placed on any terminal process or finished product
test.
This chapter summarizes measures for the control,
during manufacture, of one important feature of
product quality, the level of microbial
contamination.
Factory and Hospital Hygiene 120
14.2 DEfiNITIONS
Several terms used in industrial and hospital
production must be defined to enable the reader to
follow this chapter. The inter-relationship between
quality assurance, good manufacturing practice,
quality control and in-process control is shown
below:
14.2.1 MANUFACTURE
Manufacture is the complete cycle of production of a
medical product. This cycle includes the acquisition
of all raw materials, their processing into a final
product, and its subsequent packaging and
distribution.
14.2.2 QUALITY ASSURANCE (QA)
This term refers to the sum total of the
arrangements made to ensure that the final product
is of the quality required for its intended purpose. It
consists of good manufacturing practice plus factors
such as original product design and development.
14.2.3 GOOD MANUFACTURING PRACTICE
(GMP)
Good manufacturing practice (GMP) comprises that
part of quality assurance that is aimed at ensuring
the product is consistently manufactured to a quality
appropriate for its intended use. GMP re- quires
that:
(i) the manufacturing process is fully de- fined
before it is commenced; and
(ii) the necessary facilities are provided. In
practice, this means that:
• personnel must be adequately
trained
• suitable premises and equipment
employed
• correct materials used
• approved procedures adopted
Pharmacy Exam Guide

• suitable storage and transport
facilities available
• appropriate records made.
14.2.4 QUALITY CONTROL (QC)
Quality control refers to the part of GMP that
ensures that
(i) at each stage of manufacture the necessary
tests are conducted; and
(ii) the product is not released until it has
passed these tests. An example is the
test for pyrogens applied to sterile
pharmaceutical products.
14.2.5 IN-PROCESS CONTROL
This comprises any test on a product, the
environment or the equipment that is made during
the manufacturing process. An example of this is
testing that an autoclave is functioning correctly
(Gardner & Peel, 1998).
14.3 CONTROL OF MICROBIAL
CONTAMINATION DURING
MANUFACTURE
14.3.1 GENERAL ASPECTS
A pharmaceutical product may become
contaminated by a number of means and at several
points during manufacture. There are several ways
in which this risk can be minimized. Any such
measures require an understanding of the risks
involved.
14.3.2 HAZARD ANALYSIS OF CRITICAL CONTROL
POINTS (HACCP)
Hazard analysis of critical control points (HACCP) has
been widely used in the food industry and is
becoming more commonly used in the
pharmaceutical industry (Jahnke, 1997). HACCP is a
tool for evaluating steps in a manufacturing process.
It provides a structured thought process for GMP.
The seven steps involved are:
1 Analysis and identification of the potential risk
(hazard) represented by each step in the process.
2 Determination of the critical control points (CCP)
where it is necessary to control the hazards.
3 Definition of the limits within which each critical
parameter should be controlled.
4 Establishment (and validation) of in-process
control methods and tests to be used to determine,
for each critical control point, whether or not the
potential hazard is maintained within the defined
limits.
Factory and Hospital Hygiene 121
5 Establishment of corrective measures designed to
correct any out of control situation at each and
every CCP.
6 Confirmation that the HACCP regime (and hence
the manufacturing process adopted) is functioning
as intended.
7 Documentation of the entire system, in terms of
both HACCP steps to be followed, and of the results
obtained.
14.3.3 ENVIRONMENTAL CLEANLINESS AND
HYGIENE
Microorganisms may be transferred to a product
from working surfaces, fixtures and equipment.
Pooled stagnant water is a frequent source of
contamination. Thus it is essential that all working
areas are kept clean, dry and tidy. Any cracks where
microorganisms may accumulate must be
eliminated. All walls, floors and ceilings should be
easy to clean. This requires impervious and washable
surfaces, free from open joints or ledges. Coving
should be used at junctions between walls and floors
or ceilings. All services such as pipes, light fittings
and ventilation points should be sited so that
inaccessible recesses are avoided. A rigorous
disinfection policy must be in place. All equipment
must be easy to dismantle and clean and should be
inspected for cleanliness before use. Fall-out of dust-
and droplet-borne microorganisms from the
atmosphere is an obvious route for contamination.
Therefore ‘clean’ air is a prerequisite during
manufacturing processes and the spread of dust
during manufacture or pack- aging must be avoided.
Microorganisms may thrive in certain liquid
preparations and creams and lotions, so the
manufacture of such products should, as far as
possible, be in a closed system; this serves a dual
purpose as it also prevents evaporative loss.
Personnel are another source of potential
contamination. High standards of personal hygiene
are essential. Operatives should be free from
communicable disease and open lesions on the
exposed body surfaces. To ensure high standards of
personal cleanliness, adequate hand-washing
facilities and protective garments, including
headgear and gloves, must be provided. Staff should
be trained in the principles of GMP and in the
practice (and theory) of the tasks assigned. Staff
employed in the manufacture of sterile products
should also receive basic training in microbiology.
14.3.4 QUALITY OF STARTING MATERIALS
Raw materials, including water supplies, are an
important source of microorganisms in the
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manufacturing area and can lead to contamination
of both the environment and final product. Materials
of natural origin, plant or animal, are usually
associated with an extensive microbial flora and
require careful storage to prevent growth of the
organisms and spoilage of the material. If stable,
natural products with a high microbial count may be
sterilized. Staff handling raw materials must be given
adequate training to prevent cross-contamination.
Water for manufacturing may be potable mains
water, water purified by ion exchange, reverse
osmosis or distillation, or Water for Injection. Water
used for parenteral products must be apyrogenic
and is usually produced in a specially designed still.
Although pyrogens are not volatile, some are carried
over mechanically into the distillate with the
entrainment (spray), so a spray trap, consisting of a
series of baffles, is fitted to the distilling flask to
prevent spray and pyrogens from entering the
condenser tubes. Water prepared in this way can be
used immediately for the preparation of injections,
provided they are sterilized within 4 hours of water
collection. Alternatively, the water can be kept for
longer periods at a temperature above 65°C (usually
80°C) to prevent bacterial growth with consequent
pyrogen production. Ultraviolet radiation may be
useful in reducing bacterial count but must not be
regarded as a sterilizing agent.
14.3.5 PROCESS DESIGN
The manufacturing process must be fully defined
and capable of providing, with the facilities avail-
able, a product that is microbiologically acceptable
and conforms to specifications. The process must be
fully evaluated before starting to ensure that it is
suitable for routine production operations.
Processes and procedures must also be subject to
frequent reappraisal and should be re-evaluated
when any significant changes are made in the
equipment or materials used.
14.3.6 QUALITY CONTROL AND DOCUMENTATION
The lower the microbiological count of the starting
materials, the more readily the quality of the
product can be controlled. Microbiological standards
should be set for all raw materials as well as
microbial limits for in-process samples and the final
product. Microbiological quality assurance also
covers the validation of cleaning and disinfectant
solutions and the monitoring of the production
environment by microbial counts. This monitoring
should be carried out while normal production
operations are in progress. In addition, sterile
manufacture requires extra safeguards. Operators
Factory and Hospital Hygiene 122
must be adequately trained and their aseptic
technique monitored both by observation and
microbial testing. Air filter and sterility efficiency
must also be evaluated. The final tests that are
usually conducted on the finished product are those
for pyrogens (endotoxin test) and sterility.
Documentation is a vital part of quality assurance.
Details of starting materials, packaging materials,
and intermediate, bulk and finished products should
be recorded, so that the history of each batch may
be traced. Distribution records must be kept. This
information is of paramount importance in the event
that a defective batch has to be recalled.
14.3.7 PACKAGING, STORAGE AND TRANSPORT
Packaging serves a number of functions: it keeps the
contents in, it should keep contaminants out and it is
labelled to permit identification of its con- tents. The
product is contained within primary packaging. In
industry these packages are then placed inside
secondary packaging for storage and transport. This
secondary packaging may take the form of cartons,
boxes, trays or shrink-wrapping. Consideration must
be given to both the fabrics of the packaging and its
cleaning and also the actual process of packaging.
Where terminal sterilization is carried out, the
packaging must be suitable for the process.
Packaging of aseptically processed products into a
sterile container must be carried out in grade A
environment.
14.4 MANUFACTURE OF STERILE PRODUCTS
For production purposes an important distinction
exists between sterile products which have been
terminally sterilized and those which are not.
Terminal sterilization involves the product being
sealed in its container and then sterilized, usually by
heat, but ionizing radiation or, less commonly,
ethylene oxide may be employed. Such a product
must be produced in a clean area. A product that
cannot be terminally sterilized is pre- pared
aseptically from previously sterilized materials or by
sterile filtration and then filled into sterile
containers. Strict aseptic conditions are required
throughout. Vaccines, consisting of dead
microorganisms, microbial extracts or inactivated
viruses may be filled in the same premises as other
sterile medicinal products. The completeness of
inactivation (or killing or removal of live organisms)
must be proved before processing. Separate
premises are needed for the filling of live or
attenuated vaccines and for the preparation of other
products derived from live organisms. Non-sterile
Pharmacy Exam Guide

products and sterile products must not be processed
in the same area.
14.5 CLEAN AND ASEPTIC AREAS: GENERAL
REQUIREMENTS
14.5.1.1 D ESIGN OF PREMISES
Sterile production should be carried out in a
purpose-built unit separated from other
manufacturing areas and thoroughfares. The unit
should be designed to encourage separation of each
stage of production but should ensure a safe and
organized workflow. Sterilized products held in
quarantine pending sterility test results must be kept
separate from those awaiting sterilization.
14.5.1.2 I NTERNAL SURFACES , fiTTINGS AND
flOORS
Particulate, as well as microbial, contamination must
be prevented. To this end all surfaces must have
smooth, impervious surfaces which will:
(i) prevent accumulation of dust or other
particulate matter; and
(ii) permit easy, repeated cleaning and
disinfection.
Smooth rounded coving should be used where
the wall meets the floor and the ceiling. Suitable
flooring may be provided by welded sheets of
polyvinyl chloride (PVC); cracks and open joints
that might harbour dirt and microorganisms
must be avoided. The preferred surfaces for
walls are plastic, epoxy-coated plaster, plastic
fibreglass or glass reinforced polyester. Often
the final finish for the floor, wall and ceiling is
achieved using continuous welded PVC sheeting.
Factory and Hospital Hygiene 123
False ceilings should be adequately sealed to
prevent contamination from the space above.
Use should be made of well- sealed glass panels,
especially in dividing walls, to ensure good
visibility and allow satisfactory super- vision.
Doors and windows should be flush with the
walls. Windows should not be openable.
Internal fittings such as cupboards, drawers and
shelves should be kept to a minimum. They
must be sited where they do not interfere with
the laminar flow of the filtered air supply.
Stainless steel or laminated plastic are the
preferred materials for such fittings. Stainless
steel trolleys may be used to trans- port
equipment and materials within the clean and
aseptic areas but must remain confined to their
respective units. Equipment must be designed
so that it may be easily cleaned and sterilized or
disinfected.
14.5.1.3 S ERVICES
Clean and aseptic areas must be adequately
illuminated; lights are best housed in translucent
panels set in a false ceiling. Electrical switches and
sockets must be flush with the wall. When required,
gases should be pumped in from outside the unit.
Pipes and ducts, if they must be brought into the
clean area, must be sealed through the walls.
Additionally, in order to prevent dust accumulation,
they must be boxed in or readily cleanable.
Alternatively they may be sited above false ceilings.
Sinks should be of stainless steel with no over- flow,
and water must be of at least potable quality.
Wherever possible, drains should be avoided. If
installed they must be fitted with effective, readily
cleanable traps and with air breaks to prevent back-
flow. Any floor channels should be open, shallow
and cleanable and connected to drains outside the
area. They should be monitored microbiologically.
Sinks and drains should be excluded from aseptic
areas except where radiopharmaceuticals are being
processed when sinks are a requirement.
14.5.1.4 A IR SUPPLY
Areas for sterile manufacture are classified according
to the required characteristics of the environment.
Each operation requires an appropriate level of
microbial and particulate cleanliness; four grades are
specified in Rules and Guidance for Pharmaceutical
Manufacturers and Distributors (2002). The air
supplied to the manufacturing environment
substantially influences environmental quality.
Pharmacy Exam Guide

Filtered air is used to achieve the necessary
standards; this should be maintained at positive
pressure throughout a clean or aseptic area, with the
highest pressure in the most critical rooms (aseptic
or clean filling rooms) and a progressive reduction
through the preparation and changing rooms; a
minimum pressure differential of 10–15 Pa is
normally required between each class of room. A
minimum of 20 changes of air per hour is usual in
clean and aseptic rooms. The air inlet points should
be situated in or near the ceiling, with the final filters
placed as close as possible to the point of input to
the room. The greatest risk of contamination of a
product comes from its immediate environment.
Additional protection is needed in both the filling
area of the clean room and in the aseptic suite. This
can be provided by a workstation supplied with a
unidirectional flow of filtered sterile air. This is
known as a laminar flow cabinet. Displacement of air
may be vertical or horizontal with a minimum
homogeneous airflow of 0.45 m/sat the working
position. Consequently airborne contamination is
not added to the workspace and any generated by
manipulations is swept away by the laminar air
currents. A fuller description of high efficiency
particulate air (HEPA) filters in laminar flow cabinets
is given by Gardner and Peel (1998). The efficacy of
the filters through which the air is passed should be
monitored at predetermined intervals. Air quality
may be monitored by volumetric air sampler or
settle plate.
14.5.1.5 C LOTHING
Clothing worn in the clean area must be of non-
shedding fibres; terylene is a suitable fabric.
Airborne contamination, both microbial and
particulate, is reduced when trouser suits, close-
fitting at the neck, wrists and ankles, are worn. Clean
suits should be provided once a day, but fresh
headwear, overshoes and powder-free gloves are
necessary for each working session. Special
laundering facilities are desirable.
14.5.1.6 C HANGING FACILITIES
Entry to a clean or aseptic area should be through a
changing room fitted with interlocking doors; this
acts as an airlock to prevent influx of air from
outside. This route is for personnel only, not for the
transfer of materials and equipment. Staff entering
the changing room should already be clad in the
standard factory or hospital protective clothing. For
entry into a clean area, passage through the
Factory and Hospital Hygiene 124
changing room should be from a ‘black’ to a ‘grey’
area, via a dividing step-over sill. Movement through
these areas and finally into the clean room is
permitted only when observing a strict protocol,
whereby outer garments are removed in the ‘black’
area and clean room trouser-suits donned in the
‘grey’ area. After hand-washing in a sink fitted with
elbow- or foot-operated taps the operator may
enter the clean room.
14.5.1.7 C LEANING AND DISINFECTION
A strict disinfection policy is necessary if microbial
contamination is to be kept to a minimum. Cleaning
agents used include alkaline detergents and ionic
and non-ionic surfactants. A wide range of chemical
disinfectants is available. Clear soluble phenolics are
commonly used for interior services and fittings.
Disinfectants for working surfaces are alcohols (70%
ethanol or isopropanol) or, less commonly, chlorine-
base agents such as hypochlorites. Skin may be
disinfected with cationic detergents such as
cetrimide or chlorhexidine, usually formulated with
70% alcohol to avoid the need for rinsing. Gloved
hands may be disinfected with these detergents (so
offering residual activity) or 70% alcohol alone.
Rotation of different disinfectants reduces the risk of
the emergence of resistant strains. In-use dilutions
must not be stored unless sterilized. Disinfectants
and detergents for use in grade A/B areas must be
sterile prior to use. Smooth polished surfaces are
most readily cleaned. Floors and horizontal surfaces
should be cleaned and disinfected daily, walls and
ceilings as often as required, but the interval should
not exceed 1 month. Regular microbiological
monitoring should be carried out to determine the
efficacy of disinfection procedures. Records must be
kept and immediate remedial action taken should
normal levels for that area be exceeded.
14.5.1.8 O PERATION
The number of persons involved in sterile
manufacture should be kept to a minimum to avoid
the inevitable turbulence and shedding of particles
and organisms associated with the operatives. All
operations should be undertaken in a con- trolled
and methodical manner as excessive activity may
increase this turbulence and shedding. Containers
made from fibrous materials such as paper,
cardboard and sacking are generally heavily
contaminated (especially with moulds and bacterial
spores) and should not be taken into clean areas.
Ingredients that must be brought into clean areas
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must first be transferred to suitable metal or plastic
containers.
Containers and closures for terminally sterilized
products must be thoroughly cleaned before use and
should undergo a final washing and rinsing process
in apyrogenic distilled water (which has been passed
through a bacteria-proof membrane filter)
immediately prior to filling. Containers and closures
for use in aseptic manufacture must, in addition, be
sterilized after washing and rinsing in preparation for
aseptic filling.
14.5.1.9 C LOTHING
Clothing requirements are necessary for aseptic
areas. The operative is a potential source of
microorganisms and it is imperative that steps are
taken to prevent this contamination. The operative
must wear sterile protective headwear, totally
enclosing hair and beard, powder-free rubber or
plastic gloves, a non- fibre-shedding facemask (to
prevent the release of droplets) and footwear. A
suitable garment is a one- or two-piece trouser-suit.
Fresh sterile clothing should be provided each time a
person enters an aseptic area.
14.5.1.10 E NTRY TO ASEPTIC AREAS
Entry to an aseptic suite is usually through a ‘black-
grey-white’ changing procedure, where white
represents the highest level of cleanliness.
Movement from ‘black’ to ‘white’ is via two changing
rooms, the ‘grey’ area also serving as an entry to the
clean room.
14.5.1.11 E QUIPMENT AND OPERATION
Any articles entering the aseptic area must be
sterilized. In order to achieve this, articles should be
transferred via a double-ended sterilizer (i.e. with a
door at each end). If they are not to be discharged
directly to the aseptic area, they should be
(i) double-wrapped before sterilization;
(ii) transferred immediately after sterilizing
into a clean environment until
required;
(iii) transferred from this clean environment via
a double-doored hatch (where the
outer wrapping is removed) to the
aseptic area (where the inner wrapper
is removed at the work- bench).
Hatches and sterilizers must be designed so that only
one door may be opened at any one time. Solutions
manufactured in the clean room may be brought
into the aseptic area through a sterile 0.22-mm
Factory and Hospital Hygiene 125
membrane filter. Workbenches, including laminar
flow units, and equipment, should be disinfected
immediately be- fore and after each work session.
Equipment must be of the simplest design possible
for the operation being performed. Aseptic
manipulations must be carried out in the grade A air
of a laminar flow cabinet. Speed, accuracy and
economy of movement are essential features of
good aseptic technique. It is therefore essential that
workers are well trained and motivated and familiar
with the task in hand. Observation and
microbiological monitoring of the operator and of
the environment are very important. Air quality is
measured using settle plates or slit samplers, work
surfaces by taking swabs or by use of contact plates.
Under no circumstances must living microorganisms,
including those used for vaccine preparation and for
biological monitoring, be introduced into the aseptic
area.
14.5.1.12 I SOLATOR AND BLOW / fiLL / SEAL
TECHNOLOGY
All aseptic packaging should be carried out in a grade
A environment with a grade B background. Advances
in technology now permit the production of self-
contained workstations, or isolators, which
incorporate many of the design principles of clean
rooms and laminar flow cabinets. The isolator
protects both the product from contamination by
the operator and the operator from any hazardous
materials. Direct interaction between the operator
and the product is minimized by providing a grade A
laminar flow of air with a positive pressure, the
internal space being accessed by means of a
glove/sleeve system. A grade D background is
considered adequate for such operations.
Blow/fill/seal units are purpose-built pieces of
equipment, which carry out these three steps in a
continuous process within a controlled environment.
Containers, which are formed from thermo- plastic
granules, are blown to form the correct shape, filled
and heat-sealed. These units are fitted with a grade
A air shower and operated in a grade C environment
for aseptic manufacture and a grade D background
for products which are to be terminally sterilized.
14.6 GUIDE TO GOOD PHARMACEUTICAL
MANUFACTURING PRACTICE
Between 1971 and 1983 the essential features of
GMP were covered in the UK by three editions of the
Guide to Good Pharmaceutical Manufacturing
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 Factory and Hospital Hygiene 126

Practice, frequently referred to as ‘The Orange
Guide’. This guide was prepared by the UK
Medicines Inspectorate in consultation with
industrial, hospital, professional and other
interested parties. The principles of this national
guide were subsequently assimilated into the EC
Guide to Good Manufacturing Practice for Medicinal
Products in 1989 and are now published as Rules and
Guidance for Pharmaceutical Manufacturers and
Distributors (2002) by the Medicines Control Agency,
Department of Health. Two important recent
publications from the Pharmaceutical Press are
Quality in the Manufacture of Medicines and Other
Health Care Products (Sharp, 2000) and Quality
Assurance of Aseptic Preparation Services (Beaney,
2001), which discusses manufacturing in hospitals.
Compliance with GMP is one of the major factors
considered by the Licensing Authority when
examining an application for a license to
manufacture under the Medicines Act (1968). Similar
codes exist in the USA and other countries.
Greater stringency is required for terminally
sterilized products. Such environmental and process
controls might seem overzealous, but it is better to
minimize risk at all stages rather than to rely on final
product testing. The lower the bioburden the easier
it is to achieve the required sterility assurance level
in the terminal sterilization process. It is also
important to exclude pyrogens and particulate
matter that would not be removed by sterilization.
Where products are processed aseptically even
higher standards of cleanliness are necessary. The
importance of the knowledge and commitment of
the operatives cannot be overemphasized, both in
hospital and industry. A majority of reported
incidents of defective products have been traced to
human rather than technological error.

14.7 CONCLUSIONS
Quality assurance is not just a process; it is a way of
thinking. All staff should be well trained and
motivated and be working to a common goal: the
production of a pharmaceutical product of a quality
that is safe for the patient. The procedures should
not be seen as a chore or burden to make work
more difficult, but essential steps in the production
of a safe, satisfactory product. Self-inspection and
external audit of procedures are important
processes in maintaining standards of cleanliness.
Even after manufacture and distribution it is vital
that the products are used properly, especially multi-
use containers that are subject to potential in-use
contamination. The manufacture of non-sterile
products requires that certain standards of
cleanliness, personal hygiene, production methods
and storage must be met. Many such products are
for oral and topical use and one might wonder why
such stringent parameters need be in place.
However, there have been controlled hospital
studies and case reports associating these products
with nosocomial (hospital- acquired) infection.
Furthermore, methods of controlling pathogens also
control spoilage organisms, which could cause the
industry considerable expense. Spoilage organisms
can alter the aesthetic qualities (such as smell, taste,
and appearance), physical properties (pH, viscosity)
and efficacy of the product, in addition to producing
toxins.

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 Vaccine & Sera 127

A vaccine typically contains an agent that resembles

a disease-causing microorganism and is often made
Chapter 15 VACCINE & SERA from weakened or killed forms of the microbe, its
toxins or one of its surface proteins.
15.1 VACCINE The agent stimulates the body's immune system to
recognize the agent as foreign, destroy it, and keep a
Vaccine are preparation of living microorganisms
record of it, so that the immune system can more
and their metabolites and filtrates and certain drug
easily recognize and destroy any of these
and food.
microorganisms that it later encounters.

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 Vaccine & Sera 128

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Vaccine can be classified as:
 Living vaccine. (E.g. BCG vaccineT.B).
 Dead vaccine. (E.g. TBA vaccine Typhoid).
 Attenuated vaccine.
15.2 HISTORY
The terms vaccine and vaccination are derived from
Variolae vaccinae (smallpox of the cow), the term
devised by Edward Jenner to denote cowpox.
In 1798 he described the protective effect of cowpox
against smallpox.
Vaccination is the administration of antigenic
material (a vaccine) to stimulate an
individual's immune system to develop adaptive
immunity to a pathogen.
Antiserum is blood serum containing Antibodies.
Serology is the scientific study of plasma serum and
other bodily fluids
Vaccine & Sera 129
15.3 LIVING VACCINE
A living vaccine is a prophylactic that contains living
parts or whole germ cells into a person or animal to
create immunity.
15.3.1 EXAMPLES
Mycobacterium tuberculosis Human type.
Mycobacterium tuberculosis Bovine type. (Cattle).
Mycobacterium tuberculosis Avain type. (Birds).
Calmelte & Guerin makes BCG (T.B) vaccine.
15.4 PASTEURIZATION
Pasteurization is a process of heating food, which is
usually a liquid, to a specific temperature for a
specific time and then immediately cooling it after it
is removed from the heat.
It is named after microbiologist Louis Pasteur.
This process slows spoilage caused by
microbial growth in the food.
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Milk is an excellent medium for microbial growth,
and when stored at ambient temperature bacteria
and other pathogens soon proliferate.
Pasteurization can prevent include tuberculosis,
diphtheria, scarlet fever, it also kills the harmful
bacteria Salmonella, Yersinia, Campylobacter, Staph
ylococcus aureus, and Escherichia coli.
Mycobacterium tuberculosis is the indicator microbe
of Milk.
Pasteurization is the reason for milk's extended shelf
life.
Pasteurized milk is free from Mycobacterium
tuberculosis.
15.5 DEAD VACCINE
 The vaccine having dead microbes that
cannot replicate but can still trigger the production
of antibodies.
 Dead vaccine. (E.g. TBA vaccine 
Typhoid).
15.6 ATTENUATED VACCINE
Any physical or chemical process, by which we
reduce the virulence and increase the antigenicity, is
called Attenuation.
Dilution is also a way of Attenuation, the active
substances are very diluted.
Vaccines can be prophylactic (example: to prevent or
ameliorate the effects of a future infection by any
natural or "wild" pathogen), or therapeutic (e.g.,
vaccines against cancer are also being investigated.
An attenuated vaccine is a vaccine created by
reducing the virulence of a pathogen, but still
keeping it viable (or "live").
Attenuation takes an infectious agent and alters it so
that it becomes harmless or less virulent.
Attenuation can be done by several method:
 Heat ( low or high temperature inactive the
microbes)
 Phenol (5% phenol is used for attenuation).
 Formaldehyde (4% is used).
 Potash alum (ppt the microbe and seprated)
 Aluminum phosphate (1% is used).
Virulence can be increase by allowing the microbe in
living tissues. Virulence can be decrease when
microbe can be allow to grow on artificial media in
the lab. Bacteria culture can be weak by keeping in
the refrigerator.
Vaccine & Sera 130
E.g. Old microbes are less virulent.
Vaccine may Monovilent (contains 1 types of
microbes), Bivilent, Trivilent, Polyvilent……
Vaccine can be prepared by heating the cell of
o
microbes at 56 C at for 1 hour, that all his toxiphoric
portion is destroyed and its antigenicity remains
alive.
15.7 BACTERIAL VACCINE
15.7.1 PREPARATION OF BACTERIAL
VACCINE
It has 3 steps:
 Preparation of culture.
 Preparation of Bacterial suspension.
 Counting of microbes present in the vaccine
(Standardization).
15.7.2 BCG VACCINE
BCG vaccine provides immunity or protection against
tuberculosis (TB). The vaccine may be given to
persons at high risk of developing TB. It is also used
to treat bladder tumors or bladder cancer.
Calmelte & Guerin makes BCG (T.B) vaccine.
It consists of suspension of living tubercular bacilli of
bovine type .
The bacterium is render a virulent by cultivating it on
Glycerin-bile patato media.
The BCG vaccine can be anywhere from 0 to 80%
effective in preventing tuberculosis for a duration of
15 years.
Usual dose is 0.5 ml.
BCG vaccine can be implemented after the birth
intradermally.
A tuberculin skin test should always be done before
administering BCG.
BCG is given as a single intradermal injection at the
insertion of the deltoid.
India and Pakistan: India and Pakistan introduced
BCG mass immunization in 1948, the first countries
outside Europe to do so.
15.7.3 CHOLERA VACCINE
Cholera vaccine is a vaccine used against cholera.
The first vaccines used against cholera were
developed in the late nineteenth century.
It consists of sterile suspension of vibrio cholera
8000 cell/ ml.
Vaccine is prepared by general method containing 2
types of microbes serotype.
 Vibrio Cholera Inaba type.
 Vibrio Cholera Ogawa type.
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15.7.4 TAB VACCINE
It is typhoid-paratyphoid A and B vaccine.
A vaccine containing killed typhoid bacilli and the
paratyphoid organisms (Salmonella para-typhi A and
B) most frequently involved in paratyphoid fever.
It is used in the prophylaxis of enteric fevers.
It Contains heat-killed Salmonella typhi and S.
paratyphi A and B in the smooth specific phase and
possessing their normal complement of O antigens.
Usually preserved with phenol and aceton .
Dose: 1000 million salmonella typhi.
500-750 million para-A, 500-750 million para-B/ ml.
15.8 PLAGUE VACCINE
Plague is a deadly infectious disease that is caused
by the Yersinia pestis.
The symptoms of plague depend on the
concentrated areas of infection in each person:
bubonic plague in lymph nodes, septicemic plague in
blood vessels, pneumonic plague in lungs.
A plague vaccine is used for an induction of active
specific immunity in a susceptible organism to
plague.
It contains sterile suspension of yersinia pestis.
The growth of bacteria is suspended in Nacl
suspension, containing 0.5 %formaldehyde solution
at room temperature.
Dose: 3000 million plague organism/ ml.
15.8.1 PERTUSSIS VACCINE
Pertussis is commonly called whooping cough.
Pertussis vaccine is a vaccine used against Bordetella
pertussis.
The vaccine is prepared very freshly from patient
suffering from disease.
Dose: 20,000 million organism/ml.
15.8.2 SCARLET FEVER
Scarlet fever is a bacterial infection having strep
throat with bright rash.
This vaccine is taken as a prophylactic measure.
Vaccine is mixture of sterile filtrate of toxigenic stain
of steptococcus pyogenes.
When a small dose is injected, intracutanously to a
susceptible person, it gives a typical scarlet (violet)
rashes.
15.8.3 DIPHTHERIA VACCINE
Diphtheria vaccine is a vaccine used against
Corynebacterium diphtherie, the agent that causes
diphtheria.
It is a component of the DPT vaccine.
Vaccine is modified form of Corynebacterium
diphtherie.
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Highly toxigenic stain is selected and grown on
suitable broth to obtain abundant exotoxin, which is
converted into formal toxoid.
Diphtheria toxin is unstable if store in refrigerator
for six months, it matured.
Toxin can also be converted into Toxoid by 0.4%
formaldehyde.
Alum precipated toxoid (APT) is prepared by using
1% potash alum to formal toxoid.
Purified toxoid of aluminum phosphate (PTAP) is
now used as Diphtheria prophylactic measure.
(F.T + suspension of hydrated toxoid of aluminum
phosphate in saline).
15.8.4 TETANUS VACCINE
Tetanus (also called lockjaw) is a preventable disease
that affects the muscles and nerves, usually due to a
contaminated wound.
Tetanus vaccine is a vaccine composed of
deactivated tetanus toxins.
It is produced by growing a toxigenic stain of
clostridium tetani, in cooked meat medium placed in
well field bottle (so there will be no air).
Toxin is converted into Toxoid by 0.4%
formaldehyde.
15.8.5 STAPHYLOCOCCUS T OXOIDS
Staphylococcus toxoid is a complex of
staphylococcus toxins (alpha toxin) detoxicated with
formalin and by heat, prepared in a highly effective
and purified form.
It contains thiomersal as a antimicrobial
preservative.
The staphylococcus toxoid is used in the individual
therapy or prophylaxis of chronic obstructive
pulmonary disease and allergic diseases.
It may be also used as prophylactic measure.
15.9 VIRAL VACCINE
15.9.1 SMALL POX VACCINE
Smallpox vaccine, the first successful vaccine to be
developed, was introduced by Edward Jenner in
1798.
It is example of living vaccine.
Vaccinia virus cause cow pox.
Viriola virus cause small pox.
There are two forms of the variola virus:
 Variola major (mortality rate 30%).
 Variola minor (mortality rate 0.3%).
Dose: 1:1000 dilution.
An infection of the variola virus starts out with flu
then rashes and scabs form throughout – similar to
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chickenpox, leading to malignant diseases such as
intravascular coagulation, hypotention,
cardiovascular collapse or bleeding of skin and
intestinal tract.
The vaccine has provided protection to 95% of the
vaccinated individuals.
The vaccine will provide protection after 2 days of its
administration.
It offers the best possible protection against a
possible exposure to smallpox.
15.9.2 INFLUENZA VACCINE
Influenza vaccine boast up immunity against
Influenza virus.
Influenza vaccines may be administered as an
injection, also known as a flu shot, or as a nasal
spray.
Laidlaw discovered Influenza virus in 1933.
Vaccine is mixture of sterile suspension of Influenza
virus prepared in normal saline.
Dose: 1:1000 dilution.
Vaccine is prepared in chicken embryo.
15.9.3 YELLOW FEVER VACCINE
The vaccine consists of a live, but attenuated, strain
of the yellow fever virus.
Vaccine is prepared in chicken embryo.
The embryo is incubated for 17 days as a result it
become weaken and used as vaccine.
The chicken embryo filter through millipore, 0.5 µm
as a standard size.
The sample is taken through syringe by creating
vacuum.
15.9.4 POLIOMYELITIS (POLIO) VACCINE
Two polio vaccines are used throughout the world to
combat poliomyelitis (or polio) Salk vaccine and
Sabin vaccine.
The first vaccine by Jonas Salk was developed and
tested in 1952.
An oral vaccine by Albert Sabin was developed in
1957 using attenuated poliovirus.
Vaccine may be prepared by 3 immunological type of
poliovirus.
Vaccine & Sera 132
th
It is 1 of the smallest virus (1/100,000 of
staphylococcus).
 Dumb polio
 Abortive polio, (causing fever, headache,
vomiting).
 Paralytic polio, (causing respiratory
difficulties).
Non- Paralytic polio cause convulsions, stiffness of
neck.
Polio multiply in never cells of Monkey and Man.
The kidney of infected person/animal are removed,
grinded into cubes of 1mm and vaccine is prepared.
Oral vaccine consisting living poliovirus from monkey
kidney tissues which are cultivated and checked.
The oral vaccine is prepared in the form of syrup/
head gelatin capsule containing stock vaccine(which
is stored), described by Sabin .
Viral particle multiply in the canal and developed
immunity.
Salk vaccine: (dead vaccine) Monkey tissues
containing polio virus is treated with formaldehyde
to prevent paralysis.
15.10 ANTI-IDIOTYPIC VACCINES
Anti-idiotypic vaccines comprise antibodies that
have three-dimensional immunogenic regions,
designated idiotopes, that consist
of protein sequences that bind to cell receptors.
Idiotopes are aggregated into idiotypes specific of
their target antigen.
An example of anti-idiotype antibody
is Racotumomab.
15.10.1 USE:
Because the antibody produced using the "anti-
idiotypic" process closely resembles the original
epitope of the antigen, these antibodies can be used
to induce immune responses from cellular to
antibody-antigen for a given antigen, e. g., TAA,
when administered as a vaccine to a human.
They are mainly used for high risk cancer patients.
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TERMINOLOGIES
Abiogenesis
Abiogenesis is the process by which a living organism arises naturally from non- living matter, as opposed to
biogenesis
Acid-fast
Acid-fastness is a physical property of certain bacteria (and, less commonly, protozoa), specifically their resistance
to decolorization by acids during staining procedures e.g. Mycobacterium tuberculosis, and Mycobacterium leprae
etc.
Aerobe
A microorganism which grows in the presence of air or requires oxygen for growth. E.g. Myobacterium tuberculosis
Agar
A dried polysaccharide extract of red algae (Rhodophyceae) used as a solidifying agent in microbiological media.
Agglutination
The clumping of cells.
Agglutinin
An antibody capable of causing the clumping or agglutination of bacteria or other cells.
Anaerobe
An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. It may react
negatively or even die if oxygen is present, which means that it can perform its bodily functions better in the
absence of oxygen. E.g. Escherichia coli, Clostridium botulinum (which causes food poisoning)
O BLIGATE ANAEROBES , which are harmed by the presence of oxygen. E.g. Clostridium tetani and Bacteroides tectus
A EROTOLERANT ORGANISMS , which cannot use oxygen for growth, but tolerate the presence of it
F ACULTATIVE ANAEROBES , which can grow without oxygen but use oxygen if it is present. E.g. Escherichia coli and
Shewanella oneidensis (Gram negative)
Animalcules
Animalcule is an older term for a microscopic animal or protozoan
Antagonism
The killing, injury or inhibition of growth of one species of microorganism y another when one organism adversely
affect the environment of the other.
Antibiotic
A substance of microbial origin that has antimicrobial origin that has antimicrobial activity in very small amounts.
Antimicrobial agent
Any chemical or biological agent that either destroys or inhibits the growth of microorganism.
Antibody
Any of a class of substances (proteins) produced by an animal in response to the introduction of antigen.
Antiseptic
Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection,
sepsis, or putrefaction.
Autoclave
An apparatus using pressurized steam for sterilization
Autotroph
These are bacteria which are able to synthesize their own organic food from inorganic substances.
Photoautotrophic Bacteria
The photoautotrophic bacteria possess photosynthetic pigments in membrane bound lamellae (or thylakoids) and
utilise solar energy. The bacterial photosynthesis is different from that of green plants since here water is not used
as a hydrogen donor. Hence oxygen is not released as a byproduct. For this reason, the process is described as
anoxygenic photosynthesis. E.g. cyanobacteria
Chemosynthetic Bacteria
These are bacteria which manufacture organic compounds from inorganic raw materials utilising energy liberated
from the oxidation of inorganic substances. Following are the common types of chemo autotrophic bacteria.

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Nitrifying bacteria which derive energy by oxidizing ammonia into nitrates. Eg: Nitrobacter vulgari
Sulphur bacteria which derive energy by oxidising hydrogen sulphide to sulphur. Eg: Thiobacillus aquaesulis
Iron Bacteria which derive energy by oxidising ferrous ions into ferric form. Eg: ferrobacillus ferrooxidans
Autolysis
The disintegration of cells by the action of their own enzymes.
Bacterial Conjugation
Bacterial conjugation is the transfer of genetic material (plasmid) between bacterial cells by direct cell-to-cell
contact or by a bridge-like` connection between two cells.
Bacteriostatic
Inhibiting the growth of bacteria without killing them.
Bergey’s Manual
An international reference work which classifies and describes bacteria.
Binomial nomenclature
Binomial nomenclature (also called binominal nomenclature or binary nomenclature) is a formal system of naming
species of living things by giving each a name composed of two parts, both of which use Latin grammatical forms,
although they can be based on words from other languages. The first part of the name identifies the genus to
which the species belongs; the second part identifies the species within the genus. For example, humans belong to
the genus Homo and within this genus to the species Homo sapiens.
Biodegradable
Biodegradation is the chemical dissolution of materials by bacteria or other biological means.
Biogenesis
Biogenesis is the production of new living organisms or organelles
Biomass
The mass of living matter present in a specified area
Brownian motion
A peculiar dancing motion exhibited by finely divided particles and bacteria in suspension due to bombardment by
the molecules of the fluid.
Budding
Budding is a form of asexual reproduction in which a new organism develops from an outgrowth or bud on another
one. Common Yeast show the phenomena of budding commonly. One bud is formed at one time and the
maximum number of buds that can be formed during the life of common yeast is seven.
Carrier
A person in apparently good health who harbors a pathogenic microorganism.
Colony
The resultant of a single cell or it is a progeny of a single cell is called colony.
Colony Forming Unit (CFU)
The cell or aggregate of cells which gives rise to single colony in the plate-count technique.
Complement
A normal thermo-labile protein constituent of blood serum that participates in antigen-antibody reactions.
Contaminated Culture
The culture containing a few unknown microbes or the entrance of unknown microbe/s in the pure or mixed
culture.
Cytolysis
The dissolution or disintegration of a cell.
Disinfectant
Disinfectants are substances that are applied to non-living objects to destroy microorganisms that are living on the
objects.
Endemic
In epidemiology, an infection is said to be endemic in a population when that infection is maintained in the
population without the need for external inputs. For example, chickenpox is endemic (steady state) in the UK, but
malaria is not.
Epidemic
A widespread occurrence of an infectious disease in a community at a particular time.

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Epidemiology
Epidemiology is the study (or the science of the study) of the patterns, causes, and effects of health and disease
conditions in defined populations.
Etiology
The cause, set of causes, or manner of causation of a disease or condition.
Fermentation
Fermentation is a metabolic process converting sugar to acids, gases and/or alcohol. It occurs in yeast and
bacteria, but also in oxygen-starved human muscle cells
Fission
In biology, fission is the subdivision of a cell (or body, population, or species) into two or more parts and the
regeneration of those parts into separate cells (bodies, populations, or species). Binary fission produces two
separate cells, populations, species, etc., whereas multiple fission produces more than two cells, populations,
species, etc.
Flagellum
A flagellum is a lash-like appendage that protrudes from the cell body of certain prokaryotic and eukaryotic cells.
Fluorescence
The emission of light of a particular wavelength by a substance which has absorbed light of a shorter wavelength.
For example, the emission of green light by the molecules of fluorescein dye which have absorbed blue light.
Germicidal
An agent that kills germs, especially pathogenic microorganisms
Gram negative bacteria
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram
stain test, a counterstain is added after the crystal violet, coloring all gram-negative bacteria with a red or pink
color. E.g. Escherichia coli (E. coli), Salmonella typhus and Shigella Dysentery.
Gram positive bacteria
Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to gram-
negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain and appearing
red or pink. E.g. Clostridium tetani (cause of tetnus), and Bacillus anthracis (cause of anthrax)
Gram Stain
Gram staining (or Gram's method) is a method of differentiating bacterial species into two large groups (Gram-
positive and Gram-negative). The name comes from its inventor, Hans Christian Gram.
Heterotroph
A heterotroph is an organism that cannot fix carbon and uses organic carbon for growth. E.g. Escherichia coli
Immunity
A natural or acquired resistance to a specific disease
In vitro
(Literally “In Glass”) pertaining to biologic experiments performed in test tube or other laboratory vessels.
In vivo
In vivo is experimentation using a whole living organism
Infection
Infection is the invasion of a host organism's bodily tissues by disease-causing organisms, their multiplication, and
the reaction of host tissues to these organisms and the toxins they produce
Inhibition
The prevention of the growth or multiplication of microorganism
Inoculation
The artificial introduction of microorganism or other substances into the body or into a culture medium
Inoculum
The substance, containing microorganisms or other material that is introduced in inoculation

Lypholization
The preservation of biological specimens by rapid freezing and rapid dehydration in a high vacuum
Microbes
Any microscopic organism; a microorganism. E.g. bacteria, fungi, virus etc.

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Microbiology
The study oforganisms of microscopic size including their culture, economic importance, pathogencity etc.
Mixed Culture
The culture conitaing two or more than two types of known microbes.
Mortality Rate
Death rate
Normal Flora
The normal flora are bacteria which are found in or on our bodies on a semi-permanent basis without causing
disease.
Passive immunity
Passive immunity is the transfer of active humoral immunity in the form of ready-made antibodies, from one
individual to another.
Pathogensity
Pathogenicity is the potential capacity of certain species of microbes to cause a disease.
Pandemic
A pandemic is an epidemic of infectious disease that has spread through human populations across a large region;
for instance multiple continents, or even worldwide.
Phagocytosis
Phagocytosis is the process of engulfing a solid particle by a phagocyte or a protist to form an internal phagosome
Pilus
A pilus is a hairlike appendage found on the surface of many bacteria.
Plague
Plague is a deadly infectious disease that is caused by the enterobacteria Yersinia pestis, named after the French-
Swiss bacteriologist Alexandre Yersin.
Plasmid
A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of,
chromosomal DNA within a cell.
Pure Culture
Pure culture is the culture or growth of the organism of only one specific type of microbe.
Obligate Parasite
An obligate parasite is a parasitic organism that cannot complete its life cycle without exploiting a suitable host
Rickettsia
Obligate parasitic bacteria of arthropods; many are pathogenic for humans and other mammals.
Smear
The sample spread out on the slide evenly as thin as possible is called smear
The smear should be prepared by minimum quantity of the sample, evenly, homogenously spread out on an area
2
of about 1cm in one direction at one attempt.
Spontaneous generation
The hypothetical process by which living organisms develop from nonliving matter
Spore
In biology, a spore is a unit of asexual reproduction that may be adapted for dispersal and for survival, often for
extended periods of time, in unfavorable conditions.
Sterilization
The elimination of microbiological organisms to achieve asepsis, a sterile microbial environment
Substrate
Substrate is considered as the food for the microorganism
Syphilis
Syphilis is a sexually transmitted infection caused by the spirochete bacterium Treponema pallidum
Taxonomy
A field of science that encompasses the description, identification, nomenclature, and classification of organisms
Transcriptase
A polymerase that catalyzes the formation of RNA from a DNA template in the process of transcription.
Transduction

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Transduction is the process by which DNA is transferred from one bacterium to another by a virus
Vaccination
Vaccination is the administration of antigenic material to stimulate an individual's immune system to develop
adaptive immunity to a pathogen. Vaccines can prevent or ameliorate morbidity from infection
Vacine
A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an
agent that resembles a disease-causing microorganism and is often made from weakened or killed forms of the
microbe, its toxins or one of its surface proteins. The agent stimulates the body's immune system to recognize the
agent as foreign, destroy it, and keep a record of it, so that the immune system can more easily recognize and
destroy any of these microorganisms that it later encounters.
The term vaccine derives from Edward Jenner's 1796 use of cow pox (latin form vacca = cow)
Viable
Capable of working successfully; feasible.
Virulence
The degree of pathogenicity within a group or species of parasites as indicated by case fatality rates and/or the
ability of the organism to invade the tissues of the host. The pathogenicity of an organism - its ability to cause
disease - is determined by its virulence factors.
Viruses
A virus is a small infectious agent that replicates only inside the living cells of other organisms.
VIRIODS
An infectious particle, similar to but smaller than a virus, that consists solely of a strand of RNA and is capable of
causing disease in plants. Viroids are common plant pathogens which are a serious economic problem.
Recombinant Organism
An organism that contains a different combination of alleles from either of its parents.
Recombinant DNA
A form of artificial DNA
PYROGEN
A substance that produces fever.
ANTITOXIN
An antitoxin is an antibody with the ability to neutralize a specific toxin.

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REFERENCE BOOKS
1. Jawetz, Medical Microbiology and Immunology, 11th edition, Churchill Livingstone, London, 2001.
2. W B Hugo & A D Russell, Pharmaceutical Microbiology, Black Well Science Ltd, London, 7th Ed, 1998.
3. Lippincott, Microbiology, 4th Ed, by lippincott, William & Willkins, USA, 2004.
4. Alcamo, Introduction to Microbiology, John Bartlett Publishers, 6th Ed., 2003.
5. Collin and Lynes, Microbiological Methods, 8th Ed, Vutterworth Heineman, Oxford, 2004.
6. M Mekallee, Microbiology: Essentials and Application, McGraw-Hill Inc, 2nd Ed.
7. Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 3rd Ed, John Willey & Sons, New
York, 2006.
8. Pelczar, Microbiology, 5th Ed, McGraw-Hill Inc,2002.
9. Prescott, Harley, Microbiology, 6th Ed, Klein Wm, C Brown Publishers, 2006.

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