11th Micro Biology English-Medium Text

GOVERNMENT OF TAMIL NADU
HIGHER SECONDARY FIRST YEAR
MICROBIOLOGY
A publication under Free Textbook Programme of Government of Tamil Nadu
Department of School Education
Untouchability is Inhuman and a Crime
TN_GOVT_XI_Micro_Biology_FM.indd 1 16-02-2019 10.43.09 AM

Government of Tamil Nadu
First Edition - 2018
Revised Edition - 2019, 2022
(Published under New Education
Syllabus)
NOT FOR SALE
Content Creation
The wise
possess all
State Council of Educational

Research and Training
© SCERT 2018
Printing & Publishing
Tamil NaduTextbook and Educational

Services Corporation
www.textbooksonline.tn.nic.in
II

Contents
Chapter 1 Introduction to Microbiology�� 01

Chapter 2 Microscopy�� 15
Chapter 3 Stains and Staining Methods�� 25
Chapter 4 Sterilization�� 40
Chapter 5 Cultivation of Microorganisms�� 53
Chapter 6 Microbial Nutrition and Growth�� 72
Chapter 7 Morphology of Bacteria�� 86
Chapter 8 Microbial Taxonomy�� 111
Chapter 9 Environmental Microbiology�� 122
Chapter 10 Soil Microbiology�� 149
Chapter 11 Agricultural Microbiology�� 162
Chapter 12 Medical Microbiology�� 181
Chapter 13 Immunology�� 213
Chapter 14 Microbial Genetics�� 253
E-Book Assessment

HOW TO USE
THE BOOK ?
Chapter Outline Presents a complete overview of the chapter
Learning Goals to transform the classroom processes into

learner centric with a list of bench marks
Objectives:
Amazing facts, Rhetorical questions to lead students

to biological inquiry
Directions are provided to students to conduct activities

Activity in order to explore, enrich the concept�
Infographics Visual representation of the lesson to enrich learning �
To motivate the students to further explore the content

digitally and take them to virtual world
ICT To enhance digital Science skills among students
Glossary Explanation of scientific terms
Evaluation Assess students to pause, think and check their understanding
Career corner List of professions particular to that chapter
References List of related books for further details of the topic
Web links List of digital resources
IV

Career Opportunities for Microbiologists
Microbiologists are biological scientists microbiologists study the relationship

who study about organisms that are between microorganisms and disease
generally so small and can only be seen establishment.
with a microscope. These microorganisms Immunology – Those who work on
include bacteria, algae, yeasts, fungi, immune system related work are called
protozoa, viruses, and other microscopic immunologists. Immunologist study
forms of life. Some microbiologists the body’s defensive responses to
specialize in one type of microorganism. microorganisms. They learn how our
For example, bacteriologists concentrate immune system protects our body during
on bacteria and virologists study viruses. the infection. They suggest possible ways
Microbiologists isolate and make cultures to increase our immunity. It is one of the
of microorganisms, identify their fastest growing areas in science.
characteristics, and observe their reactions
Microbial Ecology - The microbial
to chemicals and other kinds of stimuli.
interactions with living and non-living
They also study how microorganisms
matters of the environmental habitats is
develop and reproduce as well as their
referred to as microbial ecology. Microbial
distribution in nature.
ecologists study the contributions of
microorganisms to the cycling of various
The Scope of Microbiology (Course
nutrients or elements. The ecologists are
Benefit / Advantages)
employed in reducing the pollution of the
The whole ecosystem depends on bacterial environment which is the burning issue in
activities. The modern microbiology is a all metro cities. They work on employing
large discipline with different specialities. microorganisms in bioremediation to
Microbiology has a great impact on reduce pollution effects.
fields such as medicine, agriculture, food
Food and Dairy Microbiology – Some
sciences, ecology, genetics, biochemistry
of our foods are actually the by-products
and molecular biology. There are many
of microbial growth. Example: Cheese is
possible avenues of advancement for
produced by the growth of microorganisms
microbiologists.
such as Leuconostoc citrovorum and
Medical Microbiology – Medical Streptococcus lactis. Yoghurt results
microbiologists are involved in from the growth of bacteria such as
identifying the microorganisms causing Lactobacillus bulgaricus and Streptococcus
the infectious diseases. They work on thermophilus in milk. The leavening of
identifying the pathogens and assist the bread is accomplished by Saccharomyces
medical practitioners for prescribing the cerevisiae (Baker’s yeast). Main work of
apt antibiotics in right dosages. They the food and dairy microbiologists in the
also study the ways in which the micro food industry is to prevent contamination
organisms cause the infection. Medical during processing and the transmission

of food borne diseases. Microbiologists and do research. Microbiologists can
are currently employed in all food and become directors of research in medical
dairy processing industries. They are also centres, private firms, or government
employed in Mineral water companies to agencies. Those who hold a teaching
check the quality of water. and research position in a university can
Agricultural Microbiology – It advance to the rank of full professor. They
is concerned with the impact of can also make significant discoveries in
microorganisms on agriculture. Most their research and gain the recognition
bacteria and fungi live saprophytically on of other microbiologists. Many scientists
dead and organic matter of the soil. They consider this to be the highest form of
decompose the complex organic matter advancement.
into simpler form making it available Microbial Genetics and Molecular
for the soil microorganisms. Thus they Biology – The use of micro organisms
form an important constituent of soil has been very helpful in understanding
called humus. Certain microbes increase the functions of the genes. Microbial
the fertility of the soil by converting the geneticists play an important role in
atmospheric nitrogen into ammonia, applied microbiology by producing new
nitrites and nitrates. This is brought about microbial strains that are more efficient
by the microbes such as Nitrosomonas, in synthesizing useful products. Genetic
Nitrobacter and Rhizobium sp. Agricultural techniques are used to test substances
microbiologists try to combat plant for their ability to cause cancer.
diseases that attack commercial food Microbiologists are in greater demand
crops and they also work on methods to in genetic engineering companies and
increase soil fertility and crop yields. research units.
Industrial Microbiology – Biomining – Microbes are used in
Microorganisms are used to make extracting valuable metals like uranium
products such as antibiotics, vaccines, from rocks. Thiobacillus ferrooxidans
steroids, alcohols, vitamins, amino acids unlocks energy from inorganic compounds
and enzymes. Some important drugs are like iron sulphide. During this process, it
synthesized by microorganisms such as produces sulphuric acid and iron sulphate.
streptomycin, penicillin, chloramphenicol, The use of micro organisms in mining has
tetracycline. Industrial microbiologists considerably reduced the cost of mining
work on improving the strains that to 75%. Microbiologists involved in
produce the industrially important Biomining research are highly paid in the
products and thereby increase the yield. Government sector.
The Research and Development (R&D) Medical coding – Medical coding
units in the industries provide various job is the transformation of healthcare
opportunities to microbiologists. diagnosis, procedures, medical services
Directors of Research Units and and equipment into universal medical
Universities – Many microbiologists alphanumeric codes. The diagnoses
work for universities, where they teach and procedure codes are taken from
VI

medical record documentation, such which conduct selection through entrance
as transcription of physician’s notes, examination. Candidates can choose any
laboratory and radiologic results. Medical of the specialization streams in order to
coding jobs are assigned to Life Science, choose courses related to Microbiology
Paramedical and Medical Graduates and • Agricultural Microbiology
Post Graduates.
• Food microbiology
Editor in Scientific Journals – Editing,
• Medical Microbiology
proof reading in scientific journals,
handle manuscripts on topics ranging • Pharmaceutical Microbiology
from Zoology, Biology, Plants and Animal • Microbial Genetics
sciences are few assignments that could • Environmental Microbiology
be accomplished by microbiologists.
• Aero Microbiology
Microbiologists review the research
articles that are to be published in reputed • Microbial Physiology
National and International Journals.
Different Courses in Microbiology
They are employed as Editors and
Associate Editors of Scientific Publishing • Bachelor of Science in Microbiology
Companies. • Bachelor of Science in Microbiology
Pharma companies – A microbiologist in and Microbial Technology
a pharmaceutical company is a member • Bachelor of Science in Clinical
of quality department. The role of the Microbiology
microbiologist is to ensure the quality of
• Bachelor of Science in Medical
raw materials before they are processed
Microbiology
in the production area, monitor the
microbiological quality of environment • Bachelor of Science in Industrial
and water and validate the test methods Microbiology
used in testing the finished products from • Bachelor of Arts in Microbiology
microbiological perspective. • Diploma Courses in Microbiology
• Post Graduate Diploma in Marine
Eligibility Criteria for Undergraduate
Microbiology
level courses in Microbiology
• M.Sc in Microbiology
In order to apply for under graduate level
• M.Sc in Applied Microbiology
courses in Microbiology, candidates should
complete 12th class. It is important to opt • M.Sc in Microbial Genetics and
Physics, Chemistry and Biology subjects in Bioinformatics
12th class to join for Microbiology courses.
Candidates need to score good percentage Universities offering Courses in
of marks in 12th class as the selection Microbiology
process for undergraduate level courses • Indian Institute of Technology
in this stream will be based on the marks • Banaras Hindu University
scored. There are certain top universities
VII

• Aligarh Muslim University • Indian Institute of Science
• University of Mumbai • Amity University
• Vinayaka Mission University • Kurukshetra University
• Mahatma Gandhi University
Para Medical Courses and certificate courses in Tamilnadu Government Medical

Colleges
1 Year Certificate Courses

Courses Educational Age limit
Qualification
Cardio Sonography Technician
ECG/ Tread Mill Technician
Pump Technician
Cardiac Catheterisation Lab Technician
Emergency Care Technician Pass in H.Sc.
Dialysis Technician with physics,
Chemistry,
Anaesthesia Technician Should complete 17 yrs
Botany &
Theatre Technician Should not exceed 32 yrs
Zoology (or)
Orthopaedic Technician Biology and
Audiometry Technician Microbiology
Hearing Language and Speech Technician
Clinical, Therapeutic, Nutrition & Food Ser-
vice Management Technician
E.C.G/E.M.G Course Technician
Multipurpose Hospital Worker Course Pass in SSLC
2 Years Diploma Courses

Courses Educational Age limit
Qualification
Dental Mechanic(Male)
Dental Hygienist (Female) Pass in H.Sc. with
Should complete
Diploma in Medical Lan Rechnology (Dmlt) physics, Chemistry,
17 yrs
Botany & Zoology
Diploma in Radio Diagnosis Technology (Drdt) Should not exceed
(or) Biology and
Diploma in Radio Therapy Technology (Drtt) 32 yrs
Microbiology
Diploma in Optometry
VIII

Medical Record Science
Courses Educational Qualification Age limit
Diploma in Medical Record Pass in H.Sc. with physics, Should complete 17 yrs
Technician (Six Months) Chemistry, Botany & Zoology Should not exceed 32 yrs
(or) Biology and Microbiology
Job Prospects similarities between microbiology

Candidates who have studied courses and biotechnology, the career options
related to Microbiology have good scope available for professionals in the field
for jobs in different sectors. Candidates of biotechnology are applicable to the
can take up jobs in private sectors professionals in the field of microbiology
mainly in pharmaceutical companies, as well.
research firms. Candidates can get in
Central Government jobs after M.Sc
to roles like Medical Microbiologists,
Microbiology
Agricultural Microbiologists, and Marine
Microbiologists. Candidates can join Post Graduates of Microbiology can find
for teaching jobs as well. Jobs are also plenty of job opportunities in the Central
available in public sector after doing under Government sector. Several vacancies
graduate or post graduate level courses in are available for them in the research
Microbiology. Job opportunities occur institutes run by Central Government.
in government controlled development These graduates can apply for Scientist,
laboratories, chemical industries, Research Assistant, Technical Assistant,
hospitals, food industry, pharmaceutical Field Assistant or Project Assistant
companies. Apart from this, candidates posts in these institutes whenever
can also try for jobs abroad. Candidates vacancies are available. Institute of Liver
who attain good experience in this field and Biliary Sciences, New Delhi offers
will get higher salary packages in jobs. Microbiologist job for these graduates.
They can apply for this post when
Career Prospects after completion of the notification gets published in the
B.Sc Microbiology course newspaper or website. Staff Selection
Candidates, who have completed Commission conducts Combined
their B.Sc Microbiology, can become Graduate Level Exam for recruiting
microbiologists and there is wide range graduates to various departments in
of employment opportunities available the Government. Those who have
for microbiologists. They can find job completed M.Sc Microbiology can apply
placement in research laboratories and for this exam, if they are interested to
research organizations in public sector work in the Government sector. There
and private sector. They can also find are many laboratories working under
job placement in pharmaceutical firms, the supervision of Council of Scientific
chemical firms. Since there is many and Industrial Research (CSIR). M.Sc
IX

Microbiology graduates can apply The Future of Microbiology
for various posts available for them Microbiology has a clearer mission than
in these laboratories. There are also other scientific disciplines. It is confident of
vacancies available for these graduates its value because of its practical significance.
in Government hospitals. The following brief list will give us some
idea of what the future may hold:
Teaching Profession in Government
Sector after M.Sc Microbiology • Everyday microbes are changing its
nature (mutation) and new diseases are
Candidates who want to work in the emerging. Microbiologists will have to
teaching field after M.Sc Microbiology can respond to these threats.
apply to various colleges or universities.
An M. Phil / Ph.D degree is required for • Microbiologists must find ways to stop
these graduates to apply for these posts. the spread of established infectious
They also need to clear NET exam so as to diseases.
be eligible for teaching posts available in
various universities. • Microbial diversity is another area
requiring considerable research.
Microbiology in India • Much work needs to be done on
There are number of Institutes engaged in microorganisms living in extreme
microbiological research in our country. environmental conditions. The
The Indian Institute of Petroleum, discovery of new microorganisms may
Dehradun; Tata Energy Research Institute, lead to further advances in industrial
Delhi and National Chemical Laboratory, processes and enhanced environmental
Pune have worked on microbial dewaxing control.
of heavier petroleum fractions. The
Institutes has also played a vital role on the • The genomes of many micro organisms
area of microbial enhanced oil recovery already have been sequenced, and
and production of biosurfactants. National many more will be determined in the
Institute of Nutrition, Hyderabad and coming years.
National Institute of Occupational Health, • Microorganisms are essential partners
Ahmedabad have already completed a long with higher organisms in symbiotic
time plan on monitoring and surveillance relationships. Greater knowledge
of food contaminants hazards in India of symbiotic relationships can help
while genome analysis and synthetic improve our appreciation of the living
gene design for modulation of genome world. It also will lead to improvements
expression invivo was carried out by the in the health of plants, livestock and
scientists of Indian Institute of Science, humans.
Bangalore.

Chapter 1 Microbiology includes the study of
Introduction to
Microbiology
Bacteria Algae
Chapter Outline
1.1 Groups of Microorganisms Protozoa
1.2 Contributors to Microbiology

1.3 Branches of Microbiology Viruses Fungi
Microorganisms - Bacteria, Fungi, Algae,

Protozoa and Viruses - have been around for at least
3,500 million years. Microbes affect every aspect of
life on earth. They have an amazing variety of shapes
and sizes. They can exist in a wide range of habitats.
Science knows no country, because knowledge belongs to humanity, and is the torch
which illuminates the world.
Louis Pasteur
Learning Objectives
After studying this chapter the student developments in biotechnology, genetic
will be able, engineering and nanotechnology have
• To know the features of microorgan- placed Microbiology in the limelight.
isms. Microorganisms provide the model for
interdisciplinary research and for studying
• To know the contributions of
fundamental life processes. There is
different scientists.
growing recognition of microorganisms
• To know the branches of and their potential in many applied
Microbiology. areas like Environmental science,
Agriculture, Food and Pharmaceutical
Microbiology is one of the fascinating industries. The uses of microorganisms
fields of science. Microorganisms and their are becoming increasingly attractive.
activities are the major concerns of society Some microorganisms are beneficial to
both nationally and internationally. The human and cannot live without them.
TN_GOVT_XI_Micro_Biology_CH01.indd 1 16-02-2019 9.54.21 AM

However microorganisms can be harmful usually multicellular. They range in size
in many ways and bring about undesirable and shape from single celled microscopic
changes. These microorganisms can cause yeasts to giant multicellular mushrooms
diseases that can make us sick or even kill and puffballs.
us. Although much more is known today Example: Aspergillus niger, Agaricus
about microbial life than ever before, the bisporus
vast majority of this invisible world remains
Protozoa: They are unicellular
unexplored. Microbiologists continue to
eukaryotic organisms. Their role in nature
identify new ways that microbes benefit
are varied. The best known protozoa cause
and threaten humans.
disease in human beings and animals.
Microbiology is the study of living
Example: Giardia lamblia, Plasmodium
organisms of microscopic size, which
vivax
include bacteria, fungi, algae, protozoa,
and viruses. Microbiology is concerned Algae: They range from unicellular,
with form, structure, reproduction, colonial to multicellular forms. All algal
physiology, metabolism, and classification cell contain chlorophyll and are capable
of microorganisms. It includes the study of of photosynthesis. They are found most
commonly in aquatic environments and
• their distribution in nature, damp soil.
• their relationship to each other and to Example: Spirogyra, Chlamydomonas
other living organisms, Viruses: In the study of Microbiology,
• their effects on human beings, animals we encounter “organisms” which may
and plants, represent the borderline of life. Viruses
•
their abilities to make physical and are simpler in structure and composition
chemical changes in our environment, than other living cells. A virus is made
• their reaction to physical and chemical up of nucleic acids and proteins. Viruses
agents. are obligate parasites. They grow only
within an appropriate host cell (plant,
1.1 Groups of Microorganisms animal, humans or microbe). They cannot
multiply outside a host cell.
There are many kinds of microorganisms
present in the universe. They are broadly Example: HIV, Rabies virus
classified into the following groups.
Bacteria: They are unicellular
prokaryotic organisms or simple association
of similar cells. Cell multiplication usually Prions are infectious
happen by binary fission. agents composed
Example: Escherichia coli, Bacillus entirely of protein
subtilis material. Creutzfeldt–
Jacob Disease (CJD) is one of the human
Fungi: They are eukaryotic organisms
prion diseases.
which is devoid of chlorophyll. They are

1.2 Contributors to Microbiology
Many scientists contributed to the science
of Microbiology from the 17th century
to the present day. Some prominent
microbiologists who have made
significant contribution to the study of
microorganisms are given below:
1.2.1 Antony Van Leeuwenhoek

Antony Van Leeuwenhoek (1632-1723) of
Holland (Figure 1.1) developed microscopes.
He was a Dutch merchant and a skilled lens Figure 1.1:
maker. He made a variety of lenses with Antony Van Leeuwenhoek
magnifying power 50-300X. Antony Van Leeuwenhoek wrote
He was the first person to invent simple many letters. He wrote them in
microscope. It has a single biconvex lens with a Dutch, the only language that he
magnification of about 200X (Figure 1.2). His knew. These letters, described his
microscopes resolved bodies with diameters complete scientific output. Antony
measuring below 1micron. He examined Van Leeuwenhoek in a letter dated
water, mud, saliva and found living 12th June 1716, wrote “... my work,
organisms. He called these microorganisms which I’ve done for a long time, was
as Animalcules (little animals). Bacteria like not pursued in order to gain the praise
cocci, bacilli and spirochetes were recognized. I now enjoy, but chiefly from a craving
He proposed that the size of bacteria is one after knowledge, which I notice resides
sixth the diameter of Red Blood Cells. in me more than in most other men.
He observed the growth of bacteria in And therewithal, whenever I found out
infusions. The existence of spermatozoa anything remarkable, I have thought
and RBC was revealed by him. Animal it my duty to put down my discovery
histology was established by him. He on paper, so that all ingenious people
described capillary circulation and added might be informed thereof”.
a new dimension to Biology. All kinds of
unicellular microorganisms were accurately
described by him including human oral Contribution to science as a chemist
microbial flora. He is commonly known as Louis Pasteur was working with tartaric
the ‘Father of Microbiology’. acid crystals. He could pick up the dextro
and levo rotatory crystals by seeing the
1.2.2 Louis Pasteur (1822-1895) morphology of the crystals. Later he was
Louis Pasteur was a French chemist and a called to solve some of the problems
crystallographer (Figure 1.3). His greatest in fermentation industry and turned
contribution to microbiology made him to his attention to biological process of
be the ‘Father of Modern Microbiology’. fermentation.
3

Lens
Sample
Observer’s eye
Mounting pin
Focusing screws
Figure 1.2: Leeuwenhoek’s Microscope
theory of spontaneous generation. He strongly

supported theory of Biogenesis (life orginates
from pre-existing life forms). To prove this he
carried out several experiments. Pasteur poured
meat infusions into flasks and then drew the top
of each flask into a long curved neck that would
admit air but not dust (Figure 1.4). He found
that if the infusions were heated, they remained
sterile (free from any growth) until they were
exposed to dust. After opening them on a dusty
Figure 1.3: Louis Pasteur (1822-1895)
road and resealing them, he demonstrated the
Contribution to Microbiology growth of microorganisms in all the flasks. The
unopened flasks were sterile. Thus he disproved
To wine industry
the theory of spontaneous generation.
Louis Pasteur discovered alcohol
production from grape juice was due to Pasteurization
yeast. The presence or contamination Louis Pasteur used heat to destroy undesirable
of rod shaped bacteria resulted in large microbes in fruit juices. He employed 62.8°C
amounts of lactic acid production in wine. (145°F) for 30 mins to kill microbes. This
He also found that microorganisms in process is called Pasteurization which is
fermented fruits and grains, resulting in commonly used in distillaries and dairy
alcohol production. He coined the term industry.
“fermentation”.
Discovery of diseases
Pasteur disproved spontaneous generation Louis Pasteur found that Pebrine disease
Spontaneous generation states that life could in silk worm was caused by a protozoan
arisespontaneouslyfrominanimate(non-living) parasite. He suggested that Pebrine disease
materials (Abiogenesis). Pasteur disproved the could be eliminated by using only healthy,
4

Wait
Boil
No growth
Wait
Break
Boil neck
Microbial growth
Figure 1.4: Pasteur’s swan neck flask experiment
disease free silk worms. Wool Sorter’s disease an individual against the disease. He
was named as “Anthrax” by him. He isolated developed vaccines for anthrax and rabies.
Bacillus anthracis from the blood of infected
1.2.3 Edward Jenner (1749-1823)
animals. Chicken cholera bacterium was
also isolated by Louis Pasteur using pure In ancient observation, persons who had
culture. suffered from a specific disease such as
small pox (causative agent of small pox
He proved that many
is varicella virus) or mumps, resisted the
diseases were caused by
infection on subsequent exposures. They
the presence of foreign
rarely contracted these infections for second
microorganisms (Germ
time. Edward Jenner, a country doctor in
theory of disease).
England noted a pustular disease on the
He discovered various
hooves of horses called the grease. This was
infection causing microorganisms such
carried by farm workers to the nipples of
as Staphylococcus, Streptococcus and
cows (cow pox). This was again carried by
Pneumococcus.
milk maids. They got inflamed spots on the
Vaccination hands and wrists. The people who got this
Pasteur found out that bacteria could cow pox were protected from small pox.
be attenuated by growing them in He reported that 16 farm workers who had
unnatural conditions. He coined the term recovered from cow pox (causative agent of
“attenuation”. It is a process wherein cow pox is vaccinia virus) were resistant to
bacteria lose their virulence due to small pox infection.
repeated subculturing under laboratory He took the material (pus) from the cow
conditions. He used attenuated cultures as pox and inoculated into the cut of 8 year
vaccines for immunizing and protecting old boy on 14th May 1796 (Figure 1.5). Two
5

months later Jenner inoculated the same He modified Ziehl-Neelsen Acid Fast
boy with material taken from small pox staining procedure which was introduced by
patients. This was a dangerous but accepted Ehrlich. He devised solid medium to grow
procedure at that time. This procedure was microorganism. He developed powerful
called variolation. The boy was protected method to isolate the microorganisms in
against small pox. His exposure to the mild pure culture from diseased tissue. He also
cow pox disease had made him immune perfected the techniques of identification of
to the small pox disease. In this manner the isolated bacteria.
Jenner began the Science of Immunology, He introduced Koch’s thread method
the study of the body’s response to foreign to find out the efficacy of disinfectants.
substances. Edward Jenner was regarded as He established certain rules that must be
the ‘Father of Immunology’. followed to establish a cause and effect
relationship between a microorganism
and a disease. They are known as
Koch’s Postulates. He also described the
Koch’s Phenomenon. He was regarded as
the ‘Father of Medical Microbiology’.
Infobits
Koch’s Thread Method

Figure 1.5: Dr. Edward Jenner Robert Koch carried out systematic
performing his first vaccination (1796)
experiments on disinfection, using
pure cultures of bacteria. By means of
1.2.4 Robert Koch (1843-1910) his Thread Method, he investigated the
Robert Koch was a effect on anthrax spores of the popular
German physician disinfectants at that time. Koch’s Thread
and microbiologist Method also called as carrier test. A
(Figure 1.6). He carrier such as silk is contaminated by
was the founder submerging in a liquid culture of the
of Modern Bacillus anthracis, a test organism. The
Bacteriology. Robert carrier is further dried and immersed
Koch discovered in the disinfectant solution for a given
Bacillus anthracis exposure time. Thereafter the thread is
(Anthrax bacillus), Figure 1.6: cultured in a nutrient broth. No growth
My c o b a c t e r i u m Robert Koch after incubation indicated that the
tuberculosis, and (1843-1910) product (disinfectant) is active.
Vibrio cholerae. For the first time he showed
the evidence that a specific germ (Anthrax Koch’s Postulates
bacillus) was the cause of a specific disease Four criteria were established by Robert
(splenic fever in sheep) and introduced Koch to identify the causative agents of an
scientific approach in Microbiology. infectious disease. These include
6

1. A specific organisms can always be 4. It is possible to recover the organism in
found in association with a given pure culture from the experimentally
disease. If we take typhoid as an infected animals and it is observed to
example it is caused by a bacterium be the same as originally inoculated
Salmonella typhi. pathogen. Figure 1.7 explains the
2. The organism can be isolated and Koch’s postulates.
grown in pure culture in the laboratory. Limitations
Salmonella typhi are grown in soild
Some organisms have not yet been grown
media under laboratory conditions.
in artificial culture media
3. The pure culture will produce the
Example: Mycobacterium leprae and
diseases when inoculated into a
Treponema pallidum.
susceptible animal.
Modern addition to Koch’s Postulates
Almost all the pathogenic organisms
produce the same disease in experimental Today we recognize additional criteria of
animals. Usually rats, mice, rabbits or causal relation between a microorganism
guinea pigs are used as experimental and a disease. The important one is
animals. Pneumococci produce the demonstration of abnormally high
pneumonia in animals. Salmonella species concentration of specific circulating
do not produce typhoid fever in rat, antibodies to the organism in the infected
mice or rabbit. So chimpanzee is taken host or the presence of abnormally
as experimental animal and it produces high degree of specific immunity or
fever in chimpanzee. hypersensitivity to the infecting agent in
Koch postulates
Postulate 1
The same microorganisms are
present in every case of the
disease.
Anthrax bacilli
Postulate 2
The microoraganisms
are isolated from the
tissues of a dead
animal, and a pure
culture is prepared. Postulate 4
The identical micro-
organisms are isolated
and recultivated from
the tissue specimens
of the experimental
animal.
Postulate 3
Microorganisms from the
pure culture are inoculated
into a healthy , susceptible
animal. The disease is
reproduced.
Figure 1.7: Koch’s postulates for infectious diseases

7

a recently recovered host. In addition to
culture techniques, serological techniques
are also used for diagnosis of diseases.
Usefulness of Koch’s Postulates

• It is useful in determining pathogenic
organisms.
•
To differentiate the pathogenic and
nonpathogenic microorganism.
• For the classification of organisms.
• To detect the susceptibility or resistance Figure 1.9: Original culture plate on
of the laboratory animals. which the observation of action of
penicillin was made by Alexander Fleming
1.2.5 Joseph Lister(1827-1912) inhibited (Figure 1.9). He also showed
Joseph Lister was that the culture filtrate of mold inhibited
a British surgeon the growth of Staphylococcus aureus. He
(Figure 1.8). He called this substance Penicillin, which
found out that acted on Gram positive bacteria. For
microorganisms the discovery of this antibiotic Fleming
were responsible for (Figure 1.10), Florey and Chain got Nobel
wound infections. He Prize in 1945. Penicillin eventually came
developed a system into use during world war II as a result
of antiseptic surgery. Figure 1.8: of the work of a team of scientists led by
He used bandages Joseph Lister Howard Florey of the University of Oxford.
soaked in phenol (1827-1912)
solution to prevent wound infection.
He sterilized instruments by heat and
sprayed diluted phenol over surgical area
and prevented contamination of wounds.
He was the first person to isolate bacteria
in pure culture using liquid culture.
Thus, he was considered as co-founder
Figure 1.10: Alexander Fleming (1881-1955)
of Medical Microbiology with Koch, who
later isolated bacteria on solid media.
Alexander Fleming,
1.2.6 Alexander Fleming (1881-1955) the discoverer of
penicillin warned
He was a British Bacteriologist. He
about the possibility
observed a mold (Penicillium notatum)
of antibiotic resistant bacteria due to
growing on a plate of Staphylococcus
antibiotics misuse, as early as in 1920s.
aureus. The growth of Staphylococcus
aureus around the mold colony was
8

1.2.7 Selman Abraham Waksman griseus which is not required for its
(1888-1973) growth but may help it to compete with
Waksman was from Rutger University, other bacteria for food and space in the
USA (Figure 1.11). His research was environment. Streptomycin is used in
largely on soil microorganisms. He showed the treatment of tuberculosis. Waksman
antimicrobial activity of streptomyces that got Nobel Prize in 1952. for his work on
led to the discovery of Streptomycin and Streptomycin
several other antibiotics.
Antibiotics are
usually not effective
for sore throats and
common colds. They
are commonly caused by viruses rather
than bacteria. Taking antibiotics for such
illnesses is considered more harmful
than beneficial.
Figure 1.11: Selman Abraham Waksman

(1888-1973) 1.3 Branches of Microbiology
Waksman and his co-workers isolated Microbiology can be classified into Pure and
Actinomycin in 1940, Streptothrecin in 1942, Applied Microbiology. Pure Microbiology
Streptomycin in 1943, and Neomycin in 1949. is classified based on taxonomical and
Streptomycin is produced by integrative characteristics. Table 1.1 shows
Streptomyces griseus. It is a secondary various branches of microbiology.
metabolite produced by Streptomyces
Table 1.1: Branches of Microbiology
Based on Taxonomical characteristics

Bacteriology The study of bacteria
Mycology The study of fungi
Protozoology The study of protozoa
Based on Taxonomical characteristics
Phycology (or algology) The study of algae
Parasitology The study of parasites
Immunology The study of the immune system
Virology The study of viruses
Nematology The study of the nematodes

Based on integrative characteristics
Microbial Cytology The study of microscopic and sub microscopic details

of microorganisms
Microbial Physiology The study of biochemical functions of microbial cell. It
also includes the study of microbial growth, microbial
metabolism and microbial cell structure
Microbial Ecology The study of relationship between microorganisms and
their environment
Microbial Genetics The study of gene are organisation and regulation in
microbes in relation to their cellular functions.
Cellular Microbiology A discipline bridging microbiology and cell biology
Evolutionary Microbiology The study of the evolution of microbes
Microbial Taxonomy The study of naming and classification of microorganisms
Microbial Systematics The study of the diversity and genetic relationship of

microorganisms
Systems Microbiology A discipline bridging systems biology and microbiology
Generation Microbiology The study of microorganisms which have the same
characters as their parents
Molecular Microbiology The study of the molecular principles of physiological
processes in microorganisms
Nano Microbiology The study of microorganisms at nano level
Exo Microbiology (or Astro The study of microorganisms in outer space
Microbiology)
Biological Warfare The study of microorganisms used in weapon industries
Applied microbiology
Medical Microbiology The study of the pathogenic microbes and the role
of microbes in human illness. Includes the study of
microbial pathogenesis and Epidemiology and is related
to the study of disease, Pathology and Immunology
Pharmaceutical The study of microorganisms that are related to the
Microbiology production of antibiotics, enzymes, vitamins, vaccines,
and other pharmaceutical products
10

Industrial Microbiology The study of exploitation of microbes for use in industrial
processes. Examples include industrial fermentation and
waste water treatment. This field also includes brewing,
an important application of microbiology
Microbial Biotechnology The study of manipulation of microorganisms at the
genetic and molecular level to generate useful products
Food Microbiology and The study of microorganisms in food spoilage, foodborne
Dairy Microbiology illness and food production.
Summary
Student Activity
Microbiology is the study of
microorganisms that includes bacteria, 1. Want to see spontaneous
fungi,algae,protozoa and viruses. Many generation of life?
scientists contributed to the science of Take chicken soup or meat soup
microbiology. boil it in a bottle. Keep it over
Antony Van Leuwenhoek made the shadow of your window/or in
simple microscope. For the first time, a open place with mouth open.
Antony Van Leuwenhoek described the Observe for a week. You will
microorganisms. Louis Pasteur disproved see maggots (worms) growing.
the theory of spontaneous generation. Observe and record your findings.
Germ theory of disease came from the 2. For you to enjoy-like Antony Van
work of Pasteur and Robert Koch. Vaccines Leeuwenhoek !!
for Anthrax and rabies was developed Get a palmist lens, see through it
by Pasteur. Direct relationship between a paper print. You will see letter
the suspected pathogen and disease was becomes big, bigger, and at one
established by Koch’s postulates. Koch point it is no longer magnifying
developed the technique of pure culture the letter. A simple convex lens is
on solid medium. Joseph lister developed magnifying things. Leeuweenhoek
antiseptic surgery. Alexander Fleming used such lens only. (as seen
discovered Penicillin. Waksman showed above) You know useful and
antimicrobian activity that led to the useless magnification.
discovery of Streptomycin and other
antibiotics. The branches of microbiology
can be classified into pure and applied
microbiology. Pure microbiology is
classified based on taxonomical and
integrated characteristics. Microbiology
has got vast areas open for job
opportunities.
11

12

ICT CORNER
Microbiology
Lets meet our

micro friends
STEPS:
• Use the URL or scan the QR code to open ‘NPTEL’ page.
• Click ‘History’ and ‘Scope of Microbiology’ to know the history of microbiology.
• Select history of microbiology and click ‘Start Course’ at the bottom.
• Select ‘Members of the Microbial world’ to know about it.
Step1 Step2 Step3
URL:
http://nptel.ac.in/courses/102103015/41#
13

Evaluation c. Streptomyces griseus
Multiple choice questions d. Penicillium mornefii
1. Theory of spontaneous generation 7. Which of the following antibiotics
were discovered by Selman Abraham
was disproved by whom?
Waksman?
a. Robert Koch
a. Streptomycin
b. Edward Jenner
b. Neomycin
c. Louis Pasteur
c. Actinomycin
d. All of them
d. All the above
2. Which of the following did Edward
Jenner used to protect the boy against Answer the following
small pox?
1. Name the causative agent of cow pox
a. Cow pox material and small pox.
b. Small pox material 2. Explain the method of Edward Jenner
c. Both the above used to protect people against small
d. Rabbit pox pox.
3. Among the following scientists, who 3. List two organisms that do not obey
discovered solid medium? Koch’s postulates.
a. Louis Pasteur 4. Give the usefulness of Koch’s
b. Edward Jenner postulates.
c. Robert Koch 5. What are the modern additions to
d. None of them Koch’s postulates?
4. Which of the following organisms 6. List the contribution of Alexander
does not obey Koch’s postulates? Fleming.
a. Cow pox virus 7. What is the theory of spontaneous
generation?
b. Small pox virus
8. How was spontaneous generation
c. Treponema pallidum
theory disproved?
d. M.Tuberculosis
9. Highlight the contribution of
5. Who modified Ziehl-Neelsen staining
Waksman.
technique?
10. State the characteristics of
a. Louis Pasteur streptomycin.
b. Robert Koch 11. Give a list of contribution of Louis
c. Ziehl-Neelsen Pasteur to wine industry.
d. All the above 12. Explain Koch’s postulates?
6. Which of the following fungi grow on 13. Describe the microscope made by
Alexander Fleming’s plate? Antony Van Leeuwenhoek.
a. Penicillium chrysogenum 14. What are the contributions of Antony
b. Penicillium notatum Van Leeuwenhoek to microbiology?
14

Chapter 2
Microscopy
Chapter Outline
2.1 Historical Background

2.2 Principles of Microscopy
2.3 Bright Field Microscope
2.4 Dark Field Microscope
Microorganisms are very small and cannot be viewed

by human eye. The microscope helps in observing the
microbial world which exists in a wide range of sizes. The
prokaryotes (bacteria and archae) are smaller (~ 0.4-10µm)
and the eukaryotes are larger (~ or >10µm). The word
microscope is derived from the Latin word micro, which
means small, and the Greek word skopos means to look at.
Robert Hooke, built compound

Learning Objectives microscopes with multiple lenses. In 17th
century, Dutch spectacle maker Zaccharias
After studying this chapter the student
Janssen is given the credit for making first
will be able, compound microscope. However, the early
• To know the properties of light and compound microscopes were poor in quality.
lens. In 1830, Joseph Jackson Lister (the father of
Joseph Lister who practised antiseptic surgery)
• To know the science of image made significant development which resulted
formation in brightfield microscopy. in the invention of modern compound
• To understand the design of light microscope used in microbiology today.
microscope.
• To learn and compare the principle, 2.2 Principles of Microscopy
instrumentation and working of All kind of microscopes use visible light
brightfield and darkfield microscopy. to observe specimens. Light has a number
of properties that affect our ability to
visualise objects.
2.2.1 Properties of Light
Antony Van Leeuwenhoek (1632-1723)
was the first person to use a simple Light is a part of the wide spectrum of
microscope with one lens similar to a electromagnetic radiation from the sun. It
magnifying glass. The lens is capable of is a form of energy. The most important
50X to 300X magnification. property of light is wavelength (the length
15

of light ray) (Figure 2.1). light waves or light particles. The
combined properties of particle and
One wavelength wave enable light to interact with an
Wave Crest
object in several different ways like
transmission, absorption, reflection,
refraction, diffraction and scattering
(Figure 2.3).
Wave trough
2.2.2 Lenses and its Properties
Figure 2.1: Wavelength-the distance
between two adjacent crests or two Lenses are optical devices which focus
adjacent troughs of the wave and denoted or disperse a light beam by means of
by greek letter (λ) refraction. A simple lens consists of a
single piece of transparent material.
The sun produces a continuous spectrum Light rays from a distant source are
of electromagnetic radiation with waves of focused at the focal point F. The focal
various lengths (Figure 2.2). Radiation of point lies at a distance f (focal length)
longer wavelength includes Infrared (IR) from the lens’ centre (Figure 2.4).
and radiowaves, the shorter wavelengths
include Ultra Violet (UV) rays and X-rays.
The physical behaviour of light can
be caterigorised as either light rays,
Increasing wavelength
Increasing frequency
1×10-6 nm
1×10-2 nm
(wavelength)
400 nm
700 nm
100 km
10 nm
10 cm
1 nm
X-rays UV IR Microwave Radio and TV
400 nm Visible light 700 nm
Figure 2.2:Transmission Reflection

The electromagnetic spectrum-White Refraction
light is a combination of all colours of
visible spectrum
Transmission Reflection Refraction
Diffraction Absorption Scattering
Figure 2.3: Interaction of light with matter

16

Microscope resolution
Microorganisms
Objective is the important part in the
are measured in microscope which is responsible to produce a
micrometers and clear image. The resolution of the objective is
nanometers. The most important. Resolution is the capacity of
average bacterial cell is 0.001mm in a lens to separate or distinguish between small
diameter. objects that are close together. The major
factor in the resolution is the wave length of
light used. The greatest resolution obtained
with light of the shortest wave length, that
F
is the light at the blue end of the visible
spectrum are in the range of 450 to 500nm.
The highest resolution possible in compound
f
light microscope is about 0.2µm. That means,
the two objects closer together than 0.2µm
Figure 2.4: Lens function are not resolvable as distinct and separate.
The light microscope is equipped with three
Generating an image with a lens
or four objectives. The working distance of
When an object is placed outside the an objective is the distance between the front
focal plane (the plane containing the surface of the lens and the surface of the
focal point of the lens), all the light rays
cover glass or the specimen. Objectives with
from the object are bent by the lens.
large numerical apertures and great resolving
The bent rays converge at the opposite
focal point. At the focal point, the power have short working distances.
light rays continue and converge with Numerical aperture
nonparallel refracted light rays. The
resultant reversed and magnified image Numerical Aperture (NA) is the value
is formed in the plane of convergence representing the light gathering capacity
(Figure 2.5). of an objective lens. NA was first described
Object
F Focal point F’ Focal point
Focal distance
Focal plane Focal plane Real
Biconvex lens image
Figure 2.5: Generating an image with a lens
17

by Ernst Abbe, and is defined by the The larger the numerical aperture
following expression the better the resolving power. It is
f important to illuminate the specimens
properly to have higher resolution.
The concave mirror in the microscope
creates a narrow cone of light and has a
F
D
(Diameter
small numerical aperture. However, the
of lens)
resolution can be improved with a sub
stage condenser. A wide cone of light
through the slide and into the objective
lens increases the numerical aperture
Numerical Aperture (NA) =n × sin(θ) there by improves the resolution of the
microscope.
n = the refractive index of the medium
between the specimen and objective; Types of microscopes
θ = half aperture angle or collection angle In order to view microorganism and
of the objective. (the maximum half angle microbial structures of different sizes we
of the cone of light that can enter or exit require different kinds of microscopes.
the lens). • Light microscopes resolve images with
the help of light. The specimen is viewed
Infobits as dark object against a light background
in bright field microscope. Dark field
The smallest cells on the planet are microscope uses a special condenser
some forms of Mycoplasma with and the specimen appears light against
dimensions of 0.2 to 0.3 µm, which is a black background. The other types
within the limit of resolution of light of mircoscopes are Phase contrast and
microscopes. Tiny cells that look like Fluorescence microscope.
dwarf bacteria but are 10 times smaller • Electron microscope uses a beam of
than Mycoplasma and 100 times electrons instead of light. Electrons pass
smaller than the average bacterial cell through the specimen and form a two
are called nanobacteria or nanobes dimensional image in Transmission
Electron Microscope (TEM). Electrons
(Greek nanos means one billionth).
are reflected from the specimen and
produce a three dimensional image in
The resolving power of a light
Scanning Electron Microscope (SEM).
microscope depends on the wavelength
of light used and the NA of the objective
2.3 Bright Field Microscope
lens.
The numerical aperture of a lens can The most commonly used microscope
be increased by for general laboratory observations is the
standard bright field microscope (Figure 2.6).
• Increasing the size of the lens
It contains the following components
opening and/or
• Increasing the refractive index of • A mirror or an electric illuminator is
the material between the lens and the light source which is located at the
the specimen. base of the microscope.
18

Figure 2.6: Bright field Microscope
• There are two focusing knobs, the which is many times larger than the real
fine and the coarse adjustment knobs image. This magnification occurs when light
which are located on the arm. These rays from an illuminator (light source), pass
are used to move either the stage or the through a condenser which has lenses that
nosepiece to focus the image.
direct the light rays through the specimen. The
• Mechanical stage is positioned about light rays then pass into objective lens (the lens
half way up the arm, which allows
closest to the specimen). The image is again
precise contact on moving the slide.
magnified by the ocular lens or the eyepiece.
• The substage condenser is mounted
(Figure 2.7).
within or beneath the stage and
focuses a cone of light on the slide. In • Magnification is the process of
the simpler microscope, its position is enlarging the image of the specimen
fixed where as in advanced microscope and can be calculated by multiplying
it can be adjusted vertically. the objective lens magnification power
The upper part of microscope arm holds by ocular lens magnification power.
the body assembly. The nose piece and one or Representative magnification values for
more eyepieces or oculars are attached to it. a 10X ocular are:
The body assembly contains series of mirrors Scanning objective (4X) × (10X) = 40X
and prisms so that the barrel holding the magnification
eyepiece may be tilted for viewing. Three or five Low power objective (10X) × (10X) =
objectives with different magnification power 100X magnification
are fixed to the nosepiece and can be rotated High dry objective (40X) × (10X) = 400X
to the position beneath the body assembly. In magnification
bright field microscopy; the specimen is viewed Oil immersion objective (100X) × (10X)
against a bright background. The details of the = 1000X magnification
image are defined by the surrounding light. A
series of finely ground lenses forms an image
19

Path of light rays (Bottom to top) to eye Oil Immersion
Ocular lens enlarges
primary image formed Oil immersion lens is designed to be in
by objective lenses.
Prism that direct contact with oil placed on the cover
directs rays to
ocular lens slip. An oil immersion lens has a short
focal length and hence there is a short
working distance between the objective
lens and the specimen. Immersion oil has
Objective lenses (those closest a refractive index closer to that of glass
to specimen) form the primary
image. Most compound light than the refractive index of air, so the use
microscopes have several.
of oil increases the cone of light that enters
the objective lens. Figure 2.8 explains
Stage supports
microscope slide
the working principle of oil immersion
objective lens.
Condenser lenses focus
light rays through specimen.
HOTS
• What are the two ways by which

Illuminator
the resolving power of microscope
can be enhanced?
• What are the advantages of the
low-power objective over the oil
immersion objective for viewing
Figure 2.7: The path of light in light
fungi or algae?
Microscopes
• What will happen if water is used
instead of immersion oil under a
100X objective lens?
Microscope Microscope
objective Lenses objective
Refracted Unrefracted
light rays light rays
lost to lens enter lens Immersion oil
Glass cover slip Glass cover slip
Slide Slide
Specimen Light source Specimen Light source

(a) Without immersion oil (b) With immersion oil
Figure 2.8: Oil Immersion Objective Working Principle

20

2.4 Dark Field Microscope the objective lens and in the absence of
direct background light, the specimen
This is used for examining live unstained
appears light against a dark background
microorganisms. The distinct feature is
(Figure 2.9). The microbes are visualized
the dark field condenser that contains
as halos of bright light against the darkness,
an opaque disc. The disc blocks direct
as stars are observed against the night sky
entry of light to the objective lens. The
(Figure 2.10).
light rays reflected off the specimen enter
Objective
Specimen
Abbe
condenser
(a)
Dark-fields stop
(b)
Figure 2.9: Dark Field Microscopy. The simplest way to convert a microscope to dark field
microscope is to place. (a) a dark field stop underneath (b) the condenser lens system
Infobits
Compound microscope (also known

as light microscope) produces a mono
(2D) image and stereo microscope
produces stereo (3D) image. ‘Upright’
life science microscopes are the most
numerous of all microscopes. An
inverted microscope is the kind of
microscope that views objects from an
inverted position. Digital microscopes
are becoming widespread. These provide
Figure 2.10: Dark field observation of
simple image and are convenient for bacteria Treponema pallidum specimen
electronic image capturing. from a patient with Syphilis
21

22

ICT CORNER
SEM
Lets focus
with SEM
STEPS:
• Use the URL or scan the QR code to reach ‘myscope outreach’ interactive page.
• Click ‘The Scanning Electron microscope’ under ‘Basic’ menu to know about
its parts and function.
• Follow the successive steps that lead to describe the n nuances of SEM.
• Select ‘Let’s Zoom in’ under the activity to menu and explore the SEM
stimulations.
Step1 Step2 Step3
URL:
http://myscopeoutreach.org
Summary Evaluation
The microscope is a tool to study small Multiple choice
microscopic life forms. Zaccharias questions
Janssen is given the credit for making first
compound microscope. Light microscopy 1. The credit for inventing
has undergone a renaissance during the later the first compound
years of the 20th century and early stages of microscope goes to
21st century. a. Robert Hook
There are two main types of b. Anton von Leewenhoek
microscopes (i) Light microscope and (ii) c. Kepler and Galileo
Electron microscope. Light microscope d. Zaccharias Janssen
makes use of light and Electron microscope
uses the electrons.
23

2. All the following are components of compound 4. What happens to light rays when they
microscope except interact with an object?
a. Stage clips 5. Elucidate the lens function in image
b. Fine adjustment knob formation.
c. Electron gun 6. Define the characteristics of resolution,
d. Binocular eye piece magnification and numerical aperture.
3. Numerical aperture was first described by 7. How do eukaryotic and prokaryotic
a. Robert Hook cells differ in appearance under the light
b. Anton von Leewenhoek microscope?
c. Ernst Abbe 8. Trace the pathway of light in brightfield
d. Zaccharias Janssen microscopy.
4. The resolving power of light microscope is 9. Elaborate the role of condenser and image
a. 1 cm formation in dark field microscope.
b. 1.0 µm
c. 0.2 µm
d. 2 nm
Answer the following
1. What is the importance of microscopy in
microbiology?
2. Write down the names of different types of
microscopes.
3. What principle defines an object as
“microscope”?
Student Activity
Experiment and enjoy……
Imaging Properties of a Simple Lens
Objective: In this experiment you will observe and measure the imaging properties of a
simple lens.
Apparatus: You will need a good lens (magnifying glass), a flashlight, a viewing screen
(tri-folded white copy paper), a meter stick and perhaps some modeling clay to hold
things in place. Set all these things on a flat table about 1 meter wide in an area where the
lighting can be dimmed.
Flashlight Lens Screen
Objecct Image
o i
Meter stick
24

Chapter 3
Stains and Staining
Methods
Chapter Outline
3.1 Techniques in Observing
Microorganisms
3.2 Purpose of Staining
3.3 Stains
3.4 Principle of Staining
3.5 Preparation of Materials
for Staining
3.6 Simple Staining Method
3.7 Differential Staining
3.8 Special Staining – Endospore
Staining
3.9 Commonly used Stains and its
Unstained and stained Lactobacillus sp. in curd.
Applications Lactobacillus is a genus of bacteria which can convert lactose
in milk into lactic acid by means of fermentation. Staining is
used to visualize microbial cells under a microscope.
Learning Objectives
After studying this chapter the student • To describe the procedure of simple,
will be able, Gram’s and endospore staining
• To appreciate the need for staining. methods.
• To differentiate between an acidic • To describe the appearance of
dye and a basic dye and understand Gram positive and Gram negative
the principle of staining. cells after each step of Gram staining
procedure.
• To classify organisms based on
staining reaction and differentiate • To know the importance of Gram
between simple and differential staining and endospore staining in
stains. diagnosing and identifying bacteria.
• To know smear preparation and heat • To learn a few staining solutions and
fixation. names of bacteria.
25

Have you ever thought of observing the using a match stick. A drop of culture
microorganisms present in rain water when (liquid containing microorganisms) is
you play? Have you ever wondered how milk placed on a cover slip. The cavity slide
turns into curd and which microorganisms is placed upside down on the cover
are involved? It is clearly understood from slip and inverted such that the drop is
previous unit that microorganisms can be seen hanging (Figure 3.1b).
only under microscopes. But microorganisms Since microbial cells are colourless
do not show much of its structural details and transparent, observation of
under the light microscope due to lack of microorganisms in wet preparation by
contrast and poor resolution. To improve the bright field microscope is difficult. But,
visibility of these tiny living organisms, stains dark-field and phase contrast microscopes
and staining methods are of great use. give contrast and make structures within
3.1 Techniques for Observing the cells to appear clear. Therefore, these
Microorganism microscopes are useful for examination of
unstained preparation.
A considerable amount of information can be
gained by careful microscopic examination
of microorganisms. There are two general 3.1.2 Examination of Stained
techniques used in the preparation of Preparation
microbial specimens to observe them under Staining enables better visualization of
microscope. First technique employs the microorganisms under a microscope.
unstained preparation of living cells and Microscopic examination of stained
second one employs stained preparations of cells helps to reveal the size, shape
killed microorganisms. and arrangement of microbial cells.
Microbial cell staining is important in the
3.1.1 Examination of Unstained
identification of infectious pathogens.
Preparation
Living microorganisms can be examined 3.2 Purpose of Staining
directly by wet mount or by hanging drop
preparations. Both the techniques are Staining is very useful for the following
very useful in determining size, shape reasons:
and motility of the microorganisms. The
• To make the microscopic
spirochetes (spiral bacteria) are normally
semi t ransparent microbial cell visible.
examined in wet preparation through Dark-
field microscope. Some cell inclusion bodies • To reveal the size and shape of
such as vacuoles and spores can be readily microorganisms.
observed even without staining. • To demonstrate the presence of internal
• A wet mount is made by keeping a drop and external structures of microbial
of liquid containing microorganisms cells.
(culture) on a microscope slide and • To distinguish between different types
placing a cover slip over the drop. of microorganisms.
(Figure 3.1a) • To produce specific chemical and
• A hanging drop mount is made by using physical reactions.
a cover slip and a cavity slide. Vaseline
• To preserve the stained microorganisms
is applied on each of the four corner
as specimen slide.
of the cover slip or around the cavity
26

Cover slip
Glass slide
(a) Liquid containing micro organism
Inoculation loop
Hanging drop
Bacterial culture
Cavity slide
Cover slip
(b)
Figure 3.1: a) Wet mount and b) Hanging drop preparation
3.3 Stains contain not only a chromophore group but

also another group known as auxochrome
Stains are dyes used to increase colour contrast.
that imparts the property of electrolytic
Dye is a coloured organic compound that
dissociation. Auxochrome gives salt
adheres to microbial cells, giving colour to the
forming properties to the compound.
cell. Today several stains and staining procedures
are available to study the morphological details Hence, each stain or dye is composed
of various microorganisms. The process of of three components:
imparting colour to the microbial cell is known
(i) Benzene
ring: It is the basic
as staining.
colourless structural component
Stains are organic compounds
of a stain or dye.
containing chromophore and auxochrome
groups linked to benzene ring. (ii) Chromophore: It is the functional
A chromophore group imparts colour group that gives colour.
to the compound. Compounds of benzene (iii) Auxochrome: It is the group that
containing chromophore radicals are gives ionic properties to the stain.
called chromogens. Such a compound, The term stain and dye are not the
even though it is coloured, is not a dye. In same. The basic differences between dye
order for a compound to be a dye, it must and stain are given in Table 3.1.
27

Table 3.1: Difference between dyes and • A majority of stains used in
stains. microbiology are the synthetic type
and manufactured from Aniline. For
Dyes Stains example, Crystal violet, Safranin,
Dyes are a Stains are Methylene blue and Acid fuchsin.
colouring agents colouring 2. On
the basis of chemical behavior,
used for general agents used
dyes are classified as acidic, basic and
purposes. for biological
purposes. neutral.
• An acidic dye is one in which
Dyes are the Stains are
textile colouring the colour bearing ion, the
pure. They are
agents that are chromophore, is an anion.
prepared with
prepared with lesser greater care and • A basic dye is one in which the colour
specification and bearing ion, the chromophore, is a
they may contain specification.
impurities. cation.
• A neutral dye is a complex salt of a
dye acid with a dye base.
3.3.1 Classification of Stains
Acid dyes generally combine more strongly
1. On
the basis of origin, stains can be with cytoplasmic (basic) elements of the cell,
classified as natural and synthetic. and basic dyes combine best with nucleic
(i) Natural stains: acid (acidic) elements of the cell. Table 3.2
• These stains are obtained directly shows the chemical characteristics of a stain
from natural products. For example, or dye.
Haematoxylin is obtained from the
heartwood of a tree (Haematoxylon 3.4 Principle of Staining
campechianum).
Positive Staining
• The natural stains are used mainly
In positive staining, the surface of the
for histological purposes. bacterial cell takes on the colour of the
(ii) Synthetic stains: stain. When basic stain is applied, there
is an attraction between the negatively
• These are artificially produced charged cell surface and positively charged
mainly from coal tar products and chromophore, which leads to staining of
hence popularly called coal-tar dyes. the cell (Figure 3.2).
+ Stain (C+)
Negatively charged Stain containing

cell surface positively charged
chromophore
Stained bacterial cell
Figure 3.2: Positive staining

28

Table 3.2: Chemical characteristic of stain or dye
Acid stain Basic stain Neutral stain

Chromogen of acidic stain Chromogen or coloured It is a complex salt of dye
is negatively charged, so it is part of basic stain is acid with dye base.
also known as anionic stain. positively charged, so it is
also known as cationic stain.
Used to stain the positively Used to stain negatively It stains both positive and
charged component of charged component of negative charged components
microbial cell. microbial cell. of microbial cell.
Example: Eosin, Nigrosin, Example: Methylene blue, Example: Giemsa stain,
India ink, Acid fuchsin, Safranin, Malachite green, Leishmanstain.
Congo red. Basic fuchsin, Crystal violet
Infobits
On the basis of demonstrating the living or non-living status of microorganisms,

some stains are classified as vital stains. These stains differentiate between living and
non-living microbial cells. For example, Tryphan blue selectively colour dead tissues
or cells.
Certain stains will give a different colour to the cell inclusion bodies from its
original colour. Such stains are called metachromatic stains. Metachromatic granules
of Corynebacterium diphtheriae contain polymerized inorganic polyphosphate
responsible for metachromasia with Toluidine blue or Methylene blue.
Negative Staining
In negative staining, the background is The background gets stained and the
coloured and bacteria remains colourless. cell remains colourless. This technique
It is because the acidic dyes are repelled by is useful for revealing the cell shape, size
the negatively charged bacterial surface. and demonstrating capsule (Figure 3.3).
+ Stain (C –)
Negatively charged Stain containing

cell surface negatively charged
chromophore
Stained bacterial cell
Figure 3.3: Negative staining Negative staining
29

3.5 Preparation of Materials for Staining Heat fixation
The essential steps in the preparation of In this method the slide is gently heated by
materials to be observed are passed through a flame (Figure 3.5). Heat
fixation will preserve the overall morphology
1) Preparation of smear of the cell without destroying the internal
2) Fixation structures.
3) Application
of one or more staining
solutions
3.5.1 Preparation of Smear
Smears can be made from liquid or solid
cultures or from clinical specimens. Smear is
prepared by placing a loopful of culture on a
clear glass slide with an inoculation loop. The
culture is spread on the glass slide so as to
form a thin film. This film is allowed to air dry
(Figure 3.4). Figure 3.5: Fixation of smear by passing
slide gently through the flame
Step 1:
Place the liquid on the slide Chemical fixation
It involves the use of chemical fixative
to protect the fine cellular structures of
delicate microorganisms. For this purpose,
Ethanol, Acetic acid, Formaldehyde,
Glutaraldehyde and Mercuric chloride are
Step 2: usually used.
3.5.3 Bacterial Staining Methods

Add the microbes to the Different staining methods are employed
liquid and spread over
the slide
to study the bacterial morphology and
to identify bacteria. Some methods are
Step 3:
used for general purposes and others are
Air dry or heat gently. when dry used for special purposes. There are three
briefly heat fix the cells to the slide
categories of staining methods, they are:
Figure 3.4: Preparation of smear
3.5.2 Fixation Robert Hooke was

the first to describe
Fixation kills the microorganisms and attaches
them to the slide. This prevents washing away the appearance of
of microorganism in further steps of staining stained objects under
procedure. It also preserves various parts of light microscope.
microorganisms in their natural state with Professor Joseph Von Gerlach of
only minimal distortion. The two fixation
Germany was the first to use stain in
methods that are used to fix microbial cells
are heat fixation and chemical fixation. histology.
30

i) Simple staining method iii) Special staining method.
ii) Differential staining method Different types of bacterial staining
methods are summarized in Flowchart 3.1
Staining Methods
Simple staining Special staining

Differenial staining
(For examination of shape, (For visualising the
(For differentiating
size and arrangement of bacterial external and
bacterial groups)
bacterial cells) internal structure)
Grams staining Acid Fast staining

To distinguish To distinguish Acid Fast bacteria
Gram positive and such as Mycobacterium sp from
Gram negative bacteria Non-Acid Fast bacteria
Endospore staining Flagella staining Metachromatic

Capsule staining
Demonstrates Demonstrates the staining
Demonstrates the
spore structure in presence and Demonstrates
presence of capsules
bacteria. Example: arrangement of flagella. the presence of
surrounding the cells
Schaeffer Fulton Example: Silver nitrate granules. Example:
using nigrosin stain
method staining method Albert staining
Flowchart 3.1: Types of Bacterial Staining methods
3.6 Simple Staining Method

In Simple Staining method only one stain
is used. Stain is applied to the smear in one
application. The fixed smear on the glass
slide is flooded with a staining solution
for about one minute. The solution is then
washed off with water and the slide is blot
dried. The stained slide is examined under
a microscope (Figure 3.6). The cells stain
uniformly. The simple stains used by the
microbiologists for routine purposes are
dilute solutions of Methylene blue, Crystal
Figure 3.6: Simple stain – Micrococcus
violet, Safranin and Carbol fuchsin.
sp. stained with Methylene blue
31

they are mixed and applied in one application.
Mycobacterium lep- These procedures show differences between
rae which causes the cells or parts of a cell and can be used for
leprosy is an uncul- of identification. The two most important
turable bacterium. It differential stains used by bacteriologists
is primarily diagnosed by using a spe- are Gram stain and Acid Fast stain. The
cial bacteriological stain called Acid differences between simple and differential
staining are shown in Table 3.3.
Fast stain.
3.7.1 Gram’s Staining Method
The Gram’s stain technique was developed by
Danish Bacteriologist Hans Christian Gram
in 1884. It is one of the most useful staining
methods because it classifies bacteria into
two large groups namely Gram positive and
Gram negative. In this method, the fixed
Mycobacterium leprae (Acid Fast bacterial smear is subjected to staining
bacilli) stained with modified Ziehl reagents in the order of sequence listed
Neelson stain. below:
Methylene blue is more frequently used than Crystal violet

any other stain in Bacteriology. It is used for the (Primary stain)
rapid survey of bacterial population of milk.
It is also used for the diagnosis of Diphtheria. Iodine
This stain is incorporated along with Eosin (Mordant)
in Lactose Agar to distinguish Escherichia coli
from other fecal bacteria in contaminated water.
Alcohol/Acetone
3.7 Differential Staining (Decolourising agent)
In this method more than one stain is

employed. In some method the stains are Basic fuschsin/Safranin
applied separately, while in other method (Counter stain)
Table 3.3: Differences between Simple and Differential Staining

Simple staining Differential staining
1. This
method uses only one stain. This method uses more than one stain.
2. It
imparts only one colour to all It imparts two or more different colours
bacterial cells. to bacterial cells.
3. It
reveals the size, shape and It reveals the size, shape and arrangement.
arrangement of bacterial cells. In addition, it differentiates two groups of
bacteria.
Example: Methylene blue staining Example: 1. Gram’s staining method
method. 2. Acid
Fast staining method
32

The organisms that retain the colour of 3.7.2 Procedure of Gram’s Staining
the primary stain are called Gram positive Gram’s Staining comprises of four steps:
and those that do not retain the primary
stain when decolorised and take on the Step 1: A heat fixed smear
colour of the counter stain are called is covered with a basic
Gram negative. violet dye, Example: Crystal
violet. This stain imparts
Mordants: Mordants are not dyes. They its colour to all cells. It is
are important to increase the biological referred to as a primary
specimen’s affinity for a dye. Some stains stain, since it is applied first.
never stain the cells or its components
unless treated with a mordant. The mordant Step 2: After a short time, the slide is
becomes attached to a cell or its components washed off and the smear is covered with
and then combines with the stain to form an iodine, a mordant. At this stage both
insoluble colour complex. Gram positive and Gram negative bacteria
Step Microscopic appearance Chemical reaction

of cell in cell wall
Gram positive Gram nagative
1. Application of
Crystal violet
(Primary stain)
2. Application of
Iodine
(Mordant)
3. Alcohol wash
(Decolourization)
4. Application of
Safranin
(Counter stain)
Outer membrane
Peptidoglyeon
Cell membrane
Figure 3.7: Steps, micrograph and chemical reaction of Gram Stained Bacteria
33

appear dark violet. and Gram negative bacteria. In addition,
Step 3: Next, the slide is decolurized with Gram negative bacteria contain a layer of
alcohol or an acetone alcohol solution. lipo polysaccharide (consists of lipids and
This solution is a decolurizing agent, polysaccharide) as part of their cell wall.
which removes the primary stain from the When Crystal violet and subsequently
cells of some species but not from others. Iodine is applied to both Gram positive
and Gram negative cells, the two combine
Step 4: The slide is immediately washed after
to form CV-I complex.
decolurization and the slide is then counter
stained with basic fuchsin or safranin, a The cell wall of Gram positive bacteria
basic red dye. The smear is washed again, with lower lipid content get dehydrated
blot dried and examined under microscope during alcohol treatment. The pore
(Figure 3.7). size decreases and the permeability is
reduced. Thus, the CV-I complex cannot
be extracted and the cells remain violet.
3.7.3 Principle of Gram’s Staining
The exact mechanism of action of The alcohol treatment of Gram negative
this staining technique is not clearly bacteria extracts the lipid which results
understood. However, the most acceptable in increased porosity or permeability
explanations are associated with the of the cell wall. Thus, the crystal violet
structure and composition of the cell wall. iodine [CV-I] complex is extracted and
the bacteria are decolorized. These cells
The cell wall of Gram positive bacteria
subsequently take on the colour of the
have a thicker peptidoglycan (consists
counter stain basic fuchsin or safranin
of disaccharides and amino acids) than
and appears red to pink.
Gram negative bacteria. Figure 3.8
depicts the cell wall of Gram positive
Gram-Positive Gram-negative
Outer membrane
Peptidoglycan
Peptidoglycan
Cell membrane Cell membrane
Figure 3.8: Cell wall of Gram positive and Gram negative Bacteria
34

Infobits
HOTS
There are several modifications of
1. If
the iodine step were omitted Gram’s Stain
in the Gram’s staining procedure,
• Kopeloff and Beerman’s
what colour would you expect modification.
Gram positive and Gram negative • Jensen’s modification.
bacteria to stain?
• Weigert’s modification.
a. Gram
positive : pink and • Preston and Morell’s
Gram negative : purple modification.
b. Gram
positive : purple and
Gram negative : pink medical technology, the Gram’s staining
c. Gram
positive : purple and remains an important, inexpensive and
unbeatable tool in the identification of
Gram negative : purple
pathogens.
d. Gram
positive : pink and
Examination of Gram stained organisms
Gram negative : pink usually provides the basis for classifying,
2. In
a Gram’s staining method, a identifying and characterizing bacteria.
step could be omitted and still Gram staining of clinical specimens, however
allow differentiation between provides only a preliminary indication of
Gram positive and Gram negative the identity of the etiological agent (the
organism causing the disease). Gram nature
cells. Name the step.
of common pathogenic bacteria is given in
Table 3.4.
3.7.4 Importance of Gram Staining
Gram stains of clinical specimens or
This century old staining method still of growth on culture plates are especially
remains as the universal basis for bacterial important in determining the most
classification and identification. Even effective antibiotic for the ill patients who
with today’s elaborate and expensive required immediate therapy.
Prof. Hans Christian Gram had died of pneumonia, discovered that

(September 13, 1853-November 14, 1938) certain stains were preferentially taken up
and retained by bacterial cells. Gram was a
modest man, and in his initial publication
he remarked, “I have therefore published
the method, although I am aware that as
yet it is very defective and imperfect; but
it is hoped that also in the hands of other
investigators it will turn out to be useful”.
Dr. Gram used Bismarck brown instead
of Safranin. It was a few years later,
In 1884, Prof. Hans Christian Gram while German pathologist Carl Weigert (1845-
examining lung tissue from patients who 1904), added the final step of staining
with Safranin.
35

Table 3.4: Gram nature of common pathogenic bacteria
Gram positive bacteria Gram negative bacteria
Cocci Staphylococcus aureus, Streptococcus Neisseria gonorrhoeae
pyogenes
Rods(bacilli) Mycobacterium tuberculosis, Escherichia coli, Shigella
Bacillus anthracis, Corynebacterium Salmonella, Pseudomonas
diphtheriae, Clostridium tetani aeruginosa
Spirochaetes Leptospira, Treponema
3.8 Special Staining – Endospore stained, the spore tends to retain the dye
Staining even after treatment with decolorizing
agents. The most commonly used endospore
Endospores are highly resistant structures
staining procedure is the Schaeffer Fulton
produced by some bacteria during
endospore staining method. Malachite
unfavourable environment conditions.
green, the primary stain, is applied to a
Endospore formation is a distinguishing
heat fixed smear and heated to steaming
feature of aerobic genera Bacillus and
for about 5 minutes. Heat helps the stain
anaerobic genera Clostridium. The
to penetrate the endospore wall. Then the
size, shape and position of the spore
preparation is washed for about 30 seconds
(Figure 3.9) are relatively constant
with water. Next safranin, a counterstain is
characteristics of a given species and
applied to the smear to stain the portions of
are important in identifying the species
the cell other than endospores.
within genera. The position of spore in
the cell may be terminal, central or sub- In a properly prepared smear, the
terminal. Figure 3.9 shows the position of endospores appear green within red cells
spores in a vegetative cell. (Figure 3.10). Endospores are highly
refractive. They can be detected under
the light microscope when unstained, but
cannot be differentiated from inclusions of
stored material without a special stain.
Terminal Central Subterminal
spores Spores Spores
Figure 3.9: Position of spore in a

vegetative cell.
Endospores cannot be stained by
ordinary methods, such as simple staining
and Gram staining, because the dyes do
not penetrate the wall of the endospore. If
simple stains are used, the vegetative body
of the bacillus is deeply coloured, whereas
the spore is unstained and appears as a clear
area in the organism. Figure 3.10: Schaeffer Fulton Endospore
staining method- spores stained green
By vigorous staining procedure, the and vegetative cell stained pink
dye can be introduced into the spore. Once
36

Common Bacteria with their Gram reactions
Gram positive bacilli (rod)

Corynebacterium sp.
Clostridium sp.
Gram positive cocci
Staphylococcus
Streptococcus
Gram negative diplococci

Gram nagative bacilli (rod) Neisseria sp.
Klebsiella sp.
Escherichia. coli
3.9 Commonly used Stains and its Summary

Applications Staining makes microscopic semi transparent
Lactophenol cotton blue stain is the most bacterial cell visible. It is a substance that
widely used for staining and observing adheres to a cell and impart colour. On the
fungi. Giemsa stain is a Romanowsky stain, basis of the chemical composition, stains
widely used in microbiology laboratory for or dyes are classified as acidic, basic and
staining of blood and blood parasites like neutral. Staining techniques are classified
malarial protozoans. Calcofluor white stain as simple, differential and special. Simple
is commonly used stain to directly detect staining uses a single dye and can help to
the fungal elements in tissues and in culture. identify the shape and size of an organism.
Acridine orange stain is used to confirm Differential staining use more than one
the presence of bacteria in blood cultures dye to distinguish between structures in
when Gram stain results are difficult to a cell or different types of cells. The Gram
interpret using light microscopy. The stain stain procedure divides bacteria into Gram
binds to nucleic acid and stains them. It is also positive and Gram negative bacteria.
used for the detection of cell wall deficient Specialized staining such as endospore
bacteria example Mycoplasma. Fluorochrome staining is used to detect the presence of
stains such as auramine-rhodamine stains are endospores in bacteria.
readily available to detect the bacteria in the
specimens through Fluorescent microscopy.
37

Evaluation b. Crystal violet, iodine, alcohol,
safranin
Multiple choice questions
c. Methylene blue, alcohol, iodine,
1. An dye has negative charge. safranin
a. Basic d. Crystal violet, alcohol, iodine,
b. Acidic safranin
c. Neutral d. None
9. The Schaeffer-Fulton endospore staining
2. stain is incorporated with usually shows
Eosin in Lactose agar to distinguish a. Spore green within pink cells
typical Escherichia coli in contaminated
b. Spores pink within green cells
water.
a. Crystal violet b. Acid fuchsin c. Colourless spores within
pink cells
c. Methylene blue d. Safranin
d. Colourless spores within
3. Which of the following is not an anionic green cells
dye?
a. Safranin b. Eosin Answer the following
c. Rose Bengal d. Acid fuchsin
1. Define stain.
4. Christian Gram discovered a staining
2. Give examples for basic stain.
technique to differentiate the bacteria of
similar morphology in the year. 3. Why heat fixation is important?
a. 1857 b. 1880 c. 1884 d. 1881 4. What are endospores?
5. Distinguish between a dye and a stain.
5. Which of the following is used for
negative staining of microbial cells? 6. List out few gram positive bacteria.
a. Nigrosin and Acid fuchsin 7. What is the purpose of a counterstain/
b. Rose Bengal and malachite green decolorizer in the gram stain?
c. Safranin and Eosin 8. Fill in the following table regarding the
d. Nigrosin and Indian Ink gram stain.
6. is used as a mordant Appearance after this step of
in Gram staining techniques. gram staining
a. Iodine Steps Gram Gram
b. Crystal violet positive cells negative cells
c. Methylene blue Crystal
d. Safranin violet
7. Which of the following pairs is Iodine
mismatched? Alcohol
a. Capsule-negative stain Safranin
b. Cell arrangement-simple stain
c. Cell size-albert stain 9. What is meant by negative staining?
d. Gram stain-bacterial 10. What are the uses of staining?
identification 11. Differentiate simple and differential stain.
8. The order of reagents in the gram
12. What are acidic stains? Give examples.
staining reactions are:
a. Safranin, alcohol, methylene blue, 13. Why do basic dyes stain bacterial cells?
iodine Why won’t acidic dyes stain bacterial
cells?
38

14. For what purpose would you use each 16. How will you appreciate the need of
of the following? staining?
a. Simple stain 17. Classify staining technique based on
b. Negative stain their purpose.
c. Acid- fast stain 18. Explain the principle of grams
d. Gram stain staining.
15. The gram stain has been described 19. Diagrammatically explain Gram’s
as the most important stain for staining procedure.
microbiologist. Explain why? 20. How to visualise an endospore.
ICT CORNER
Gram Staining of Bacteria
Know the Gram

Staining process
STEPS:
• Use the URL OR Scan the QR code to reach ‘Virtual Interactive bacteriology laboratory’.
• Click ‘module’ and select ‘steps’ and read the procedure to follow.
• Select ‘start’ to enter the ‘Gram Stain’ process and follow the procedure.
• Leave the slide to dry and heat fix with Bunsen burner and view under microscope
OBSERVATIONS :
• Select other examples and record your observation on Gram +ve and Gram –ve bacterial
stains.
Step1 Step2 Step3 Step4
URL:
https://www.cellsalive.com/toc_micro.htm
39

Chapter 4
Sterilization
Chapter Outline
4.1 Need for Sterilization

4.2 Methods of Sterilization
4.3 Physical Methods of Sterilization
4.4 Sterilization by Heat
4.5 Radiation
4.6 Filtration
The inoculation loop is sterilized with flame or any
other heat source, until it becomes red hot before and
after each use.
Learning Objectives
After studying this chapter the student The process of sterilization is used in
will be able, Microbiology
• To understand the concepts of •
for preventing contamination by
sterilization to maintain aseptic
extraneous organisms
conditions. • in surgery for maintaining asepsis
• To compare the effectiveness of dry •
in food and drug manufacture for
heat (red heat, flamimg, incineration, ensuring safety from contaminating
hot air oven), and moist heat (boiling, organisms
autoclaving, pasteurization).
The choice of methods of sterilization
• To learn the uses of pasteurization depend on the purpose for which it is
in the field of food industry. carried out: the material to be sterilized and
• To describe the role of radiation in the nature of the microorganisms that are to
killing pathogens. be removed or destroyed.
• To describe how separation of microor-
ganism is achieved through filtration.
As early as the stone
Microorganisms are ubiquitous. They can age, humans used
contaminate, infect or decay inorganic physical methods
of microbial control
and organic matter. Hence, it becomes
to preserve foods,
necessary to kill or remove them from
like drying (desiccation) and salting
materials or from areas around us. This (osmotic pressure).
is the objective of sterilization.
40

Sterilization is defined as the process 4.2 Methods of Sterilization
of complete removal or destruction of all Growth and multiplication of
forms of microbial life, including vegetative microorganisms can be controlled by
cells and their spores. removing, killing or inhibiting them using
various physical or chemical agents.
4.1 Need for Sterilization
The aim of all sterilization strategies is to kill 4.3 Physical Methods of Sterilization
or remove the unwanted microorganisms. The various physical methods of sterilization
In certain cases, microbes are regarded are given in flowchart 4.1
as potential pathogens and therefore
it is essential to eliminate these forms 4.4 Sterilization by Heat
(vegetative and spores) of microbial life. Heat is the most rapid and best method of
All microbiological techniques require sterilization. It is the method of choice that
appropriate and adequate sterilization. the material to be sterilized is stable enough to
withstand the required temperature necessary
Sterilization of culture media,
to kill the microbes. The time needed for
containers and instruments is essential
sterilization depends on the initial number
in microbiological work for isolation
of organisms present, type of materials to be
and maintenance of microorganisms. In
sterilized (hence washed and cleaned items
surgery and medicine, the sterilization
are easier to sterilize than dirty ones) and also
of instruments, drugs and other supplies
on the temperature used. Spores need higher
is important for the prevention of
temperatures while vegetative bacteria can be
infection.
destroyed at lower temperatures.
Physical methods
Heat Radiation Filtration
• Depth filter
• Membrane filter
Dry heat Moist heat Non ionizing Ionizing
• Air filter
• Red heat • Temperature • Infrared • X-rays

• Flaming below 100°C • Ultraviolet • Gamma rays
• Incineration • Temperature
• Hot air oven at 100°C
• Temperature
above 100°C
Flowchart 4.1: Physical Methods of Sterilization
41

a) Red heat
Infobits
Inoculating wires, points of forceps and
searing spatulas are sterilized by holding
Heating process in canning was first
them in the flame of a bunsen burner until
used by Nicholas Appert in 1890. He they are seen to be red hot.
described a safe means of preserving all
kinds of food substances in containers b) Flaming
or in cans. Appert is known as father This method is used for sterilizing scalpels,
of cannning. needles, mouths of culture tubes, slides and
cover slips. It involves passing the article
through the bunsen flame without allowing
Heat resistance varies among different it to become red hot.
microorganisms. These differences can be
expressed in terms of thermal death point. c) Incineration
Thermal Death Point (TDP) is the lowest This is an excellent method for destroying
temperature at which all the microorganisms materials such as contaminated clothes,
in a particular liquid suspension will be cotton wool stoppers, animal carcasses and
killed in 10 minutes. pathological materials. It involves burning
of materials in incinerators.
Another factor to be considered
in sterilization is the duration of time d) Hot air oven
required. This is expressed as Thermal This is the most widely used method of
Death Time (TDT). TDT is the minimal sterilization using dry heat. The oven is
time required for all microorganism in a usually heated by electricity and it has a
particular liquid culture to be killed at a thermostat that maintains the chamber air
given temperature. Both TDP and TDT constantly at the chosen temperature.
are useful guidelines that indicate the It has a fan or turbo-blower to assist
degree of treatment required to kill a given the circulation of air and to ensure rapid,
population of bacteria. uniform heating of the load. In Hot Air
Decimal Reduction Time (DRT) is Oven, the air is heated at a temperature
related to bacterial heat resistance. DRT of 160oC for one hour. Figure 4.1 shows
is the time, in minutes, in which 90% of a laboratory hot air oven.
population of microorganism at a given
temperature will be killed.
Heat is employed either as dry heat or
moist heat. Double jacket chamber
4.4.1 Sterilization by Dry Heat

Thermometer
Dry heat is frequently used for the
sterilization of glassware and laboratory Shelves
equipments. In dry heat sterilization, Temperature indicator

microbial cells are apparently killed by valve
oxidation of their constituents and protein
denaturation. Dry heat is applied in the
following ways:
Double jacket door
Figure 4.1: Hot Air Oven

42

This is the best method of sterilizing dry harmful microorganisms. It should be
glass ware such as test tubes, petri dishes, noted that pasteurization process kills
flasks, pipettes and instruments such as only vegetative cells but not the spores.
forceps, scalpels and scissors. It is also used Pasteurization named in honour of its
to sterilize some pharmaceutical products developer Louis Pasteur. Table 4.1 gives
such as liquid paraffin, dusting powder, fats comparison between Sterilization and
and grease. Pasteurization.
Quality control of dry heat sterilization:
Raw milk can
The spores of a nontoxigenic strain of harbour dangerous
Clostridium tetani are used to test the microorganisms,
efficiency of dry heat sterlization. such as Salmonella,
4.4.2 Sterilization by Moist Heat Escherichia coli and
Moist heat kills microorganisms primarily by Listeria, that can pose serious health
the coagulation of proteins (denaturation), risk, and children are particularly
which is caused by breakage of the hydrogen susceptible to the potential infection of
bonds that hold the proteins in three unpasteurized or raw milk
dimensional structure.
There are three methods employed in Pasteurization can be done in the
moist heat sterilization. following methods,
• Temperature below 100°C. • Low Temperature Holding
Method (LTH)
• Temperature at 100°C.
In this method milk, beer and fruit
• Temperature above 100°C. juices are maintained at 62.8°C for 30
minutes.
a) Temperature below 100°C:
• High Temperature Short Time
Pasteurization
Method (HTST)
The process of heating a liquid food Products are held at 72°C for
or beverage either at 62.8°C for 30 15 seconds.
minutes or 72°C for 15 seconds to
enhance their shelf life and destroy • Ultra High Temperature (UHT)
Table 4.1: Comparison between Sterilization and Pasteurization

Sterilization Pasteurization
Sterilized products have a longer shelf life Pasteurized products have shorter shelf
life
Discovered by Nicolas Appert Discovered by Louis Pasteur
Eliminates all forms of microorganisms Eliminates pathogenic
microorganisms only
Can be accomplished in many ways Can be accomplished with heat
Applied in food industry, medical, Mainly applied in food industry
surgery and packaging
43

Milk can be treated at 141°C for in autoclave is 121°C at 15 lbs (pounds)
2 seconds (This method employ pressure for 15 minutes (Figure 4. 2a & b).
temperature above 100°C). Autoclaving is used in sterilizing culture
b) Temperature at 100°C: media, instruments, dressings, applicators,
solutions, syringes, transfusion equipment,
i) Water at 100°C (Boiling):
pharmaceutical products, aqueous solutions
Boiling is one of the moist heat and numerous other items that can
sterilization methods. It kills vegetative withstand high temperatures and pressures.
forms of bacterial pathogens, almost all The same principle of autoclaving applies
viruses and fungi (including their spores) for the common household pressure cooker
within 10 minutes, usually much faster. used for cooking food.
• Most vegetative bacteria will die
Factors influencing sterilization by heat:
in 5-10 minutes when immersed
in boiling water, but some spores Sterilization by heat depends upon
will survive at this temperature for various factors such as time, temperature
several hours. employed, number of microorganisms,
spores and nature of material to be
• Articles sterilized by this method
sterilized.
cannot be stored for a long time.
Quality control of moist heat s terilization:
ii) Steaming at 100°C (Tyndallization):
To check the efficiency of moist heat
It is a process discovered by John Tyndall sterilization, the indicator commonly used
in 19th century for sterilizing substances to is the paper strips containing spores of
kill the spores of bacteria. The process of Bacillus stearothermophilus.
exposure of materials to steam at 100°C
for 20 min for three consecutive days is 4.5 Radiation
known as tyndallization. First exposure Radiation is commonly
kills all the vegetative forms and in the employed for sterilizing
intervals between heating, the remaining heat sensitive materials
spores germinate into vegetative forms such as disposable
which are killed on subsequent heating. plastic products and
Tyndallization is also called fractional materials that cannot
sterilization or intermittent boiling. withstand moisture.
The most effective type of radiation to
c) Temperature above 100°C:
sterilize or reduce the microbial burden
Moist heat sterilization can be carried in the substance is through the use of
out at temperature above 100°C in electromagnetic radiations. Figure 4.3
order to destroy bacterial endospores. shows different types of electromagnetic
This requires the use of saturated steam radiations. Radiation has various effects
under pressure. This is achieved using on cells, depending on its wavelength,
autoclave. intensity and duration of explosure
Autoclave (Flowchart 4.2). Radiation that kills
microorganism is of two types namely
Sterilization using an autoclave is most
ionizing and nonionizing.
effective when the organisms are either
contacted by the steam directly or contained a) Non-ionizing radiation
in a small volume of aqueous liquid Infra-red rays and ultra-violet rays are non
(primarily water). The temperature used ionizing radiation.
44

i) Infra-red radiation Radiation
These are electromagnetic rays with
wavelengths longer than those of visible
light. These are low energy type. It kills
microorganisms by oxidation of molecules Non ionizing
Ionizing radiation
as a result of heat generated. Infrared radiation
radiation is used for rapid mass sterilization
of pre-packed items such as syringes and
catheters. • Infrared radiation • X-Rays
• UV radiation • Gamma Rays
Flowchart 4.2: Radiation
Release valve Pressure gauge
Safety valve
Cover
Cover tightening nuts
Body
Jacket
Bucket
Stand
Heating electrode
Leg
Figure 4.2: (a) Laboratory autoclave (b) Components of autoclave
Types of radiation in the electromagnetic spectrum

Non-ionizing Ionizing
Radio Infrared Ultraviolet

Visible light
Extremely
Type of radiation low
Microwave x-ray
frequency
Gamma rays
Non-thermal Thermal Optical Broken bonds
Effects Induces low Induces high Excites Damages

currents currents electrons DNA
??? Heating Photo-

chemical
effects
Static Power AM FM radio Microwave Heat tanning Medical
Source field ladio TV oven lamp booth x-rays
line
Figure 4.3: Types of radiation in electromagnetic spectrum

45

ii) Ultra-violet radiation Cobalt 60 source is used in the cold
The ultraviolet (UV) portion of the sterilization of antibiotics, hormones,
electromagnetic spectrum includes sutures and plastic disposables supplied
all radiations from 150-3900A°, UV such as syringes and in pasteurization of
radiation around 2600A° is most lethal meat.
to microorganisms. UV has a very little
4.6 Filtration
ability to penetrate matter. Thus, only
Filtration is an effective and reasonably
the microorganisms on the surface of an
economical method of sterilization. It is used
object, exposed directly to the ultraviolet
to sterilize heat-sensitive fluids, and air. It is
light are susceptible to destruction. UV
particularly useful for solutions containing
radiations are used to sterilize operation
toxins, enzymes, drug, serum and sugars.
theaters, laboratories and entry ways.
Sugar solutions used for the cultivation of
b) Ionizing radiation microorganisms tend to caramelise during
Ionizing radiations (X-rays, Gamma rays autoclaving and so they are best sterilized by
and Cosmic rays) are an excellent sterilizing filtration. Filtration is also used extensively in
agents and they penetrate deep into the beer and wine industries. Filters with known
objects. These radiations do not produce pore sizes which are sufficiently small to hold
heat on the surface of materials. Hence, back bacteria are employed. Recently filters
sterilization using ionizing radiations is that can remove viruses are also available.
referred as cold sterilization. It will destroy Filtration is an excellent way to remove
bacterial endospores and vegetative cells, the microbial population from solution
both Prokaryotic and Eukaryotic; however containing heat sensitive material.
ionizing radiation is not always effective There are two types of filters namely
against viruses. Gamma radiation from (Figure 4.4):
i) Membrane filter (surface filtration) and
Deinococcus radiodu-
rans is an extremo- ii) Depth filter
philic bacterium. It
is one of the most Membrane filters
radiation-resistant or- Membrane filtration is used for preparing
ganisms known. heat-labile culture media components. It
Figure 4.4: Principle of filtration

46

is also useful in removing bacteria from random paths through the filter that trap
heat-sensitive pharmaceutical products many particles. Depth filter are made up
and biological solutions. of diatomaceous earth (Berkefeld filters)
Membrane filters are made up of which are used as water purifiers. Examples
either cellulose acetate, cellulose nitrate, of types of depth filters (Figure 4.6)
polycarbonate, polyvinylidene fluoride or contains unglazed porcelain (Chamberl
other synthetic porous materials. These filters and filters) and asbestos (Seitz Filter).
remove microorganisms by screening them Air filtration
out, such as a sieve separates large sand particles
from small ones. Membranes with pore size of Air also can be sterilized by filtration.
0.2µm in diameter are used to remove most
vegetative cells but not viruses. These filters HOTS
are used to sterilize pharmaceutical products,
ophthalmic solutions, culture media, oils,
Give a reasonable method of sterilization
antibiotics, and other heat sensitive solutions
for the following.
(Figure 4.5a, b & c).
1. Operation theatre 2. Serum 3. Pot
Depth filters of soil 4. Plastic Petri Dishes 5. Rubber
Depth filters are the oldest type of filters gloves 6. Disposable syringes. 7. Metal
and they consist of overlapping layers of instruments 8. Flask of nutrient agar
fibrous sheets of paper, asbestos or glass 9. Milk 10. Papers with spores.
fibers. The overlapping fibers create
Membrane transferred
Sample to be to culture medium
filtered (b)
Membrane filter LM 2.5 m
retains cells
To vacuum
Incubation
Colonies
(a) (c)
Figure 4.5: (a) Membrane filter apparatus (b) Light microscope image of
microorganism filtered through membrane filter (c) Membrane filters showing
microbial colonies on culture media
47

Figure 4.6: Types of depth filters
Outside
Safety glass
Exhaust viewscreen
HEPA fiter
Blower
Supply
HEPA filter
Light
High-velocity
air barrier
Figure 4.7: Laminar air flow
The air is freed from infection by passing size. Some operation theaters and rooms
it through High Efficiency Particle occupied by burn patients receive filtered
Arrester (HEPA) filter. Laminar air flow air to lower the numbers of airborne
biological safety cabinets are one of the microbes. HEPA filters remove almost all
most important air filtration systems microorganisms above 0.3µm in diameter.
(Figure 4.7). It employes HEPA filters Various physical methods of sterlization
which remove 99.97% of 0.33µm particles is summarized in Table 4.2
48

Table 4.2: Physical methods used to control microbial growth
Mechanism Preferred for
Method of action Comment sterilizing
Heat
1 Dry heat
a. Direct Burning Very effective method of Inoculating loops
flaming contaminants sterilization
to ashes
b. Incineration Burning to Very effective method of Paper cups,
ashes sterilization contaminated dressings,
animal carcasses, bags,
and wipes
c. H
ot –air Oxidation Very effective method of Empty glassware,
sterilization sterilization, but requires instruments, needles,
temperature of 160°C for and glass syringes
about 1 hour
2 Moist heat
a. B
oiling or Protein Kills vegetative bacterial Dishes, basins, pitchers,
flowing denaturation and fungal pathogens various equipment
steam and almost all viruses
within 10 min; less
effective on endospores
b. Autoclaving Protein Very effective method Microbiological media,
denaturation of sterilization; at about solutions, linens,
15 lbs of pressure (121°C), utensils, dressings,
all vegetative cells and equipment, and other
their endospores are items that can withstand
killed in about 15 min temperature and
presure
c. Pasteurization Protein Heat treatment for milk Milk, cream, and
denaturation (72°C for about 15 sec) certain alcoholic
that kills all pathogens beverages(beer and
and most nonpathogens wine)
3 Radiation
a. Ionizing Destruction Not widespread in Used for sterilizing
of DNA routine sterilization pharmaceuticals and
medical and dental
supplies
b. Nonionizing Damage to Radiation not very Control of closed
DNA penetrating (non environment with UV
penetrating)
49

50

Summary c. Hot-air sterilization
Physical methods of microbial control d. Pasteurization
include heat, radiation, drying and f iltration.
2. Which of the following is most effective
Heat is the most widely used method
for sterilizing Petri dishes?
of microbial control. It is used in both
forms: moist and dry. The thermal death a. Chlorine
time (TDT) is the time required to kill all b. Ethylene oxide
microbes at a specific temperature. The c. Autoclaving
thermal death point (TDP) is the lowest d. Nonionizing radiation
temperature at which all microbes are killed
in a specified duration of time. 3. kills organisms by coagulation
and denaturing their proteins
Autoclaving, or steam sterilization, is
the process by which steam is heated under a. Dry heat
pressure to sterilize a wide range of materials b. Moist heat
in a comparatively short time. c. Both a & b
Dry heat kills the microorganisms under d. None of the above
specified time and temperature. Dry heat
is applied in the following ways: Red heat, 4. In which method, temperature of
incineration and Hot air oven. 160°C for 1 hour is employed?
Ionizing radiation (cold sterilization) by a. Red heat
X rays and gamma rays is used to sterilize b. Infrared radiation
medical products and meat. It damages DNA c. Hot air oven
and cell organelles by producing disruptive
d. Flaming
ions. Ultraviolet light, or nonionizing
radiation, has limited penetrating ability. It 5. Which of the following temperature and
is therefore restricted to sterilize suface of time are employed in autoclave for
the materials. sterilization of materials?
Decontamination by filtration removes a. 16 lbs 120°C for 18 min
microbes from heat sensitive liquids and b. 18 lbs 180°C for 20 min
circulating air. The pore size of the filter
c. 22 lbs 170°C for 35 min
determines what kinds of microbes are
removed. d. 15 lbs 121°C for 15 min
6. Wavelength used for the absorption of
Evaluation
UV spectrum is
a. 4000A°
1. Which of the
following does not kill b. 2600A°
endospores? c. 20A°
a. Autoclaving d. None of the above
b. Incineration
51

Student Activity
1. Define Pasteurization.
1. Collect samples of raw milk
2. What is Incineration?
(unpasteurized) and boiled milk,
3. Define membrane filters? place them in open containers
4. What is Sterilization? separately. Observe the changes
5. Explain the principle of moist heat after a few hours in both and infer.
sterilization? 2. Making a working model of
6. Differentiate the mechanism of depth and membrane filters and
operation employed in autoclave and demonstrating their uses.
hot air oven.
7. Discuss ionizing radiation.
8. How do you sterilize heat sensitive
materials?
9. Define Tyndallization.
10. Describe the sterilization in an
autoclave.
11. Explain the methods of sterilization
by dry heat.
12. Explain the methods of radiation.
52

Chapter 5
Cultivation of
Microorganisms
Chapter Outline
5.1 Significance of Culturing
Microorganisms
5.2 Bacteriological Media and its Types
5.3 Pure Culture
5.4 Growth and Colony Characteristics
The microorganisms on the handprint of an eight-year-old
of Bacteria and Fungi
boy. After incubation the plates showed coloured colonies of
bacteria and fungi as you see above.
The cultivation of microorganisms under laboratory
condition makes the microscopic cells to grow and form
individual colonies macroscopically.
Learning Objectives in environment as pathogens and normal

microflora. Excellent supporting factors
After studying this chapter the student are available in nature for microorganisms
will be able, to survive in the environment. This
• To know the importance of bacterial leads to microbial proliferation as an
media for growth of microorganisms. extended community in nature. The term
To understand various types ‘cultivation of microorganisms’ means
of media for differentiation and growing microorganisms in the laboratory
diagnosis of important pathogenic with ample supply of specific nutrients
microorganisms. (Figure 5.1). Obligate intracellular
parasites like viruses, Rickettsias and
• To know pure culture techniques
Chlamydias are cultivated within living
• To understand the methods involved cells.
in isolating pure culture of bacteria,
Survival and growth of microorganisms
which includes pour plate, spread
depend upon the favourable growth
plate and streak plate.
environment. Laboratory cultivation
• To differentiate the growth plays a crucial role in the isolation,
characteristics of bacteria and fungi. identification and classification of
microorganisms. Cultivation of bacteria
Microorganisms are omnipresent and they and fungi by artificial formulated medium
exist in soil, air, water, spoiled food, decayed is one of the important milestones in the
animal and plant residues. They are found history of Microbiology.
53

Robert Koch devised the solid medium understand the nutritional requirements
(by using gelatin) to grow and isolate the of that microorganism and then supply
microorganisms. the essential nutrients in proper form
and proportions in culture medium.
5.1 Significance of Culturing Flowchart 5.1 describes the types of media.
Microorganisms A common bacteriological medium has
Carbon and Nitrogen sources along with
• To isolate microorganisms from any
buffering agents. Most of the media are
samples
prepared using dehydrated components.
• To study the morphology and The basic components are peptone, beef
biochemical characteristics of extract, meat extract, yeast extract and agar
microorganisms (Table 5.1).
• To maintain the stock culture
Table 5.1: Common ingredients of a
• To identify disease causing
culture media
microorganisms
• To study the role of microorganisms S.No Ingredients Source of
in the production of industrially Peptone Carbon,
important products a. (protein nitrogen,
hydrolysates) energy
5.2 Bacteriological Media and its Types Beef extract Aminoacids,
Generally microorganisms occur as mixed b. (Extract of vitamins,
culture in nature. Human beings, animal lean beef) minerals
bodies and other natural resources harbour Yeast extract Vitamin B,
microbes in mixed population. By using c. (Brewer’s Carbon,
appropriate media, microorganisms can yeast) Nitrogen
be grown separately in pure form and can Solidifying
d. Agar
be studied. For successful cultivation of agent
a given microorganism, it is necessary to
Types of Media
Physical Chemical Special purpose
* Liquid * Synthetic * Basal * Anaerobic

* Semi-solid * Non-synthetic * Enriched * Transport
* Solid * Selective * Antibiotic
* Differential sensitive
Flowchart 5.1: Types of media

54

Uses of agar: pectin from the Agar. It is used in Molecular
• It is one of the principle ingredients in the Biology laboratory for the separation of
preparation of solid or semisolid media. DNA molecules by gel electrophoresis.
• It is used as a solidifying agent in
culture medium. Agar was first described
for use in Microbiology
• It is extracted from certain seaweeds
in 1882 by the German
belonging to genera of red algae like
microbiologist Walther
Gelidium and Gracilaria (Figure 5.1).
Hesse, an assistant working in Rober
Koch’s laboratory, as suggested by his
wife Fannie Hesse.
A cheap substitute for agar in
microbial culture media is Guar gum,
which can be used for the isolation and
maintenance of thermophiles.
HOTS
Why is agar preferred to gelatin as a

Figure 5.1: Gelidium – Red algae
solidifying agent in culture media?
• It is a sulphated polymer mainly
consisting of D-galactose. 5.2.1 Physical Nature of Agar Medium
• Agar is a highly preferred solidifying The concentration of agar plays a major
agent because it does not affect the role in determining the consistency
growth of microorganisms. Agar is also of the medium. A medium with agar
used in the food and pharmaceutical concentration of 2% or greater is said to be a
industries. solid medium and that of 0.5% is said to be
• The purified form of the agar is called a semisolid medium (jelly like appearance).
Agarose. It is prepared by removing the Tabel 5.2 lists the concentration of agar in
Table 5.2: Concentration of agar in media

Nature of Medium Concentration Example Uses
Solid 2% Nutrient agar To isolate microorganisms
on petridish and forming
agar slant
Semisolid 0.5% SIM (Sulphur Indole Agar stab to observe
Motility medium) motility
Liquid 0% Nutrient broth To observe biochemical
reaction.
55

media. However liquid media (broth) does
not contain agar. Figure 5.2 shows types of Infobits
media depending on physical nature.
Veggitone is a vegetable based product
containing peptones. It is made from
raw materials such as peas and fungal
proteins that are digested using fungal
and bacterial enzymes.
5.2.3 Special Purpose Medium

i) Basal medium
This medium promotes the growth of many
types of microorganisms which do not
require any special nutrient supplement.
It is a routine laboratory medium with
Carbon and Nitrogen sources along
Figure 5.2: Solid, liquid and with some minerals. Example: Nutrient
semi-solid media Agar or Nutrient Broth. It is also called
general purpose medium. It is used for
5.2.2 Chemical Nature of Medium subculturing the pathogens. It is a non-
• Synthetic medium selective medium, which is designed to
support the growth of a wide spectrum of
Chemically defined synthetic Medium
heterotrophic organisms. (Figure 5.3)
is used for various experiments. This
medium is prepared exclusively from
pure substances with known chemical
composition and concentrations. This
is widely used in research to find the
type of compound metabolized by the
experimental organism.
• Non-synthetic medium
The medium in which the exact chemical
composition and the concentration of each
Figure 5.3: Growth of bacteria on
ingredient is not certainly known is called Nutrient agar
non-synthetic medium. In this medium,
crude materials such as meat extract, yeast ii) Enriched medium
extract, various sugars, molasses and corn In enriched medium, substances like blood,
steep broth are used. This supports the egg or serum are added along with the
growth of a variety of microorganisms. It basal medium. It is used to grow fastidious
is otherwise called as complex medium. organisms that are very particular in their
nutritional needs. Fastidious organisms
56

have elaborate requirements of specific 7% Sodium chloride that inhibits the
nutrients like vitamins and growth growth of other bacterial population
promoting subtances and or not easily but allows the growth of Staphylococci
pleased or satisfied by ordinary nutrients (Figure 5.5). Moreover it has Phenol
available in nature. Example: Blood agar red dye to indicate acid production.
is used to identify haemolytic bacteria Staphylococcus utilizes Mannitol and
(Figure 5.4) and Chocolate agar used to produces acid which changes the colour
identify Neisseria gonorrhoeae. of the Phenol red indicator to yellow.
Salmonella-Shigella (SS) agar is selective
for Salmonella (Figure 5.6).
Figure 5.4: Blood Agar showing alpha,

beta & gamma – haemolytic colonies
Figure 5.5: Growth of Staphylococcus
aureus on Mannitol salt agar
In 1919, James Brown
used blood agar as
diagnostic medium to
study the haemolytic

patterns of bacteria.
iii) Selective medium

Selective medium contains one or more
agents (selective components) that
inhibit unwanted organisms but allow
the desired organisms to grow. Growth
of unwanted microbes is suppressed by
adding bile salts, antibiotics and dyes.
Example: Mannitol salt agar is selective
Figure 5.6: Growth of Salmonella
for Staphylococci. This medium contains on SS agar
57

It is nothing short of
amazing and humbling
fact that even after
120 years of trying to
grow microbes in the laboratory, we
have succeeded in culturing only 0.1%
of the microorganisms around us.
iv) Differential medium

Differential medium distinguishes between
different groups of bacteria and permit Figure 5.8: Growth of lactose fermenting
bacteria on EMB Medium
tentative identification of microorganisms
based on their biological charaterstictics as Eosin Methylene Blue (EMB) agar
they cause a visible change in the medium. medium is also a differential medium. It
We can differentiate haemolytic and is used to differentiate lactose fermentors
non-haemolytic patterns of bacteria using from non-lactose fermentors. It has lactose
blood agar. Differential medium is otherwise sugar and two dyes namely Eosin –Y and
called indicator medium as it distinguishes Methylene blue. These dyes act as inhibitory
one organism from another growing on the agent towards Gram positive bacteria.
same plate by the formation of pigments due Example: Lactose fermentors such as faecal
to its biochemical and physiological nature. Escherichia coli show metallic sheen and
Example: MacConkey agar medium has non lactose fermentors such as Enterococcus
neutral red dye. Lactose fermentors form do not show metallic sheen. (Figure 5.8).
pink coloured colonies and non fermentors
form colourless translucent colonies on it Lactose fermentation
(Figure 5.7).
Acidic pH (pH drops below 6.8)
Makes Eosin-Y and Methylene blue to

form a complex
Inhibits Gram positive bacteria
Figure 5.7: Growth of microorganisms

on MacConkey agar (Lactose fermenting Colonies of lactose fermentors has
bacterial colonies appears pink) metallic sheen
58

vi) Antibiotic sensitivity medium
Chromogenic
Antibiotic sensitivity medium is a
medium is used
microbiological growth medium that is
for the simple
commonly used for antibiotic sensitivity
and fast detection
testing. Example: Muller- Hinton agar
of transformed bacteria by using
medium. It is a non-selective and non-
chromogenic substrates. The
differential medium. It allows the growth
chromogenic mixture contains
of most type of microorganisms. It contains
substrates such as Salmon-GAL, X-GAL.
starch which absorbs toxins released from
Certain bacterial enzymes cleave the
bacteria. Hence toxins do not interfere
chromogenic substrate resulting in the
with antibiotics. Agar concentration of
coloured colonies.
1.7% is used in this media which allows
better diffusion of antibiotics (Figure 5.9).
HOTS
Why is EMB medium called a

selective, differential as well as
complex medium?
v) Enrichment medium
Enrichment medium is a liquid medium. It
is used to grow a particular microorganism
that is present in much smaller number Figure 5.9: Antibiotic sensitivity on
along with others present in sufficiently Muller Hinton agar
large numbers. An enrichment medium vii) Anaerobic medium
provides nutrients and environmental Anaerobic medium is a medium used for
conditions that favour the growth of a the cultivation of anaerobes, Example:
desired microorganisms. It is used to culture i) Robertson cooked meat medium: This
microorganisms present in soil or faecal is used for the isolation of Clostridium
samples that are very small in number. ii) Thioglycolate broth: In this medium
Example: Selenite F Broth is used to isolate Sodium thioglycollate is used as a reducing
Salmonella typhi present in low density in agent which maintain a low Oxygen tension
faecal sample. It is cultured in an enrichment by removing the molecular Oxygen from
medium containing Selenium. Selenium the environment.
supports the growth of the desired organism
and increase it to detectable levels compared viii) Transport medium
to intestinal flora. Sodium selenite inhibits Transport medium is used for the
many species of Gram positive and Gram temporary storage of specimens that are
negative bacteria including Enterococci and being transported to the laboratory for
c oliforms. cultivation. It maintains the viability of all
59

organisms in the specimen without altering 5.3 Pure Culture
their concentration. It mainly contains
In nature, microorganisms usually exist as
buffers and salts. Example: Stuart’s transport
complex multispecies community. A single
medium that lacks Carbon, Nitrogen and
species has to be characterized in order
organic growth factors. Other examples of
to know the morphology, pathogenicity
transport media are Cary Blair and Amies.
and molecular genomic pattern of the
organism. For characterizing a species we
Infobits
have to isolate the organisms in pure form.
Viral Transport Medium is used to carry Pure culture or axenic culture is a culture
a specimen containing viruses. Universal containing only one type of organism.
Transport Viral Medium (UTVM). The descendents of a single organism in
This liquid medium is stable at room pure culture is called a strain. A strain
temperature. It is used for collection, forms a single colony. Colony is a cluster of
transport, and maintenance and long microorganisms in which all the characters
term freeze storage of viruses. of the family remain same. With the advent
of the pure culture techniques many
Exceptions in cultivation of microbes in microorganisms are being identified.
artifical medium
5.3.1 Methods Employed in the
Some bacteria like Mycobacterium leprae Isolation of Microorganisms
and Treponema pallidum cannot be
cultivated in artificial medium. Though there are many methods
designed for isolation of microorganisms,
ix) Media used for isolation of fungi pour plate method, spread plate method
Apart from the bacteriological media, and streak plate method are widely used
fungal media are used to study fungal in the field of Microbiology.
morphology pigmentation and sporulation.
Sabouraud’s Dextrose Agar (SDA) is used i) Pour plate method
as a common medium to isolate fungus. • It is the used for the isolation and
There are several other important fungal counting of colony forming bacteria in
media used for fungal cultivation. Examples
the specified sample.
Niger Seed Agar and Potato Dextrose Agar
• In this technique a sample is diluted
HOTS several times to reduce the density of
the microbial population.
1.
Which medium is used to carry • A very small amount of diluted sample
the sample when the sick person is (1ml or 0.1ml) is mixed with the molten
unable to come to the laboratory? agar at a temperature of 45°C.
2.
Give a special medium to check • The mixture is poured into the sterile
the growth of anerobes in a burn petridish (In 1887, Juluis Richard Petri,
wound infection with dead tissues. a worker in Koch’s laboratory, designed
the Petriplate.) in an aseptic condition
60

Infobits
Nowadays media are available as contact plates, agar strips, media cassettes, contact
slide and settle plates which are used for microbial air monitoring and compressed
gas lines in food and beverage production plants. These media are also used for the
enumeration of typical food contaminants such as coliforms, yeast and molds.
Colour coded MC-MEDIA pads are available for rapid and convenient microbial
testing for Escherichia coli, yeast, mold, coliform and aerobes.
and plates are incubated at a specific ii) Reduced growth of obligate aerobes
temperature for a given period of time. in the depth of agar.
• Plates are incubated in an inverted iii) Colonies embedded within the
manner. agar are much smaller than that of
• After incubation, the colonies are formed s urface and may be confluent or

in a discrete pattern both on the surface i nvisible.
of agar and also embedded within the
Basic five ‘I’ steps
medium.
one should follow
• Pour plate can be also used to deter- in culturing micro
mine the number of cells in a popula- o
rganisms
tion.(Figure 5.10)
a. Inoculation
Disadvantages of pour plate method b. Incubation
i) Loss of viability of heat sensitive c. Isolation
organisms coming into contact with d. Inspection
hot agar. e. Identification
Molten Bacterial
agar Suspension
at 45ºC-50ºC
Mix, Incubate
Colonies grow on
surface and within agar
Figure 5.10: Pour plate method
61

ii) Spread plate method iii) Streak plate technique
• Spread plate method is an easy and • The streak plate
direct method of isolating a pure technique is one of the
culture. most commonly used
• In this technique a specified amount methods for isolating
of diluted inoculum (0.1ml or less) pure culture of bacteria.
of microbial culture is seeded on agar • In this method, a
plate. loopful of inoculum from a sample
• After inoculation of the sample on the is taken and it is streaked across the
agar medium, the inoculum is evenly surface of the sterile solid medium.
spread on the surface with the help of • Different streaking patterns can be
a sterile glass L rod (a bent glass rod) used to separate individual bacterial
• Microorganisms are evenly distributed cell on the agar surface.
in the entire surface of agar. • After the first sector is streaked the
inoculated loop is sterilized and
• The dispersed microorganisms develop
inoculum for the second sector is
into isolated colonies.
obtained from the first sector.
• In this method, the plates are incubated
• Similar process is followed for streaking
at a specified temperature for a given
the further areas in the sectors.
period of time.
• Since the inoculum is serially diluted
• After incubation the plates are observed
during streaking patterns the dilution
for the growth of discrete colonies.
gradient is established across the
• The number of colonies are equal to surface of the medium.
the number of viable organism. This • After streaking, plates are incubated
method can be used to count the at a specific temperature for a given
microbial population (Figure 5.11). period of time.
Spread plate method
1 Sample (0.1 mL) poured 2 Spread sample evenly 3 Bacterial colonies

onto solid medium over the surface grow on the surface
of the medium
Bacterial
dilution
Incubation
Figure 5.11: Spread plate method

62

• After incubation, plates are observed 5.4 Growth and Colony Characteristics
for growth of colonies (based on the of Bacteria and Fungi
streaking pattern and density of culture
In the previous section we have learned
growth of microbes are abundant in
the various types of media and specific
the first sector in comparison with
purpose of each medium. Morphology
the formation of separated discrete
is the basic criteria for the isolation,
colonies in the fourth sector of the
identification and classification of
agar medium).
microorganisms. Colony characteristics
• Each isolated colony is assumed to be are the basic tool in the field of taxonomy.
grown from a single bacteria and thus
Bacteria grow in both solid and
represent a clone of pure culture.
liquid medium, but identification will
• Successful isolation depends on spatial be easy on the solid medium. In solid
separation of single cells (Figure 5.12). medium bacteria form colonies. In liquid
medium growth of bacteria are generally
Infobits not distinctive because there is uniform
turbidity or sediment at the bottom or
Micro manipulator: It is a device used pellicle is formed on the surface.
along with a microscope to pick a single
Some basic attributes such as shape,
bacterial cell from a mixed culture.
size, colour, pigmentation, texture, elevation
It has micropipette or microprobe and margin of the bacterial colony in the
so that a single cell can be picked up. growth medium are explained below.
A B C
Heavy confluent growth

Discrete colonies
Initial Second set of 1
inoculum streaks
4
Heavy growth
2
Third set of Fourth set of Incubation 3
streaks streaks
Light growth
D E
Figure 5.12: Steps in streak plate isolation method

63

Form
Punctiform Circular Filamentous Irregular Rhizoid Spindle (lens)
Elevation
Flat Raised Convex Pulvinate Umbonate
Margin
Entire Undulate Filamentous Lobate Erose Curled

(even) (wavy) (lobes) (serrated)
Figure 5.13: Colony morphology of bacteria
5.4.1 Colony Morphology of Bacteria be dry, moist, mucoid, brittle (dry breaks
on Solid Media apart), viscid (sticks to loop, hard to get
Shape: The shape of colony may be off), viscous, or butyrous (buttery).
circular, irregular, filamentous, rhizoid. Opacity of the bacterial Colony: Colonies
Elevation: It is the side view of the colony. may exhibit different optical density. It may
It may be flat, raised, umbonate (having be transparent (clear), opaque (not clear),
a knobby protuberance) crateriform, translucent (almost clear), or iridescent
convex pulvinate (cushion shaped) (changing colour in reflected light).
Margin: The margin of the bacterial colony Colony Odour: Some bacteria produce a
may be entire (smooth) irregular, undulate characteristic smell, which sometimes helps
(ovary), lobate, curled, filiform. The in identifying the bacteria. Actinomycetes
irregular shape of the colony give irregular produce an earthy odour which is quite
margin (Figure 5.13). often experienced after rain. Many fungi
produce fruity smell while Escherichia coli
Colony Size: The diameter of the colony
produce a faecal odour.
is measured in millimeter. It is described
in relative terms such as pinpoint, small, Smooth colonies
medium and large. of Streptococcus
Appearance of colony on the surface: The pneumoniae are
bacterial colonies are frequently shiny/ usually virulent, where
smooth in appearance. Colonies may be as rough colonies are non-virulent. But
veined, rough, dull, wrinkled, or glistening. in Mycobacterium tuberculosis colonies
Texture of the colony: Texture means with rough surface indicates a good
consistency of the bacterial growth. It may factor of virulence.
64

Colony Colour: Many bacteria develop
colonies which are pigmented.(Table 5.3) Certain water soluble
Some bacteria produce and retain water pigments are fluorescent
insoluble pigments and the colonies in nature Example: Py-
appear coloured by taking the pigment overdin. Agar medium
intracellularly (Figure 5.14). But some around the colonies glows white or blue
bacteria produce water soluble pigment green when exposed to ultraviolet light.
which diffuse into the surrounding
agar. Example: Pyocyanin pigment of 5.4.2 Nature of bacterial growth in
Pseudomonas aeruginosa is a water soluble liquid medium
pigment and give blue colour to the 1. If the entire broth appears milky and
medium. cloudy it is called turbid.
2. If deposit of cells are present at the bottom
of the tube, the term sediment is used.
3. If the bacterial growth forms a
continous or interrupted sheet over the
broth it is called pellicle (Figure 5.15).
5.4.3 Growth and Colony Characteristics

of Fungi
Fungi are eukaryotic organisms. They exist
in both unicellular-yeast like form and in
filamentous multicellular hyphae or mold
Figure 5.14: Pigmentation of bacterial
colonies on culture medium form and some are dimorphic. Generally
Figure 5.15: Microbial growth in Liquid medium

65

Table 5.3: Pigmentation of chromogenic bacteria
Bacteria Pigment colour
Serratia marcescens Red
Staphylococcus aureus Golden yellow
Micrococcus luteus Yellow
Pseudomonas aeruginosa Green
fungi prefer to grow in the acidic medium. • Growth and colony characteristics of
Sabourad Dextrose Agar (SDA) plates and yeast Candida
Potato Agar plates are used for general Yeasts are grown on Sabourad
cultivation of fungi. The acidic nature of Dextrose Agar aerobically. Yeasts
SDA agar reduce the growth of bacteria. grow as typical pasty colonies and
The characters to be noticed in colony give out yeasty odour. The colony
of fungi are colour of the surface and morphology varies with different
reverse of the colony, texture of the surface yeasts. Yeasts colonies generally have
(powdery, granular, ecolly, cottony, velvety smooth texture and are larger than
or glabrous), the topography (elevation, bacterial colonies on SDA medium
folding, margin) and the rate of growth.
(Figure 5.16a).
Infobits • Growth and Colony characteristics
of mold Mucor
Dimorphic fungi are fungi that can
exist in both mold and yeast form The genus Mucor is typically coloured
depending on environmental and white to brown or grey and is fast
physiological conditions. Example: growing. Older colonies become grey
Histoplasma capsulatum, a human to brown due to the development of
pathogen, grows as a mold form at spores. (Figure 5.16b).
room temperature and as a yeast form at
human body temperature.
Figure 5.16: Fungal growth on Sabouraud Dextrose Agar media a) yeast growth
b) mold growth
66

67

ICT CORNER
Streak Plate Technique
Isolation of pure
culture of bacteria
STEPS:
• Use the URL or scan the QR code to reach ‘Virtual Interactive
Bacteriology Laboratory.
• Click module and read the description and steps.
• Do the streak plating process from the top left part to the bottom
left order.
• Heat and cool the loop between each steps.
Step1 Step2 Step3
Step4
URL:
http://learn.chm.msu.edu/vibl/content/
streakplate.html
68

Summary c. Basal media
In natural environments microogran- d. General purpose media
isms exist as mixed cultures. Survival and 2. is an example
growth of microorganisms depends upon for differential media
the availability of favourable growth envi- a. Blood agar
ronment. Cultivation of microorganisms b. EMB agar
in the laboratory plays an important role c. Both a and b
in isolation, identification and classifica-
d. None
tion of microorganisms. A medium is an
3. A medium in which precise
environment which supplies the nutrients
ingredients are clearly defined.
necessary for the growth of the micro-
organisms. Various kinds of media have a. Synthetic medium.
been prepared to satisfy the need of the b. Non synthetic medium.
microorganism to be isolated as a pure c. Complex medium
culture. Based on the physical, chemical d. Natural medium
and special purposes, media are classified 4. A microbial inoculum of faecal
and are used to identify a particular or- specimen is subjected to isolation
ganism from a clinical specimen or envi- of typhoid bacilli species. Which
ronment. In Microbiology there are many medium can be used to select the
methods used for isolation of microor- bacilli?
ganisms. The methods commonly used
a. Selective medium
for isolation are pour plate, spread plate
b. Basal medium
method and streak plate method.
c. Enriched medium
The growth of organisms on media
d. Differential medium
is a basic criteria in the isolation,
identification, and classification of 5. is the method
microorganisms. Colony characterization in which inoculum is not placed over
of both bacteria and fungi highly depends the surface of agar plate.
upon the nutrients, temperature and pH. a. Pour plate method
b. Spread plate method
Evaluation c. Streak plate method
Multiple choice questions d. All the above
1. In a culture, the 6. Name the method in which the
desired organism inoculums is mixed with the molten
is low in number agar medium in the test tube and
when compared with unwanted poured into the sterile petridish
microorganism. Which media can be a. Pour plate method
used to isolate the desired organism? b. Spread plate method
a. Selective media c. Streak plate
b. Enriched media d. All the above
69

7. The culture with only one type of a. Growth will be clear
organism in the colony is called b. Growth will not be clear
c. Growth will be either clear or
a. Pure culture disturbed.
b. Mixed culture d. None of the above
c. Semi mixed culture
12. If chromogenic bacteria produce
d. Contaminated culture intracellular water insoluble pigment,
8. Identify the reason for the meager it will stain
growth of aerobic colonies in pour a. Growth of a colony
plate isolation method b. Agar medium
a. Less oxygen availability c. Both a and b
b. More oxygen availability d. None of the above
c. Carbon-di-oxide availability
13. If the water soluble pigment of the
d. None of the above pigmented bacteria diffuses into the
9. If a microbial inoculum is with more medium,
contaminations, which method will a. Medium gets pigmented
be used for isolation? b. Colony get stained
a. Spread plate method c. Both a and b
b. Pour plate method d. None of the above
c. Streak plate method
d. All the above Answer the following
1. Define semisolid media with an
10. The plate has a culture of A and B with
example.
definite circular morphology If A is
producing an inhibitory substance 2. State basal media with an example.
towards B, what will happen to the
3. What is synthetic medium? Give
colony morphology of B?
suitable example.
a. Change in the colony pattern of A
4. State a few aspects of enrichment
b. Change in the colony pattern of a B
medium.
c. Change in the colony pattern of
both A and B 5. State 3 fungal media used for isolation
d. No change of fungi.
11. If a plate observing for colony 6. Define pure culture.

morphology is subjected to 7. How do you differentiate pure culture
contamination, what will happen to from mixed culture?
the colony?
70

8. Why are the colonies growth on 16. Give a list of pigment producing
surface in pour plate method are quite bacteria.
larger than those within the medium?
17. Explain the opacity of a bacterial
9. Why is it important to invert the colony.
petridish during incubation?
18. Explain the special purpose media in
10. State the various forms in the appea- detail (any 5).
rance of the colony. Name the pigments 19. Why should we use a streak plate to
produced by Pseudomonas aeroginosa. grow a bacterium rather than on agar
11. Colony characteristics will be studied medium slant or in broth medium?
and identified clearly by using the 20. Expain the colony morphology of
nutrient agar medium in agar rather bacteria with diagrams.
than agar slant. Why?
12. Write about the elevation of the Student Activity
bacterial colony?
1. The student will list out the
13. Explain streak plate/pour plate/spread substances which contain agar in
plate method. their routine life and the role of
agar in it.
14. Why is agar mainly used as a
2. Students will prepare chart/scrap
solidifying agent even though other
book containing pictures of different
solidifying agents are available?
types of media and colony types of
15. How do you differentiate enrichment bacteria and fungi.
medium from selective medium? 3. Collect decayed/spoilt food for
macroscopic observation.
71

Chapter 6
Microbial
Nutrition
and Growth
Chapter Outline
6.1 Microbial Nutrients

6.2 Nutrient Requirement of
Microorganisms
6.3 Nutritional Types of
Microorganisms
6.4 Photosynthesis
6.5 Microbial Growth Mold is a type of fungi that grows on food and other
organic matters. It breaks down the complex substances into
6.6 Measurement of Microbial Growth simpler ones and extracts nutrient for its growth from them.
Learning Objectives
acquire energy from various organic and
After studying this chapter the student inorganic compounds, light and CO2. The
will be able, requirement of energy depends on their
need and metabolic ability.
• To know the essential nutrients
required by bacterial cell.
6.2 Nutrient Requirement of
• To differentiate between macronu- Microorganisms
trients and micronutrients.
• To describe an organism based on Microorganisms requires macronutrients,
the sources of carbon and energy. micronutrients and growth factors, for their
growth. These nutrients help in constructing
• To compare the photosynthesis the cellular components like proteins,
process in plant, algae and bacteria. nucleic acids and lipids.
• To understand the phases of growth
in bacterial growth curve. Macronutrients
• To know the methods of counting Elements that are required in large amounts
bacteria. are called macronutrients. Nitrogen (N),
Carbon (C), Oxygen (O), Hydrogen (H),
6.1 Microbial Nutrition Sulphur (S) and Phosphorus (P), Potassium
(K), Calcium (Ca), Magnesium (Mg) and
All living organisms on this planet require
energy for the normal functioning, growth Iron (Fe) are macroelements.
and reproduction. Likewise, microorganisms
72

Nitrogen is needed for the synthesis of Micronutrients
amino acids, nucleotides like purines and Nutrients that are needed in trace quantities
pyrimidines which are part of nucleic acids are called micronutrients. Example: Zinc
(DNA and RNA). (Zn), Molybdenum (Mo), Cobalt (Co),
Phosphorus is a part of phospholipids, Manganese (Mn).
nucleotides like ATP and phosphodiester
bonds of nucleic acids.
Besides macro and micronutrients,
Carbon, Hydrogen and Oxygen are the
some microorganisms need growth factors
backbone of all organic macromolecules
like amino acids, purines and pyrimidines
like peptidoglycan, proteins and lipids and
and vitamins. Example: Biotin is required by
nucleic acids.
Leuconostoc sp and folic acid is required by
Sulphur is needed for the synthesis Enterococcus faecalis.
of thiamin, biotin, and aminoacids like
cysteine and methionine. HOTS
Potassium, Calcium, Magnesium and
Iron exist as cations in the cell. These Is there a microbe that can grow in
element plays vital role in the metabolic a medium that contains only the
activity of microorganisms. Potassium (K+) following compounds in water: calcium
carbonate, magnesium nitrate, ferrous
is needed for the activity of many enzymes
chloride, zinc sulphate and glucose.
Example: Pyruvate Kinase. Defend your answer.
Calcium (Ca2+) is involved in the heat
resistance of bacterial endospores.
6.3 Nutritional Types of
Magnesium (Mg2+) binds with ATP Microorganisms
and serves as a cofactor of enzymes like
Microorganisms can be classified into
hexokinase. nutritional classes based on how they satisfy
Iron (Fe2+ or Fe3+) is present in the requirements of carbon, energy and
cytochromes and act as cofactors for electrons for their growth and nutrition.
cytochrome oxidase, catalase and peroxidase. Based on the carbon source,
microorganisms are able to utilize, they are
Diatoms (A group classified into Autotrophs and Heterotrophs.
of algae) need silicon
Autotrophs: These are organisms that
to construct their
beautiful cell walls. utilize CO2 as their sole source of carbon.
Heterotrophs: These are organisms that
use preformed organic substances from
other organisms as their carbon source.
Based on energy source, microorganisms
are classified into Phototrophs and
Chemotrophs.
73

Phototrophs: These are organisms that
utilize light (radiant energy) as their energy
source.
Chemotrophs: These are organisms that
obtain energy by oxidation of organic or
inorganic compounds.
Microorganisms are classified into
Lithotrophs and Organotrophs based
on the source from which they extract
electrons. Lithotrophs are organisms that
use reduced inorganic substances as their Figure 6.1: Microscopic view of
Cyanobacteria
electron source whereas Organotrophs
obtain electrons from organic compounds 2. Photoheterotrophs: These organisms
(Table 6.1). make use of light as energy source and
organic compounds as electron and
All microorganisms fall into any one
carbon source. Example: Purple and
of the four nutritional classes based on
Green Non sulphur bacteria
their primary source of carbon, energy and
electrons. 3. Chemoautotrophs: These are
ecologically important microorganisms.
1. Photoautotrophs: Eukaryotic algae, They oxidize inorganic compounds
Cyanobacteria (Blue Green Algae) like nitrate, iron and sulphur to obtain
(Figure 6.1) and Purple and Green energy and electrons.
Sulphur bacteria belong to this class. 4. Chemoheterotrophs: These organisms
They are capable of using light energy use organic compounds to satisfy their
and have carbondioxide as the sole needs of energy, electron and carbon.
source of carbon. (Table 6.2)
Table 6.1: Classification of microorganism based on carbon, energy and electron sources
Carbon, Energy and Electron sources
Carbon sources
Autotrophs CO2 as sole carbon source
Heterotrophs Organic substances from other organisms
Energy sources
Phototroph Light energy
Chemotrophs Chemical energy source (Organic or Inorganic)
Electron sources
Lithotrophs Reduced inorganic substances
Organotrophs Organic compounds
74

a. Blood lake in Texas-the blood red colour is due to the excess presence
of purple sulphur bacteria.
b. Giant tube worms seen in deep sea hydrothermal vents survive on
nutrients given by chemolithotrophic bacteria.
a) b)
6.4 Photosynthesis Eukaryotes (plants and algae) and

Photosynthesis is a process prokaryotes (cyanobacteria and purple,
of capturing light energy and green bacteria) are capable of carrying out
converting into chemical photosynthesis. Cyanobacteria perform
energy. The chemical photosynthesis in a similar manner to
energy produced in the plants.
form of ATP and NADPH
is used to synthesise organic Photosynthesis in Bacteria
compounds (carbohydrates); to be used as
food. This ability makes photosynthesis, a There are four groups of photosynthetic
significant process taking place on earth. bacteria. They are green sulphur bacteria
Table 6.2: Nutritional classes of Microorganisms

Nutritional class Energy/Electron/Carbon source Organisms
Litho Light energy Cyanobacteria, Purple and
photoautotrophs Inorganic e- donor Green sulphur Bacteria
CO2
Organo Light energy Purple and Green
photoheterotrophs Organic e- donor Nonsulfur bacteria
Organic carbon source
Ltiho Inorganic chemical compounds as Nitrifying bacteria, Iron
chemoautotrophs energy source bacteria
Inorganic e- donor
CO2
Organo Organic compounds as energy, Most pathogenic bacteria,
chemoheterotrophs electron and carbon source. fungi and protozoa.
75

(Example: Chlorobium) and green non 6.5 Microbial Growth
sulphur bacteria (Example: Chloroflexus)
In bacteria, growth can be defined as an
purple sulphur bacteria (Example:
increase in cellular constituents. Growth
Chromatium) and purple non sulphur
results in increase of cell number.
bacteria (Example: Rhodospirillum). These
photosynthetic bacteria can fix atmospheric When bacteria are cultivated in liquid
CO2 in a similar fashion like cyanobacteria medium and are grown as batch culture
but using only one photosystem and using (Growth occurring in a single batch of
H2S as the electron donor instead of H2O. medium with no fresh medium provided),
Process of photosynthesis in bacteria cell multiplication happens till all the
The electron transport system in purple nutrients are exhausted. After sometime,
and green bacteria consists of only one nutrient concentrations decline and
Photosystem PSI (P870). They do not bacterial cells begin to die. This growth
possess photosystem II. When P870 gets pattern can be plotted in a graph as the
excited upon capture of light energy, it logarithm of viable cells versus incubation
donates the electron to bacteriopheophytin. time (Figure 6.4). The growth curve has
Electrons flow through quinones and
four distinct phases.
cytochromes and are reverted back to P870.
This process is cyclic (since the electron 1. Lag phase
excited from P870 comes back to P870)
2. Logrithmic phase/Exponential phase
and generates ATP. A reversed electron
flow operates in purple bacteria to reduce 3. Stationary phase
NAD+ to NADH. Electrons are extracted
4. Death phase
from external electron donors like hydrogen
sulphide, hydrogen, elemental sulphur and
1. Lag Phase
organic compounds to synthesise NADH.
Since H2O is not used as electron donor, When bacteria are introduced into fresh
oxygen is not evolved which explains medium, no immediate cell multiplication
the anoxygenic nature of the organisms and increase in cell numbers occur.
involved (Figure 6.3). The sulphur evolved The cell prepares itself for cell division
during this reaction is deposited as sulphur by synthesizing cell components and
globules either outside or inside the cells. increase in cell mass. Since there is a lag
CO2 + H2S  (CH2O)n + S. Table 6.3 in cell division, this phase is called lag
compares the photosystheic process in phase.
plants, algae and bacteria.
2. Logrithmic Phase/Exponential
Phase
HOTS During this phase, microorganisms rapidly
divide and grow at a maximal rate possible
1. What will be the electron flow utilizing all the nutrients present in the
sequence of noncyclic and cyclic medium. The growth rate is constant during
the exponential phase. The organism divides
photo phosphorylation?
and doubles in number at regular intervals.
2. Chemical energy produced in The growth curve rises smoothly.
photosynthesis is either ATP
NADPH or ATP NADH. Why?
76

Stationary
phase
Logarithmic Death
growth (decline)
phase phase
Logarithm of viable cells
Lag phase
0 5 10
Time (hr.)
Number of viable and

Few or no cells Viable cells Nonviable cells
nonviable cells in population
Figure 6.4: Bacterial growth curve showing phases of growth in laboratory conditions
3. Stationary Phase medium is added and utilized (spent)

As the nutrients get depleted, the cell medium are removed continuously at
growth stops and the growth curve become a constant rate. A microbial culture
horizontal. The total number of viable cells remains in exponential state for longer
remains constant which is due to a balance periods, for days and even weeks. This
between cell division and cell death. enables the researcher to learn about the
physiological processes and enzymatic
activities of organisms.
4. Death Phase
There are two ways by which continuous
Nutrient deprivation and build up of
wastes lead to the decline in cell numbers. culture is operated.
The microbial population dies rapidly and a) Chemostat
logarithmically and the growth curve also b) Turbidostat
stops down.
Chemostat
Batch culture The chemostat operates so that the sterile
It is the growth of microorganisms in a fixed nutrient medium enters the culture
volume of culture medium in which nutrient vessel at the same rate as the spent
supply is not renewed and wastes are not medium is removed. The chemostat
removed. It is a closed system. This can be can control growth rate and cell density
used to study the various growth phases of simultaneously and independently of
microorganisms. each other. Two factors play an important
role in achieving this dilution rate and
Continuous culture
concentration of the limiting nutrient (a
A continuous culture is an open system carbon or a nitrogen source like sugars or
with constant volume to which fresh
77

aminoacids). Growth rate can be controlled by adjusting the dilution rate and cell density
is controlled by modifying the concentration of the limiting nutrient (Figure 6.5).
Fresh medium Reservoir of

sterile medium
Control valve
Value controlling
Air Flow of medium
supply
Air
filter
Outlet for
Culture spent
vessel medium
Light
source
Photo cell
Receptacle Turbidostar
Figure 6.5: The Chemostat Figure 6.6: The Turbidostat
Turbidostat 1. Temperature
This type of continuous culture system Temperature is one of the most important
has a photocell that measures the environmental factor affecting the
turbidity of the culture vessel. This growth and survival of microorganisms.
automatically regulates the flow rate Temperature can affect microorganisms
of the culture medium. Turbidostat b ecause the enzyme catalysed reactions
does not contain limiting nutrients are sensitive to fluctuations in
(Figure 6.6). temperature.
6.5.1 Factors Influencing Growth For every microorganism, there is a
minimum temperature below which no
The growth and activities of
growth occurs, an optimum temperature at
microorganisms are greatly influenced
by the physical and chemical conditions which growth is most rapid, and a maximum
of their environment. Among all temperature above which no growth occur.
factors, four key factors play major These three temperatures are called cardinal
roles in controlling the growth of temperatures.
microorganisms. They are
Temperature classes of microorganisms
1. Temperature
Microorganisms are broadly distinguished
2. pH into four groups in relation to their
3. Water activity temperature optima.
4. Oxygen • Psychrophiles
78

• Mesophiles Mesophiles
• Thermophiles These are microorganisms that grow in
optimum temperature between 20-45°C,
• Hyperthermophiles they have a temperature minimum of
Psychrophiles 15-20°C and a maximum temperature
A psychrophile can be defined as an of 45°C. All human pathogens are
organism with an optimal growth mesophiles.
temperature of 15°C, maximum growth Thermophiles
temperature of 20°C and a minimum
Organisms whose growth temperature
growth temperature at 0°C. These
optimum is between 55-65°C are called
organisms are found in polar regions like
thermophiles. They have minimum
Arctic and Antarctic oceans. They are
growth temperature of 45°C. These
rapidly killed as the temperature rises
organisms are found in compost stacks,
because the cellular constituents start to
hot water lines and hot springs. They
leak due to cell membrane disruption. Some
contain enzymes that are heat stable and
examples of psychrophiles are Moritella,
protein synthesis systems function well at
Photobacterium and Pseudomonas.
high temperature.
Snow alga – Chlam- Infobits

ydomonas nivalis
grows within the snow Taq polymerase, a DNA polymerase
and its brilliant red enzyme which is of great applied
coloured spores are importance used in DNA amplification.
responsible for the formation of pink It is isolated from Thermus aquaticus, a
snow. thermophile.
Hyperthermophiles
Organisms whose growth optimum
temperature is above 80°C are called
hyperthermophiles. These are mostly
bacteria and archaebacteria. They
are found in boiling hot springs and
HOTS hydrothermal vents on seafloor.
Why do unopened pasteurized milk 2. pH

spoil even under refrigeration? pH is defined as the negative logarithm of the
hydrogen ion concentration. pH scale extends
from pH 0.0 to pH 14.0 and each exchange
of 1 pH unit represents a 10 fold change
Psychrotolerant
in hydrogen ion concentration. pH greatly
Organisms that can grow at 0°C, but influences microbial growth. Each organism
have temperature optimum growth has a definite pH range and well defined pH
temperature range of 20°C-40°C are called growth optimum. Most natural environments
psychrotolerant. have pH values between 5 and 9.
79

Organisms are classified into 4. Oxygen
Acidophiles, Neutrophiles and Alkalophiles Most of the microorganisms require oxygen
based on their optimum growth pH. for their optimal growth but some of them
Acidophiles are organisms that grow survive very well in total absence of oxygen
best at low pH (0.0–5.5) Example: Most and are killed when exposed to air.
fungi, bacteria like Acidithiobacillus, Based on their need and tolerance for
rchaebacteria like Sulfolobus
A and oxygen, microorganisms are classified into
Thermoplasma. the following types.
Neutrophiles are organisms that grow (1) Obligate aerobes exhibit growth only at
well at an optimum pH between 5.5 and 8.0. full oxygen level (21% O2 on air) because O2
Most bacteria and protozoa are neutrophiles. is needed for their respiration and metabolic
activities Example: Micrococcus, most Algae,
Organisms that prefer to grow at pH
Fungi and Protozoa.
between 8.5-11.5 are called alkalophiles.
These microorganisms are typically found (2) Microaerophiles are aerobes that
in soda lakes and high carbonate soils. require oxygen at levels lower than that of
air. Example: Azospirillum, Campylobacter,
Example: Bacillus firmus.
Treponema
3. Water Activity and Osmosis (3) Obligate anaerobes does not require
Water activity, (aw) is the ratio of vapour oxygen for their respiration and growth. This
pressure of the solution to the vapour group cannot tolerate O2 and are killed in its
pressure of pure water (aw values vary presence. Example: Methanogens, Clostridium.
between 0 and 1). Water activity is inversely (4) Aerotolerant anaerobes can grow
related to osmotic pressure. Organisms in the presence of oxygen though O2 is
that can grow in low aw values are called not required for their growth. Example:
osmotolerant. Example: Staphylococus Streptococcus pyogenes.
aureus.
Only a few organisms are capable of (5). Facultative anaerobes can grow either
under oxic or anoxic conditions: Example:
tolerating high salt concentration and still
Escherichia coli. (Figure 6.7)
growing optimally in low water activity. Such
organisms are called halophiles. Halophiles 6.6 Measurement of Growth
can grow in 1-15% Sodium chloride (NaCl)
Different methods are employed for
concentrations. Organisms that can grow in
measuring the cell growth of microorganisms.
very salty environments are called extreme Cell growth is indicated by increase in the
halophiles. (They can grow in 15-30%) NaCl number of cells or increase in weight of cell
concentration. Example: H alobacterium. mass. There are direct and indirect methods
of measuring microbial growth.
Crenation: 1. Direct Measurements
Shriveling of cytoplasm
Total count and viable count are the two
in the cell is called
methods widely employed to count cell
crenation. This effect
numbers.
helps to preserve some foods.
80

Total count: Disadvantages
The total number of cells in a population 1. Dead cells are also counted
can be measured by counting a sample 2. Special microscopes like phase contrast
under the microscope. This is called microscope are needed if unstained
direct microscopic count. This is done samples are used.
by using a specialized counting chamber 3. Small cells are difficult to count
called Petroff Hausser chamber which is
a specially designed slide with a grid. The 2. Viable Count
liquid sample is placed on the grid which A viable cell is one that is able to divide and
has a total area of 1mm2 and divided into 25 form a visible colony on the nutrient media.
large squares. The number of cells in large Viable cells are counted by methods pour
square is counted and the total number of plate and spread plate.
cells is calculated by multiplying it with a
conversion factor based on the volume of Pour plate method
the chamber (Figure 6.8). In this method, a known volume (0.1 or
1.0ml) of the culture is pipetted into a sterile
Advantages
petri plate, then molten nutrient medium is
This is a quick method of estimating cell poured over and incubated. Colonies will
numbers. appear throughout the agar medium and are
counted to obtain viable count.
Obligate Obligate Facultative Aerotolerant

aerobes anaerobes anaerobes anaerobes Microaerophiles
A B C D E
Figure 6.7: The effect of oxygen on the growth of various types of bacteria
Count cells
in this square
1.00 mm
1.00 mm
0.05 mm
0.25 mm 1.00 mm
(a) (b)
Figure 6.8: (a) Petroff-Hausser counting chamber

(b) Microscopic observation of bacterial cells
81

Spread plate method
In this method, a known volume of the culture (0.1ml) is plated and spread over solidified sterile
agar medium, using a sterile spreader. The total number of colonies appearing on the plate after
incubation represents the total number of viable cells in the culture.
3. Measurement of Cell Mass
A cell suspension appears turbid or cloudy due to active cell growth. When light is passed through
this cell suspension, microbial cells scatter light striking them. As the concentration of cells and
turbidity increases, more light is scattered and less light is transmitted through the suspension. The
amount of unscattered light can be measured using a spectrophotometer, the values of which are
indirectly related to cell numbers.
82

ICT CORNER
Culture Media
Preparation of
Bacteriological Media
STEPS:
• Use the URL or scan the QR code to reach ‘Virtual Interactive Bacterial Laboratory’.
• Click module at the bottom and read the description and steps.
• Follow the steps and open activities under’ Common Bacteriologic Media’ one by
one and explore it.
• Record your observation of Differential Media. Click examples and record the
specimen suitable for particular media
Step1 Step2
Step3 Step4
URL:
http://learn.chm.msu.edu/vibl/content/
differential.html
83

Summary 2. Magnesium is needed
Microorganisms need macro and a. For cell wall synthesis
micronutrients for their growth. Based b. As cofactor for enzymes
on the energy source, organisms c. For photosynthesis
are grouped into Phototrophs and
Chemotrophs. Based on carbon source, d. For protein synthesis
they are classified into autotrophs and 3. One of the following is an example
heterotrophs. Organisms are grouped into for chemoautotroph
lithotrophs and organotrophs based on
their electron source. The four nutritional a. Cyanobacteria
classes of microbes are photoautotrophs, b. Purple and green non sulphur
Photoheterotrophs, chemoautotrophs bacteria
and chemoheterotrophs. c. Iron bacteria
Cyanobacteria are prokaryotes that can d. Protozoa
perform photosynthesis. Chlorophyll is 4. The phase of growth in which the
the pigment needed to capture light energy growth rate is equal to the death rate
(photons). In cyanobacteria and green is
plants, non cyclic photophosphorylation
a. Stationary phase
takes place to generate ATP and NADPH b. Death phase
during photosynthesis whereas cyclic c. Exponential phase
photophosphorylation takes place in purple d. Lag phase
and green bacteria involving only one 5. Organisms that are capable of growing
Photosystem (PS I). in 0°C are called
In a batch culture, bacteria show a a. Thermophiles
characteristic growth pattern which consists b. Hyper thermophiles
of lag phase, log phase and stationary phase c. Barophiles
and decline phase. In a chemostat, cultures d. Psychrophiles
can be maintained in an exponential phase
6. Halophiles are organisms that can
for long periods. The most important factors
grow in
affecting microbial growth are temperature,
pH and oxygen level. Total count and viable a. Low water activity
b. High salt concentration
count are the two widely used methods to
c. Low temperature
measure cell numbers. d. High pH
Evaluation 7. An example of microaerophilic
organism is
a. Bacillus b. Azospirillum
1. An example of photoautotroph
c. Pseudomonas d. Escherichia.coli
a. Cyanobacteria
b. Algae
c. Green plants
d. All of the above
84

8. The specialized chamber used for the 14. Describe the role of macro and
counting of microbial cells is micronutrients in microorganisms.
a. Haemocytometer How do you think bacteria acquire their
b. Counting chamber nutrients from their environment?
c. Petroff Hauss chamber 15. Explain the classification of microbes
d. Counting slide based on their nutrition. If H2S is toxic
to living organisms, how do purple and
Answer the following green bacteria survive and use H2S in
such environments.
1. Give notes on the nutritional classes of
16. Describe the photosystems of
microorganisms.
cyanobacteria.
2. Classify microorganisms based on
17. Draw a schematic representation
energy and carbon source.
of Z scheme of non cyclic
3. What are light and dark reactions in photophosporylation.
photosynthesis? 18. Compare photosynthesis between
4. What is bacteriochlorophyll? Give plants, cyanobacteria and purple green
its role. bacteria.
5. Define chemoautotroph. 19. Explain the principle and uses of
6. Define photosynthesis. chemostat and turbidostat with
7. Give examples of photosynthetic diagrams.
bacteria. 20. Describe the classification of
8. What do mean by cardinal temperature? microorganism based on their oxygen
9. Give notes on photosynthetic pigments. requirement.
21. Explain the principle and uses of
10. What are halophiles?
chemostat and turbidostat with
11. Give reason for the ability of diagrams.
thermophiles to grow in high
22. Explain the relation of osmosis to water
temperatures.
activity.
12. How bacterial cells are counted using
23. Define growth. Explain the phases of
counting chamber?
growth of bacteria with neat diagram.
13. Classify microorganisms based on their
temperature requirement.
Student Activity
• Expose a container with water to sunlight for a week. Observe the growth of
cyanobacteria on water which explains the photoautotrophic mode of nutrition.
• Store a loaf of bread for a week after the expiry date. You can observe the growth
of fungi/molds which demonstrates the mode of nutrition of chemoheterotrophs.
• Collect rusted iron pipes which contain chemolithotrophic Thiobacillus sp which can
oxidize iron for their nutrients.
• Place two bowls of cooked rice/vegetables–one inside the refrigerator at 6°C, and
another at room temperature at 30-35°C. Give reasons for the quick spoilage of the
rice stored at 30-35°C. Check the pH of milk using a pH paper.
85

Chapter 7
Morphology of Bacteria
Chapter Outline
7.1 Bacterial Size, Shape and

Arrangement
7.2 Structures External to Cell Wall of
Bacteria
7.3 Cell Envelope of Bacteria
7.4 Structures Internal
to Cell Membrane
The distinction between prokaryotes and eukaryotes
of Bacteria is considered to be the most important distinction
7.5 Eukaryotic Cell among groups of organisms. Eukaryotic cells contain
membrane bound organelles, such as mitochondria, while
Structure prokaryotic cells do not.
Learning Objectives
• To differentiate between Gram
After studying this chapter the student positive and Gram negative bacteria.
will be able, • To know the structures and functions
• To know the size, shape and internal to cell membrane.
arrangement of bacteria. • To differentiate between prokaryotic
• To list a few examples of bacteria and eukaryotic cell structure.
with their shapes.
• To understand and describe the role of Living organisms are differentiated from
the structures external to the cell wall. non living matter by their (1) ability to
reproduce (2) ability to ingest or assimilate
• To understand the structure, function
food and metabolize them for energy
and arrangement of bacterial
and growth (3) ability to excrete waste
flagella.
products (4) ability to react to changes
• To describe the role of capsule, slime in their environment (irritability) and
layer, pili, flagella and fimbriae in a (5) susceptibility to mutation. The living
prokaryotic cell.
organisms include a variety of micro and
• To describe the structure and function macro organisms of different size, shape,
of cell wall, outer membrane and cell morphology and behaviour. They include
membrane. tiny bacteria, protozoans, worms, plants
• To know the significance of Cell and animals.
Envelope.
86

Generalized structure of a bacterium
Infolding of
plasma Capsule Cell wall
membrane DNA coiled
into nucleoid
Basal body
Flagellum
Ribosomes
Cytoplasmic
inclusion Plasma Cytoplasm
Pili
membrane
Figure 7.1: Generalized structure of a bacterium
Bacteria, cyanobacteria (blue green made until the development of electron

algae) microalgae, protozoa, yeasts and microscope, which depicted the internal
fungi represent the microorganisms. structure of these organisms. The absence
Prokaryotes are organisms with primitive of membrane bound internal structures in
type of nucleus lacking a well defined bacteria and their presence in fungi, algae,
membrane (Figure 7.1). The nuclear protozoa, plant and animal cells was taken
material is a DNA molecule in prokaryotes as criterion to differentiate prokaryotes and
compared to chromosomes of higher eukaryotes.
organisms. Eukaryotes are organisms
7.1 Size, Shape and Arrangement of
with cells having true nuclei enclosed in
Bacteria
a nuclear membrane and are structurally
more complex than prokaryotes. There 7.1.1 Size of Bacteria
exists varying degree of localization of Bacteria are minute living bodies and
cellular functions in eukaryotes that occur represent one of the lowest orders of living
in distinct membrane bound intracellular cells. The determination of size of the
organelles like nuclei, mitochondria, different forms is originally carried out
chloroplasts. The cells of living organisms by comparison with known RBC. A more
are either prokaryotic or eukaryotic in accurate estimation is now obtained by
nature and there is not any intermediate the use of a special micrometer eye-piece,
condition. The size, shape, morphology containing a graduated scale. The unit of
and the internal cellular organizations are measurement of bacteria is called micron
different in these two groups. (µ or µm). 1 micron is equal to 1 thousand
Satisfactory criteria to differentiate of millimeter. Resolution of unaided eye is
bacteria, fungi and algae could not be 200µm. The size of bacteria is constant but
87

100 pm 1 nm 10 nm 100 nm 1 µm 10 µm 100 µm 1 µm
Eye
Light Microscope
Electron Microscope
Proteins
Lipids Organelles
Small
Atom
Molecules Virus Bacteria Eukaryotic Cells
Figure 7.2: Metric unit of measurement
depends upon environmental and growth 1 metre (m) = 1000mm (millimeter)

condition. Medically important bacteria 1mm (10-3m) = 1000 µm (micrometer)
ranges from 0.2 – 1.5 µm in diameter and 1 µm (10-6m) = 1000nm (nanometer)
3-5µm in length (Figure 7.2). 1nm (10-9m) = 1000pm (picometer)
1A0 (10-10m) (angstrom)
Infobits
The smallest bacteria is Mycoplasma genitalium, which has a diameter of 200-300nm.

The largest and longest bacterium is Thiomargarita namibiensis (750µm) found in
the ocean sediments in the continental shelf of Namibia. They are large enough to
be visible to the naked eye. The previously known largest bacterial cell Epulopiscium
fishelsoni is found only in the intestinal tract of certain topical fish over 500µm long.
Epulopiscium means “guest at the table of fish”.
Epulopiscium fishelsoni Mycoplasma genitalium
88

7.1.2 Cell Shape and Arrangement of and spirochetes (agent of syphilis).
Bacteria Although these two are similar in shape
The shape of a bacterium is governed by spirochetes are flexible in nature. Spiral
its rigid cell wall. Typical bacterial cells bacteria are far too thin to be seen with
are spherical (called cocci), straight rods the standard Brightfield microscope
(called bacilli) and helically curved rods but are readily observed by Darkfield
(called spiral). These shapes are constant m
icroscope (Figure 7.3).
for the particular species or genus but there
Filamentous bacteria
are bacterial cells that are pleomorphic in
nature. They exhibit a variety of shapes. Bacteria tend to form long strands
composed of many cells. In these cases,
• Cocci appear in several characteristic an occasional single cell may be seen after
arrangements, depending on the it breaks away from a long filament. These
plane of cellular division and whether organisms resemble the threadlike strands
daughter cells remain together with of fungi but their internal structure is
the parents even after cell division. The typical of bacteria. Filamentous soil bacteria
cells may occurs in pairs (diplococci), include Streptomyces species.
in groups of four (tetracocci), in clusters
(Staphylococcus), in a bead like chain Pleomorphic bacteria
(Streptococci) or in cuboidal arrangement A few bacteria lack rigid cell walls, and their
of cells (Sarcinae). flexible plasma membrane allows them to
• Bacilli are rod shaped organism change shape. These are called pleomorphic
(Singular, bacillus = stick) usually bacteria (pleo-more; morph-form).
ranging between 1 and 10 µm in length. Example: Mycoplasma.
Some bacilli are so short and stumpy that
they appear ovoid and are referred to as 7.2 Structures External to Cell Wall of
Bacetria
coccobacilli. Bacilli are not arranged in
patterns as complex as those of cocci 7.2.1 Appendages
and mostly occur as singles or in pairs Flagella
(diplobacilli, Example: Bacillus subtilis) Flagella (singular flagellum) are threadlike,
or in the form of chains (Streptobacilli). long, thin helical filaments measuring 0.01-
Some form trichomes, which are similar 0.02nm in diameter. These appendages
to chains. In other Bacilli such as extend outward from the plasma
Corynebacterium diphtheria the cells membrane and cell wall. Flagella are so
are lined side by side like matchsticks thin that they cannot be observed directly
(pallisade arrangement). Some bacilli with a bright field microscope, but must be
are curved into a form resembling a stained with special techniques (example:
comma. These cells are called vibrios as Fontana’s silver staining technique) that
in Vibrio cholera. increase their thickness. The detailed
• Spiral bacteria: They are divided into structure of a flagellum can only be seen in
two groups, spirilla (singular spirillum) the electron microscope.
89

Coccus
Coccobacillus
Bacillus Vibrio
Spirillum
Spirochete
Different Bacterial Shapes
COCCI COCCI
Single Diplococci Streptococci Tetrads Staphyiococci Sarcinae
BACILLI
Single Diplobacilli Streptobacilli Palisade
SPIRILLA
Single
Figure 7.3: Shapes and arrangement of bacteria
90

The bacterial flagellum is composed 2. In lateral arrangement, flagella are
of three parts: a basal body (associated arranged randomly all over the
with the cytoplasmic membrane and cell surface of the cell. Bacteria with
wall), a short hook and a helical filament lateral flagellar arrangement are
(which is usually several times as long as called peritrichous. (Table 7.1)
the cell). Filament is external to cell wall Various types of mobility are observed
and is connected to the hook at cell surface; based on the arrangement of the
the hook and basal body are embedded in flagella. Serpentine motility is seen with
the cell envelope (Figure 7.4). Hook and Salmonella, darting motility with Vibrio
filament are composed of protein subunits and tumbling motility with Listeria
called as flagellin. monocytogenes. Some bacteria like Cytophaga
One can generalize that all spirilla, about exhibit a gliding motility, which is slow
half of the bacilli and a small number of sinuous flexing motion. This occurs when
cocci are flagellated. Some bacteria do not the cells come in contact with solid surface.
have flagella. Flagella vary both in number Some bacteria have the ability to move
and arrangement on the cell surface. Flagella toward or away from chemical substance.
are arranged generally in two patterns. This movement is called chemotaxis.
1. In polar arrangement, the flagella Positive chemotaxis is the movement of a
are attached at one or both ends of cell in the direction of a favorable chemical
the cell. Bacteria with polar flagellar stimulus (usually a nutrient). Negative
arrangement are further classified chemotaxis is the movement away from
into monotrichous, lophotrichous, a chemical substance (usually harmful
and amphitrichous. compound). Some photosynthetic bacteria
exhibit phototaxis, movement in response
to light rather than chemicals.
Flagellum
Hook
Filament
Outer membrane
Basal Body
Rod
Peptidoglycan portion
of cell wall
Figure 7.4: Structure of bacterial flagella

91

Table 7.1: Arrangement of bacterial flagella
Structure Flagella type Example
Monotrichous(single Vibrio cholera
flagella on one side)
Lophotrichous(tuft of Pseudomonas fluorescens

flagella on one end)
Amphitrichous(single or Aquaspirillum serpens

tuft on both ends)
Peritrichous(flagella Salmonella typhi

throughout the cells)
in certain species of Gram negative bacteria.

HOTS
Pili play no role in motility. Pili originate
from the plasma membrane and are made up
A.
If a bacterium loses its flagella, of a special protein called pilin (Figure 7.5).
does it survive?
Pili play a major role in human infection
B. If you remove the cell wall from a by allowing pathogenic bacteria to attach
flagellated bacterium, the organism to epithelial cells lining the respiratory,
loses the ability to move. Explain. intestinal or genitourinary tracts. This
attachment prevents the bacteria being
The presence of motility is one piece of washed away by body fluids, thus helps in
information used to identify a pathogen in establishment of infection. One specialized
the laboratory. One way to detect motility is type of pilus (sex pilus) helps in the transfer
to stab a tiny mass of cells into soft (semi solid) of genetic material between the bacterial
medium in a test tube. Growth spreading cells. This process is called conjugation.
rapidly through the entire medium is Fimbriae
indicative of motility. Alternatively, cells can
Fimbriae (singular: fimbria) is another term
be observed microscopically by a hanging
used for short pili that occur in great number
drop method.
around the cell. They enable bacteria to
Pili attach to surfaces and to each other, so that
Pili (singular pilus) are straight, short and the bacteria form clumps or films called
thin and more numerous than flagella pellicles on the surface of liquid in which they
around the cell. They can be observed only are growing. Fimbriae are found in Gram
by electron microscopy. They are found only positive as well as in Gram negative bacteria.
92

Flagellum
Fimbriae
Sex pilus
Flagella
Fimbriae
Pilus
Figure 7.5: Structures of pili and fimbriae
Table 7.2 compares the pili and fimbriae. homopolysaccharide (made up of a single
kind of sugar) or heteropolysaccharide
7.2.2 Extracellular Polymeric Substance (made up of several kinds of sugars). These
(EPS) are synthesized from sugars within the cell,
Many bacteria secrete high molecular transported and polymerized outside the
weight polymers that adhere to the exterior cell. The capsule of some bacteria is made
of the cell wall to form a capsule or slime of polypeptides. The capsule of Bacillus
layer. Glycocalyx is often used to refer to any anthracis has polymer of D-glutamic
polysaccharide material outside the cell wall. acid. Capsules are highly impermeable.
Capsules and slime layer are considered to Capsules can be demonstrated using
be glycocalyxes (Table 7.3). special staining technique utilizing Indian
ink or with Nigrosin stain. The presence of
Capsules capsule in fresh isolates gives a moist and
Some bacterial cells are surrounded by a shiny appearance to the bacterial colonies
viscous substance forming a covering layer on an agar medium. Capsular material
or envelope around the cell wall called is antigenic and may be demonstrated by
capsule (Figure 7.6). Capsule is usually serological methods.
made up of polysaccharide. It may be
Table 7.2: Comparison of pili and fimbriae

Characteristics Pili Fimbriae
Appearance Hair like, straight Tiny bristle like fibers arising from the
appendages. surface of bacterial cell.
Length Longer than fimbriae Shorter than pili
Numbers per cell 1-10/cell 200-400/cell
Presence Present only in Gram Present in both Gram positive and
negative bacteria Gram negative bacteria
Made -up of Pilin protein Fimbrillin protein
93

Table 7.3: Difference between Capsule and Slime layer
Capsule Slime layer
Capsule is a glycocalyx layer, consisting Slime layer is a glycocalyx layer that
of firmly associated polysaccharide consists of loosely associated glycoprotein
molecules with the cell wall. molecules.
It is a well-organized layer, difficult to be It is an unorganized layer and can be easily
washed off. washed off.
It is tightly bound to the cell wall. It is loosely bound to the cell wall.
It is thicker than slime layer. It is a thin glycocalyx layer.
It acts as a virulence factor that helps to It mainly helps in adherence. It protects
escape phagocytosis. the cell from dehydration and nutrient
loss.
Slime layer
Some bacteria are covered with a surface
layer that is loosely distributed around
the cell and diffuses into the medium, this
surface layer is referred to as slime layer.
(Figure 7.7) The slime layer is a structure
Capsule that is easily washed off. Slime layer protects
bacteria from loss of water and nutrients.
Slime has little affinity for basic dyes and is
ivisible in Gram stained smears.
Figure 7.6: Structure of capsule
Slime layer
The role of the capsule varies depending
on the bacterium.
• A thick capsule protects cells from
dehydration.
• Capsules protect the pathogenic
bacteria from being engulfed and
destroyed by white blood cells Figure 7.7: Structure of slime layer
(phagocytes).
7.2.3 Other Appendages
• Capsules are virulence factors of
many pathogenic bacteria, such Sheath
as Streptococcus pneumoniae, Sheathed bacteria are bacteria that grow
Haemophilus influenzae and Bacillus as long filaments in the form of chain or
anthracis. Encapsulated bacterial cells trichome. These bacteria are enclosed by a
generally have greater virulence. hollow tube like structure known as sheath
94

(Figure 7.8). Within the shealth, the bacteria Function:
are capable of growth and division. Aquatic • It provides mechanical support.
bacteria mostly form sheath. Examples
• In a few bacteria, shealth is
of sheathed bacteria include Leptothrix
strengthened by the deposition of
discophora (also known as iron bacteria),
ferric and manganese hydroxides.
Sphaerotilus and Clonothrix.
Prosthecae
They are semi rigid extensions of cell wall and
cell membrane. Some bacteria may contain
more than one prosthecae ( Figure 7.9).
Aerobic bacteria in fresh water and marine
Bacterial
environment possess prosthecae. Some of the
cels prosthecate bacteria are Caulobacter, Stellar,
Sheath
Prosthecobacter and Hyphomicrobium.
Figure 7.8: Sheathed bacterium First colonies

Glycoca
Cells sticks
Organic surface to coating
Infobits coating
Surface
Biofilms: As cells divide, th

dense mat bounde
by sticky extracellu
Microbial adhesion to animate or inanimate Glycocalyx slime
surfaces can be mediated by polysaccharides
capsules or slime. These adherence polymers are
collectively called as adhesions. Microorganism
Catheter surface
tend to adhere to any surface and the layer they
produce is called Biofilm. Biofilm can be harmful Cell cluster
or beneficial to humans. Biofilm formation is a
critical issue for almost all surfaces in health care
and food preparation settings. Biofilms may form
on a wide variety of surfaces, including living tissues, medical devices, industrial or
portable water piping system, etc., Biofilm formation is a multi-step process starting
with attachment to a surface, then formation of three dimensional structure and finally
ending with maturation and detachment. During biofilm formation many species of
bacteria are able to communicate with one another through specific mechanism called
quorum sensing.
First colonies
Glycocalyx
Cells sticks
Organic surface to coating
coating
Surface
As cells divide, they form a Additional microorganisms are attached to
dense mat bounded together developing film and create a mature
by sticky extracellular deposits community with complex functions
Glycocalyx slime
95
Catheter surface

Cell cluster
Function:
Holdfast • Stalk helps in attachment of cells to
solid surface.
Prosthecae
7.3 Cell Envelope of Bacteria
Flagellum
Cell envelope is an external covering that
lies outside the cytoplasm. It is composed
of two or three basic layers: the cell wall,
Swarmer cell the cell membrane and in some bacteria
the outer membrane.
Figure 7.9: Prosthecate bacteria
7.3.1 Structure of Prokaryotic Cell Wall
Function:
Prokaryotic cells almost always are
• Prosthecae increase surface area for bounded by a chemically complex cell wall.
absorption of nutrients from the dilute Cell wall lies beneath the external structures
aquatic environment. (capsules, sheaths and flagella). Cell wall
• Helps in adhesion. lies external to the plasma membrane
• Some prosthecae develop bud at the tip (cell membrane). Cell wall of eubacteria
and helps in asexual reproduction. is made up of peptidoglycan or murein,
whereas that of Archaeobacteria is composed
Stalk of proteins, glycoproteins or polysaccharides.
A few genera such as Methanobacterium,
It is a nonliving ribbon like tubular structure.
have cell walls composed of pseudomurein,
It is formed by excretory product of bacteria.
a polymer whose structure superficially
Some of the stalked bacteria are Gallionella,
resemble eubacteria peptidoglycan of
Planctomyces (Figure 7.10).
eubacteria but differs markedly in chemical
composition.(Note: Ordinary or typical
Flagellum bacteria are sometimes called eubacteria to
distinguish them from the phylogenetically
distinct group known as archaeobacteria).
Separating
daughter Peptidoglycan is a cross linked polymer
cell
of enormous strength and rigidity. It is
a polymer composed of many identical
subunits (Figure 7.11). Peptidoglycan differs
Stalk somewhat in composition and structure
Holdfast from one species to another, but it is basically
a polymer of N-acetylglucosamine(NAG),
N-acetylmuramic acid(NAM), L-alanine,
D-alanine, D-glutamate, and a diamino acid
Figure 7.10: Stalked bacteria (LL- or meso-diaminopimelic acid, L-lysine,
L-ornithine, or L- diaminobutyric acid).
96

Lipopolysaccharide
O-Polysaccharide
Core Polysaccharide
Lipid A
Outer Membrane
Lipoprotein
Peptidoglycan Periplasm
Peptidoglycan
Cell Membrane Cell Membrane
Membrane proteins Membrane proteins
Cell envelope of Gram positive Cell envelope of Gram positive

bacteria bacteria
Figure 7.11: Cell envelope of Gram positive and Gram negative bacteria
Cell wall may contain other substances membrane and the outer membrane but
in addition to peptidoglycan. For instance, no cell wall.
Staphylococcus aureus and Streptococcus
Functions of cell wall
fecalis contain teichoic acids (polymer
of acidic polysaccharides) covalently • It gives shape to bacteria like a bicycle
linked to peptidoglycan. Cell wall of Gram tyre that maintains the necessary shape
positive bacteria contain very little lipid and prevents the more delicate inner
but Mycobacterium and Corynebacterium tube (the cytoplasmic membrane)
cell walls are rich in mycolic acid (or Cord from bursting when it is expanded.
factor) which make them acid fast. When • It protects bacteria from osmotic
stained, the cells cannot be decolorized lysis in dilute solutions (hypotonic
easily despite treatment with dilute acids. environment).
Mycoplasma lack cell wall.
• It protects cell from toxic substances.
Protoplast is a bacterial cell consisting
of cell material bound by a cytoplasmic
HOTS
membrane.
Spheroplast is a bacterial cell with
How do bacteria maintain their shape?
two membranes namely the cytoplasmic
97

7.3.2 Structure of Outer Membrane Special protein channels called porins
Eubacteria and Archaeobacteria (Gram span the membrane. The points of contact
positive and Gram negative) differ with between outermembrane and cytoplasmic
respect to their cell walls. Gram negative membrane are known as adhesions. Outer
cell walls are more complex. An outer membrane is anchored to peptidoglycan
membrane surrounds a thin underlying layer by means of Braun’s lipoprotein.
layer of peptidoglycan (Table 7.4). Periplasmic space between the cell
Outer membrane is bilayered, consisting membrane and the outer membrane.
mainly of phospholipids, proteins and Functions of outer membrane
lipopolysaccharide (LPS). • It serves as an impermeable barrier to
LPS is composed of three parts which prevent the escape of important enzymes
are covalently linked to each other. They are (such as those involved in cell wall
1. Lipid A which is firmly embedded in the growth) from the periplasmic space.
m
embrane, • It serves as a barrier to various external
2. Core polysaccharide that is located at chemicals and enzymes that could
the membrane surface and damage the cell. For example, the walls
of many Gram positive bacteria can be
3. Polysaccharide O antigens that extend
easily destroyed by treatment with an
like whiskers from the membrane surface
enzyme called lysozyme, which selectively
into the surrounding medium
Table 7.4: Difference between Gram positive and Gram negative bacteria
Gram positive bacteria Gram negative bacteria
Gram reaction The bacteria that retain the The bacteria that cannot retain
colour of the primary stain the primary stain but takes on
(crystal violet) are Gram the colour of the counterstain
positive safranin are called Gram negative
Cell wall The cell wall is thick The cell wall is thin (8-12nm
(20-30nm thick) thick)
Peptidoglycan layer Thick (multilayered) Thin (single layered)
LPS content None High
Lipopolysaccharide
Periplasmic space Absent Present
Outer membrane Absent Present
Lipid and Low (acid fast bacteria High due to the presence of outer
lipoprotein content have lipids linked to membrane
peptidoglycan)
Teichoic acids Present in many Absent
Example: Streptococcus, Staphylococcus, Escherichia coli, Pseudomonas,
Corynebacterium, Bacillus, Haemophilus, Salmonella,
Clostridium Shigella.
98

dissolves peptidoglycan. However, Gram such as energy reactions, nutrient
negative bacteria are refractory to this processing and synthesis.
enzyme because large protein molecules • It regulates transport, the passage of
of enzyme cannot penetrate the outer nutrients into the cell and the discharge
membrane. Only when outer membrane of wastes. It is a selectively permeable
is damaged the enzyme can penetrate. membrane.
• Porins allow the smaller molecules, • It is also involved in secretion or
such as amino acids, monosaccharides discharge of a metabolic product into
to pass across. extracellular environment.
• Adhesions are export sites for newly • Cell membrane is an important site
synthesised LPS and porins, and are for a number of metabolic activities.
sites at which pili and flagella are Most enzymes of respiration and ATP
made. synthesis reside in the cell membrane
since prokaryotes lack mitochondria.
7.3.3 Structure of Cytoplasmic
Membrane Significance of cell envelope
• It has toxic properties (Example: LPS)
Immediately beneath the cell wall is the
cytoplasmic membrane also known as • It stimulates antibody production by
plasma membrane or cell membrane. It is immune system
composed of phospholipids and proteins. • The cell walls of many pathogens have
The phospholipids form a bilayer. Integral components that contribute to their
proteins are embedded within this pathogenicity. Example mycolic acids of
bilayer. Surface proteins or peripheral Mycobacterium tuberculosis
proteins are loosely attached to the • Cell wall is a site of action of several
bilayer. The lipid matrix of the membrane antibiotics.
has fluidity, allowing the components to
• Many of the serological properties of
move around laterally. In eubacteria, the
Gram negative bacteria are attributable
phospholipids are phosphoglycerides, in
to O antigens; they can also serve as
which straight chain fatty acids are ester
receptors for bacteriophage attachment.
linked to glycerol. In archaeobacteria, the
lipids are polyisoprenoid branched-chain 7.4 Structures Internal to Cell
lipids, in which long-chain branched Membrane of Bacteria
alcohols (phytanols) are ether linked to
Cytoplasm is called as the internal matrix
glycerol.
of the cell inside the cell membrane. Its
Functions of the cell membrane major component is water (70-80%). It also
• Prokaryotes do not have intracellular contains proteins carbohydrates, lipids,
membrane bound organelles as present inorganic ions, and certain low molecular
in eukaryotic organelles. Thus cell weight compounds. Inorganic ions are
membrane provides a site for functions present in much higher concentrations in
cytoplasm than in most media.
99

Cytoplasm is thick, aqueous, semi- Nucleus
transparent and elastic. The major structures The nuclear area has the hereditary
in the cytoplasm of prokaryotes are nucleoid material of most bacteria. It contains a
(containing DNA), ribosomes and reserve single, circular, long, continuous, thread
deposits called inclusions. Prokaryotic like double stranded DNA called the
cytoplasm lacks certain features of bacterial chromosome. Some bacteria
eukaryotic cytoplasm such as a cytoskeleton with linear chromosome also exist. It
and cytoplasmic streaming. carries the information required for the
cells structure and function. They are not
Ribosomes
surrounded by a nuclear envelope and
All living cells contain ribosomes. They are devoid of highly conserved histone
are the sites of protein synthesis. High proteins. The nuclear area can be spherical,
number of ribosomes represents the high elongated or dumbbell shaped. In actively
rate of protein synthesis. Prokaryotic growing bacteria, as much as 20% of the
ribosomes are freely found in the cell volume is occupied by DNA, because
cytoplasm, whereas eukaryotic ribosomes such cells presynthesize nuclear material
are attached to the cell membrane. for future cells. The chromosome is
Prokaryotic ribosomes consists of protein attached to the cell membrane. Proteins in
and a type of RNA called ribosomal RNA. the plasma membrane are believed to be
They are smaller and less dense than the responsible for the replication of the DNA
eukaryotic ribosomes. The ribosomes and segregation of the new chromosomes
of prokaryotes are 70S where as that of to daughter cells in cell division.
eukaryote are 80S (Figure 7.12).
Prokaryotic Ribosome Eukaryotic Ribosome
70S 80S
50S
5S RNA 5S RNA
subunit 60S
subunit 5.8S
23S RNA 28S RNA
30S 40S 18S RNA

16S RNA
subunit subunit
Figure 7.12: Prokaryotic and Eukaryotic Ribosomes
100

Plasmids Endospores:
Apart from the bacterial chromosome, Some species of bacteria produce
bacteria also contain small circular, double metabolically dormant structures called
stranded DNA molecules called plasmids spores. They are highly durable and
(Figure 7.13). Plasmids are self replicating dehydrated resting bodies produced inside
extra chromosomal genetic elements. the cells. They are formed by bacteria only
Plasmids may carry genes for activities when there is lack of water or depletion
such as antibiotic resistance and tolerance of essential nutrients in the environment.
to toxic metals. Examples: Fertility plasmid Endospores are coated with a specific
(F plasmid), Resistance plasmid (R plasmid) chemical compound diaminopimelic acid. It
and colicin plasmid (Col plasmid). binds with the Calcium and forms Calcium
dipicolinate which removes the water from
Bacterial DNA Plasmids
it and makes the spore resistant to extreme
conditions Example: Bacillus anthracis and
Clostridium tetani possess endospores.
Mesosomes
Generally prokaryotes do not have cytoplasmic
organelles like mitochondria and chloroplast.
Figure 7.13: Plasmids in Prokaryotes
It contains mesosome as their organelle. They
are the invaginations of the cell membrane
Molecular Chaperones
and they are in the form of tubules, vesicles or
They are the helper proteins which recognize lamellae. They are seen in both Gram positive
the newly formed polypeptides and fold and Gram negative bacteria, generally more in
them into their proper shape of secondary Gram positive bacteria. They are located next
and tertiary structure. Many chaperones to the septa or cross walls in dividing bacteria
are involved in proper folding of bacteria. (Figure 7.14). They may be involved in cell
They were first identified in Escherichia coli wall formation during division or play a role
mutant. Example: Heat shock proteins are in chromosome replication and distribution
produced in Escherichia coli cells subjected to daughter cells. If they are located near to the
to live at high temperatures, or in any other surface they are called peripheral mesosomes
stressful unfavorable conditions. and if they are located deep into the cytoplasm
they are called central mesosomes.
Inclusions Cell wall
The cytoplasm of prokaryotic cells has

several kinds of reserve deposits known Plasma
as inclusions. Cells may accumulate Membrane
c ertain nutrients when they are plentiful Mesosome

Sac
and use them when they are deficient.
Some inclusions are common to a wide Tubules
variety of bacteria whereas others are

limited to certain species (Table 7.5). Figure 7.14: Bacterial Mesosome
101

Table 7.5: Different types of inclusion bodies in bacteria
Type of inclusion Example of organisms

bodies possessing Significance
Polyhydroxybutyrate Bacillus Reserve of Carbon and energy
(PHB) megaterium sources. Sudan dye is used to observe
lipid inclusions
Polyphosphate Corynebacterium Reserve of phosphate
(volutin granules) or diphtheriae
metachromatic granules
Sulphur globules Phototrophic bacteria Elemental sulphur, reserve of
Like purple and green electrons in phototrophs. Reserve of
Sulphur bacteria energy source in lithotrophs
Example: Thiobacillus
Gas vesicles Aquatic bacteria, They are protein shells filled with
Cyanobacterium gases. They provide buoyancy and
keep the cells floating in vertical
water column
Parasporal crystals Genus Bacillus It is a proteinaceous compound, It is
toxic to certain insects
Magnetosomes Aquaspirillum They are like intracellular chains of
magnetotacticum magnetite particles. They help the
bacteria to swim to nutrient rich
sediments. It protects the cell against
H2O2 accumulation
Carboxysomes Photosynthetic They contain the enzyme Ribulose
Bacteria, cyanobacteria 1-5 bisphosphate carboxylase which
Autotrophic bacteria is involved in Carbon dioxide
fixation during photosynthesis
Phycobilisomes or Cyanobacteria They have a long polypeptide with
cyanophycin Granules equal proportion of Arginine and
Aspartic acid. They store Nitrogen
Chlorosomes Green bacteria They contain bacteriocholorophyll
pigments which are involved in
bacterial photosynthesis
HOTS
Why are endospores so difficult to

destory?
102

7.5 Eukaryotic Cell Structure It is primarily the structure of cell
walls and membranes, and the absence
As mentioned earlier, eukaryotic
of organelles (specialized cellular
organisms include algae, protozoa, fungi,
s tructures that have specific functions),
higher plants and animals. The eukaryotic
that d istinguish prokaryotes from
cell is typically larger and structurally
eukaryotes (Table 7.6).
more complex than the prokaryotic cell
(Flowchart 7.1). The general, eukaryotic microbial cells
have a cytoplasmic membrane, nucleus,
Prokaryotes and Eukaryotes are
mitochondria, endoplasmic reticulum,
chemically similar, in the sense that
Golgi apparatus, vacuoles, cytoskeleton,
they both contain nucleic acids,
and glycocalyx. A cell wall, locomotor
proteins, lipids, and carbohydrates
appendages and chloroplasts are found only
(Figure 7.15). They use the same kinds
in some groups. The structure and functions
of chemical reactions to metabolize
of the eukaryotic cells are discussed in
food, build proteins, and store energy.
(Table 7.7).
Eukaryotic Cell Structure
External Structures Cell Envelope Internal Structures
Appendages • Cell Wall Nucleus

• Flagella • Cytoplasmic Organelles
• Cilia membrane • Endoplasmic
Glycocalyx reticulum
• Capsule • Golgi apparatus
• Slime layer • Ribosome
• Lysosomes
• Mitochondria
• Chloroplasts
Cytoskeleton
• Microtubules
• Microfilaments
Flowchart 7.1: Eukaryotic Cell Structure
103

(b) Eukaryotic Cell
Cytoplasm
Nucleus containing
Nucleoid chromosomes
Cytoplasm
Cell membrane
(a) Prokaryotic Cell Smooth Lysosomes
endoplasmic
Ribosome reticulum
Flagellum
Glycocalyx
(capsule)
Cell wall Cell
membrane
Metachromatic Mitochondria
granules Pili
Rough
endoplasmic
Golgi complex reticulum
Nucleolus Ribosomes
Figure 7.15: Structure of prokaryotic and eukaryotic cell
Table 7.6: Differences between prokaryotic and eukaryotic cell
S. No Characteristic Prokaryotic Eukaryotic

1 Size of cell Typically 0.2-2.0nm in Typically 10-100 nm diameters
diameter
2 Nucleus No nuclear membrane True nucleus, consisting of
or nucleoli nuclear membrane and nucleoli
3 Membrane Absent Present. Example: lysosomes,
enclosed organelles Golgi complex, endoplasmic
reticulum, mitochondria and
chloroplasts
4 Flagella Consist of two protein Complex, consist of multiple
building blocks micro tubules
5 Glycocalyx Present as a capsule or Present in some cells that lack cell
slime layer wall
6 Cell wall Usually present and is When present, chemically simple
chemically complex
(typical bacterial
cell wall includes
peptidoglycan)
7 Plasma membrane No carbohydrates and Sterols and carbohydrates that
generally lacks sterols serve as receptors are present
8 Cytoplasm No cytoskeleton or Has cytoskeleton and shows
cytoplasmic streaming cytoplasmic streaming
9 Ribosomes 70S 80S (70S in organelles)
(Continued)
104

Table 7.6: Differences between prokaryotic and eukaryotic cell (Continued)
10 Chromosome Single circular Multiple linear chromosomes

(DNA) chromosome, lacks with histone arrangement
histone
11 Cell division Binary fission Mitosis
12 Sexual No meiosis (transfer Involves meiosis
recombination of DNA fragments
only)
Table 7.7: Functions of Eukaryotic organelles
Eukaryotic organelles Functions

Plasma membrane Mechanical cell boundary, selectively permeable
barrier with transport systems, mediates cell to cell
interactions and adhesion to surfaces, secretion
Cytoplasmic matrix Environment for other organelles, location of many
metabolic processes
Microfilaments, intermediate Cell structure and movements from the cytoskeleton

filaments, and Microtubules.
Endoplasmic reticulum Transport of materials, protein and lipid synthesis

Ribosome Proteins synthesis
Golgi apparatus Packaging and secretion of materials for various
purposes, lysosome formation
Lysosomes Intracelluar digestion
Mitochondria Energy production through use of the tricarboxylic
acid cycle, electron transport, oxidative
hosphorylation, and other path ways
Chloroplasts Photosynthesis, trapping light energy and formation of
carbohydrate from CO2 and water
Nucleus Repository for genetic information, control center for cell
Cell wall and pellicle Strengthen and give shape to the cell
Cilia and flagella Cell attachment and Cell movement
Vacuole Temporary storage and transport, digestion (food
vacuoles), water balance(contractile vacuole)
105

Different Shapes of Bacteria
Staphylococcus aureus Streptococcus pyogenes

Streptococcus pneumoniae
Klebsiella pneumoniae
Vibrio cholerae
Escherichia coli; Salmonella
Bordetella pertussis Corynebacterium diphtheriae Helicobacter pylori
Clostridium botulinum Clostridium tetani Neisseria gonorrhoeae Treponema pallidum
106

ICT CORNER
Bacteria
Know the various

shapes of Bacteria
STEPS:
• Use the URL or scan the QR code to download the Bacteria interactive educational
VR 3D app.
• Select sphere, rod and spiral to observe the structure of bacteria shapes.
• Select ‘structure’ tab and note the internal structure of bacteria.
• Click cell wall and note the difference between different shapes.
Step1 Step2 Step3
URL:
https://play.google.com/store/apps/
details?id=com.rendernet.bacteria&hl=en
107

Summary in all functions. The genetic material
of bacteria is DNA and the genes
Most prokaryotes have one of three
are arranged on larger, circular
general shapes coccus (round), bacillus
chromosomes. Additional genes are
(rod), or spiral, based on the configuration
carried on plasmid. Bacterial ribosomes
of the cell wall. Two types of spiral cells
are dispersed is the cytoplasm in chains
are spirochetes and spirlla. Shape and
and are also embedded in the cell
arrangement of cells are key factors for
membrane.
describing prokaryotes. Arrangements of
cells are based on the number of planes in Bacteria may store nutrients in their
which a given species divides. cytoplasm, in form of inclusions. Inclusion
vary in structure and the materials that are
Cocci can divide in many planes to
stored. A few bacteria produce dormant
form pairs, chains, packets, or clusters.
bodies called endospores, which are the
Bacilli divide only in the transverse plane.
hardiest of all life forms, survival for
If they remain attached, they form chains or
hundreds or thousands of years. The genus
palisades.
Bacillus and Clostridium are spore forming
Some bacterial cell walls are covered deadly pathogens.
by capsules or slime which protect the cell
Eukaryotes are cells with nucleus
from phagocytosis, drying and nutrient loss.
and membrane bound organelles. Cell
Fimbriae and Pili are involved in attachment
structures common to most eukaryotes
and in transfer of genetic information
are the cell membrane, nucleus, vacuoles,
between bacterial cells. Flagella are involved
mitochondria, endoplasmic reticulum,
in cell motility.
golgi apparatus and a cytoskeleton. Cell
The cell envelope is the complex wall, chloroplast and locomotory organelles
boundary structure surrounding a are present in some eukaryote groups.
bacterial cell. In Gram negative bacteria,
the envelope consists of outer membrane, Evaluation
the cell wall, and the cell membrane.
Gram positive bacteria have only cell wall
and cell membrane. Gram positive bacteria 1. The arrangement of flagella on cell
have thick murein and teichoic acid. The surface can sometimes help in the
cell walls of Gram negative bacteria are identification of an organism for
thinner and have wide periplasmic space. example, Escherichia coli to has
The outer membrane of Gram negative flagella throughout the cell surface
cells contains LPS toxic to mammalian that is referred to as.
hosts. The cell membrane is typically a. Lophotrichous
composed of phospholipids and proteins, b. Monotrichous
and it performs many metabolic functions c. Peritrichous
as well as transport activities.
d. None of the above
The cytoplasm of bacterial cell
serve as a solvent for material used
108

2. The movement of an organism toward c. Murein
or away from a chemical substance in d. All the above.
its environmentis called. 9. has fluidity
a. Tracking a. Cell wall
b. Chemotaxis b. Cell membrane
c. Outer membrane
c. Tumbling
d. All the above
d. Tumbling of none of the above. 10. The organelle of prokaryote involved
3. Bacterial cell wall is composed of in active cell division.
a. Lipid a. Mesosomes
b. Murein b. Mitochondria
c. Ribosomes
c. Cellulose
d. Endoplasmic reticulum
d. Chitin
11. The metachromaticgranules are seen
4. Cell wall shows in the bacteria
a. Semipermeability a. Escherichia coli
b. Complete permeability b. Corynebacterium diptheriae
c. Differential permeability c. Pseudomonas aeroginosa
d. Bacillus antharcis
d. Impermeability
12. The extra chromosomal DNA is called
5. Gram positive differ from Gram
a. Plasmid
negative in having
b. Episome
a. Thick wall
c. Nucleus
b. Absence of wall lipids
d. Nucleoid
c. Complete wall
13. The siderophores has high affinity
d. Simple wall
towards.
6. Lipopolysaccharide is found in cell a. Iron
wall of
b. Magnesium
a. Gram positive bacteria
c. Chloride
b. Gram negative bacteria
c. Both Gram positive and Gram d. Copper
negative 14. The molecular chaperones are
d. Algae involved in
7. Cell wall of archaeobateria contain a. Folding of proteins
a. Peptidoglycan b. Folding of carbohydrates
b. Murein c. Folding of lipids
c. Pseudomurein d. Folding of fatty aids
d. All the above
8. Endotoxin present in
a. Outer membrane
b. Plasma membrane
109

Answer the following 13. Discuss why a cell lyses without cell wall
1. What is Glycocalyx? 14. Give the significance of cell
2. What is a capsule? What are its envelope.
functions? 15. What is the role of siderophores?
3. What is a pilus, what is its function? 16. Differentiate between capsule and
4. What is chemotaxis? slime/pili and fimbriae.
5. Explain the arrangement of flagella 17. Diagrammatically explain structure

in bacteria with example. of cell wall.
6. Define carboxysomes 18. Differentiate Gram positive and

Gram negative bacteria.
7. State volutin granules
19. Explain any five cytoplasmic inclusions
8. What is called magnetosomes?
20. Differentiate between Prokaryotes
9. What is the role of ribosomes and Eukaryotes.
involved in protein synthesis?
Student Activity
10. Define periplasmic space.
1. Students will prepare clay model
11. What is LPS composed of? of bacteria.
12. List functions of cell wall 2. Students will collect the
a. Cell membrane pictures of different Eukaryotic
microorganisms.
b. Outer membrane
110

Chapter 8 Approaches to classifying organisms
A two-kingdom system – Linnaeus
Microbial Plantae Animalia
Taxonomy A five-kingdom system – Whittaker
Monera Protista Fungi Plantae Animalia

Chapter Outline
A six-kingdom system – Woese
Eu- Archae-
Protista Fungi Plantae Animalia
8.1 Diversity of Living Organisms is bacteria bacteria
Fascinating A three-domain system–Woese
8.2 Binomial Nomenclature Bacteria Archaea Eukarya
8.3 Whittaker’s System of Classification The correct identification of micro organisms is of

8.4 Taxonomic Systems fundamental importance to microbial systematists as well
as to scientists involved in many other areas of applied
8.5 The Three Domain System research and industry (Example: agriculture, clinical
8.6 The Past and Present Status of microbiology and food production).
Bacterial Taxonomy
Learning Objectives the characteristics of different organisms

to identify and group them. To understand
After studying this chapter the student life, it is essential to understand taxonomy.
will be able, The method of grouping related organisms
is the basis of classification (Figure 8.1).
• To understand the concept of
The objectives of taxonomy are:
taxonomy, taxon and phylogeny.
• To appreciate the contribution of • To establish the criteria for identifying
Linnaeus and Whittaker. organisms
• To learn the characteristics of • To arrange related organisms into
Kingdom Monera, Protista, Fungi, groups
Plantae and Animalia. • To provide evolutionary information
• To know some special methods used of the organism
in classification of microorganisms. The system of naming living organism
is called Nomenclature.
8.1 Diversity of Living Organisms is
Fascinating
Classification
The branch of science which deals with
the classification, nomenclature and
identification of all living organisms is
called Taxonomy. (Greek taxis means
arrangements and nomos means law or to Identification Nomenclature
distribute). Because of large number and
great diversity of organisms, biologists use Figure 8.1: The three facets of taxonomy
111

8.2 Binomial Nomenclature A variant strain that differ
physiologically and biologically from other
Swedish botanist Carolus Linnaeus in
strains in a particular species is called as
1735 introduced a formal system of
“Biovar”. Variations in a species is biological
classification which divided all living
in nature. One biovar in a species may
organism into two kingdoms-Animalia
grow on sucrose, while another cannot. If
and Plantae. He introduced “two name”
the biovars are very similar except for one
system, the first name, genus and second
property, they belong to the same genus
name species. The name often gives
and species, though vary in biological
information on something special about
growth properties.
it. Taxa (the basic taxonomic group)
are constructed from strains which are The strain that differ morphologically
successions of cultures derived from are called as Morphovar or Morphotypes.
an initial colony. The basic taxonomic Serovars or Serotypes are those strains
group is called the species (a collection that differ in their antigenic properties.
of strains having similar characteristics). It refers to immunological variations in a
The special bacterial strain which is species. An example of differing serovars
the permanent reference specimen for is Salmonella. Cell surface of Salmonella
the species is called the “type strain” varies slightly from one serovar to
(Figure 8.2). another. Because of this cell surface
change, a person who has been infected
HOTS by or become resistant or immune to one
serovar will not be immune to a second
Why is type strain refered as the most type, because the immune system cannot
important strain in a bacterial species? recognize a similar bacterium with a new
surface cover.
Figure 8.2: Taxonomic hierarchy-an example of hierarchy in microbial taxonomy

112

8.3 Whittaker’s System of Classification
Infobits
It is the five kingdom classification. In
The Microbial Type Culture the 20th century, advances in cell biology
Collection and Gene Bank (MTCC), a and interest in evolutionary biology
national facility established in 1986 is led scientists to question the two or
funded jointly by the Department of three-kingdom classification schemes. In
Biotechnology (DBT) and the Council 1969, Robert H. Whittaker proposed a
of Scientific and Industrial Research system which recognizes five kingdoms of
(CSIR), Government of India. The living things: Monera (Bacteria), Protista,
MTCC, housed at the Institute of Fungi, Plantae and Animalia (Table 8.1).
Microbial Technology (IMTECH), Whittaker’s system of classification is
Chandigarh, has established itself based on 1) complexity of cell structure
as a distinguished culture collection 2) mode of nutrition 3) body organization
centre for microbial resources in 4) phylogenetic or evolutionary relationship.
India. It is an affiliate member of Monera: This kingdom includes
the World Federation for Culture all prokaryotic organisms. Unicellular
Collections (WFCC) and is registered microorganism such as Mycoplasma,
with the World Data Centre for Bacteria, Actinomycetes and Cyanobacteria
Microorganisms (WDCM). The main are grouped under kingdom Monera.
objectives of this national facility are to
act as a depository, to supply authentic Phylogeny is
microbial cultures and to provide the evolutionary
related services to the scientists history of organisms
working in research institutions, that refer to the
universities and industries. relationship between life forms.
Table 8.1 Properties of Whittaker’s five kingdoms

Kingdom Monera Protista Fungi Plantae Animalia
Cell type Prokaryotic Eukaryotic Eukaryotic Eukaryotic Eukaryotic
Cell unicellular unicellular Multicellular Multicellular Multicellular
organization and
unicellular
Cell Wall Present in most Present in Present Present Absent
some absent
in others
Nutritional Phototrophic, Heterotrophic Heterotrophic Phototrophic Heterotrophic
heterotrophicor and
Class chemoautotrophic phototrophic
Mode of Absorptive Absorptive or Absorptive Mostly Mostly
nutrition ingestive Absorptive ingestive
113

organisms have typical eukaryotic cell
Infobits organization.
Hints of life: Fungi: This kingdom includes non green,
non photosynthetic eukaryotic fungi.
The Precambrian was the age
molds, mushroom, toad stools, puffballs
of microorganisms. They were
and bracket fungi are grouped under this
macroscopically expressed in a
kingdom. They are multicellular and consist
colonial structure called stromatolite.
of specialized eukaryotic cells arranged in a
It is a layer produced by live or fossilized
filamentous form.
mats of photosynthetic prokaryotes
(cyanobacteria) associated with warm Plantae: It includes all multicellular
lagoons or hot springs. The ancient plants of land and water. They use
stromalite belongs to anoxygenic photosynthesis to synthesize their organic
phototrophic filamentous bacteria molecules.
and modern stromalite belongs to Animalia: This kingdom includes all
oxygenic photo trophic cyanobacteria. multicellular eukaryotic animals. They
are also referred to as Metazoans. Animals
Protista: This kingdom includes ingest their food through one of any
eukaryotic unicellular Protozoans, slime ingestion portal and then use digestive
molds and algae. The kingdom is made enzymes to break food particles into
up of more than 250000 species. These absorbable fragments (Figure 8.3).
Eukaryotic Eukaryotic Eukaryotic

multicellutar multicellutar multicellular
photosynthosizo ingest absorb
nonmotile motile nonmotile
sexual sexual sexual
Animals
Plants Fungi
Eukaryotic
unicellular
or multicellular
absorb, ingest, or
photosynthesize
Protista sexual and
asexual
Prokaryotic
unicellular
absorb or photosynthesize
motile or nonmotile
Monera asexual
Figure 8.3: Whittaker’s Five Kingdom classification

114

8.4 Taxonomic Systems are subject to rapid change due to change in
environment. In classifying prokaryotes,
Classical Taxonomy
metabolic reactions, genetic relatedness
Classical taxonomy is a method of and other specialized properties are used
classification based on morphology, (Figure 8.4).
physiology, biochemical and ecological
characteristics of the microorganisms. HOTS
• Morphological Characteristics: The
structural characteristics are the usual If two microorganisms have an
tools which help in classification. Cell identical mol% G+C value for their
morphology gives little information DNA, are they necessarily related?
about phylogenetic relationship. The Explain
first step in identification of bacteria is If two micro organisms have very
differential staining. different mol% G+C values for their
• Physiological and metabolic DNA, are they necessarily unrelated?
characteristics: These characteristics Explain
are useful because they are directly
related to nature and activity of microbial Numerical Taxonomy
enzymes and transport protein. Since
The objective classification system
proteins are gene products, analysis
deals with the grouping by numerical
of these characteristics provides an
methods of taxonomic units based
indirect comparison of microbial
on their character and does not use
genomes.
subjective evaluation of their properties.
• Biochemical characteristics: Enzy- To be more objective about grouping
matic activities are widely used to bacteria, the scientists determine many
differentiate bacteria. Bacteria can characteristics (usually 100 to 200)
be separated into separate species by for each strain studied, giving equal
various biochemical tests. Example: weightage for each character. Then
Carbohydrate fermentation ability of by using computer %similarity is
bacteria. calculated (%S of each strain to every
• Ecological characteristics: Many other strain). For any two strains, this is
properties are ecological in nature NS
since they alter the relation of %S 5
NS 1 ND
microorganism to their environment.
where, NS is the number of
Microorganisms living in various parts
characteristics that are the same (positive
of the human body markedly differ
or negative) for the two strains, and ND
from one another and from those
is the number of characteristics that are
growing in freshwater, terrestrial and
different. Those strains having a high
marine environments.
%S to each other are placed into groups;
Prokaryotes have only a few structural and those groups having a high %S to
characteristics and these characteristics each other are in turn placed into larger
115

Dispersion & Dilution DNA extraction
Non-selective & 16S rRNA gene amplification

Selective agars with universal primers
Incubate under appropriate Cloning & partial

atmospheric conditions sequencing
for various times
Search for homology
Colony count in database
Construction of specific
Identification scheme probes for subsequent
analysis
Figure 8.4: General scheme for classification and identification in microbial taxonomy
groups. Numerical taxonomy also yields Molecular Taxonomy

classification that has a high degree of Molecular techniques in
stability and predictability. the field of biology has
helped to understand
In Numerical Taxonomy, genetic relationship
which was defined by between the numbers
Peter H.A.Sneath and of different taxonomic
Robert Sokal, each categories.
characteristic is given equal weightage DNA and protein sequencing,
and it is converted into numerical form immunological methods, DNA-DNA or
and compared by means of a computer. DNA-RNA hybridization methods are
Atleast 50 and preferably several very helpful in studying different species.
hundred characterictics are compared. The data or information from such
The presence and absence of studies are used to construct phylogenetic
selected characters in the group of tree (a branching diagram showing the
organism is calculated by simple evolutionary relationship among various
matching coefficient (SSM), called biological species based on similarities
Jaccard coefficient. and difference in their physical or genetic
characteristics).
116

A classification technique that is identification & taxonomic classification
widely used is DNA base composition of microbial species.
which is expressed as the percentage
of Guanine plus Cytosine (G+C). It is 8.5 The Three Domain System
a fixed property that reveals the degree This system of classification was introduced
of species relatedness. Ribosomal by C.Woese, O. Kandler and M.L.Wheelis,
RNA sequencing is used to determine is an evolutionary model of phylogeny
the diversity of organisms and the based on cells rRNA sequences(differences
phylogenetic relationship. Basically in the sequences of nucleotides) studies.
ribosomes consists of two subunits, each They grouped all living organisms into
of which is composed of protein and a three domains: Bacteria, Archaea and
type of RNA. Specific base sequences Eukaryota (Figure 8.5).
called as signature sequences are found Bacteria and Archaea are two different
in all groups of organisms. These unique groups of prokaryotes. The domain
DNA sequences are 5-10 bases long and Bacteria comprise the vast majority
found in 16s rRNA location and unique to of prokaryotes. The domain Archaea
major groups of prokaryotic organisms. contains prokaryotes that live mostly
Nucleic acid based detection methods in extreme environments. The domain
help in the detection of genomic materials. Eukaryota contains living organisms that
The 16s rRNA gene sequencing has been includes Kingdom Protista, Kingdom
established as the “gold standard” for Fungi, Kingdom Plantae and Kingdom
Figure 8.5: A phylogenetic tree based on rRNA analysis. Organisms are classified into
three domains: Bacteria, Archaea and Eukaryotes as proposed by Carl Woese et al.
117

Animalia. This system of classification is 8.6 The Past and Present State of
currently accepted by most biologist. Bacterial Taxonomy
The three domain system is based The first classification scheme for
on the current state of knowledge. As bacteria was published in 1773 based on
knowledge of organisms increase in the morphological characteristics. One of the
future, classification will undoubtedly unique, broadscope and widely accepted
continue to change. classification scheme was published
in 1927 by David Bergey & colleagues
is Bergey’s Manual of Determinative
Infobits Bacteriology.
The Bergey’s Manual of Systematic It provides identification schemes for
Bacteriology’s first edition was identifying Bacteria and Archae based on
published initially in four volumes. their morphology, differential staining
Volume 1 included Gram negative and biochemical tests. Whereas in 1984, a
bacteria of general, medical or industrial more detailed work was entitled. Bergey’s
importance, Volume 2 included manual of Systematic Bacteriology
Gram positive bacteria other than provides information on Bacteria and
actinomycetes, Volume 3 included Archaea based on rRNA sequencing.
Cyanobacteria, Archaebacteria and The classification in Bergey’s Manual is
remaining Gram negative bacteria and accepted by the most microbiologists
Volume 4 included Actinomycetes. as the best consensus for prokaryotic
taxonomy.
The current grouping edition 2
(2012) has five volumes based on 16S The present classification scheme based
rRNA sequencing: on genetic relatedness has more practical
value. This is expected to provide greater
Volume 1 (2001) includes
stability and predictability. It would lead
Archaea and the deeply branching and
to improved identification schemes, and
phototrophic Bacteria.
to aid our understanding of the origin of
Volume 2 (2005) includes Proteobac- present day genera and species.
teria.
Volume 3 (2009) includes Firmicutes. Summary
Volume 4 (2011) includes The branch of science which deals with
Bacteroidetes, Spirochaetes, Tenericutes the classification, nomenclature and
(Mollicutes), Acidobacteria, identification of all living organisms is called
Fibrobacteres, Fusobacteria, taxonomy. The system of naming living
Dictyoglomi, Gemmatimonadetes, organisms is called as nomenclature. Carolus
Lentisphaerae, Verrucomicrobia, Linnaeus divided all living organisms into
Chlamydiae, and Planctomycetes. two kingdoms- Animalia and Plantae.
Volume 5 (in two parts) (2012) He introduced Binomial Nomenclature
includes Actinobacteria. for naming living organisms. Whittaker
118

proposed five kingdom classification based linked to its environment and the
on various properties of living organisms. correlation has to be established by
Currently accepted classification proposed description of environmental parameters
by Woese, Kandler and Wheelis is the three whenever sampling is carried out. It is
domain classification. Modern developments also important to study the phenotypic
of sequencing technologies and recognition characteristics and link them to the
of rDNA sequences are of now cornerstone observations obtained from genotyping
for identification purposes. techniques. The link between habitat
Overall, it is important to recognize and diversity then becomes easier to
that microbial diversity is very much understand for future studies.
Student Activity
The student must understand the characteristics of each domain under the five
kingdom classification and fill in the chart below.
119

120

Evaluation 5. Binomial nomenclature means
writing the name of microorganism
in two words is
1. Which of the following a. Order and family
is a reasonable repre- b. Family and genus
sentation of phyloge-
c. Species and variety
netic diversity?
d. Genus and species
a. The Chain of Being
b. The Ladder of Life
c. The 5-Kingdom Tree
1. Define: Taxonomy and what is the
d. The 3-Domain Tree
here inter related parts of taxonomy?
2. Microorganisms belonging to the
2. Define: Classification, Nomenclature
same are expected to have the most
and Identification.
characteristics in common with
eachother. 3. What is meant by binomial system?
a. Order 4. Who developed the Bionomial system
b. Species in the year?
c. Family 5. What is taxonomic rank and why are
we using this?
d. Kingdom
6. What is the difference between
3. What was the first and most useful
biovars, serovars and morphovars?
microscopic tests for classifying
bacteria? 7. What is type strain and why it is called
a. Gramstain as type strain?
b. Flagellar stain 8. Write down the techniques which
are used to identify the taxonomic
c. Simple stain
characters of an organism?
d. Capsular stain
9. Explain in detail about the molecular
4. Which of the following is the characteristics which are used to
arrangement of organisum into identify the taxonomic orders?
groups or taxa?
10. What are the five kingdom classifica-
a. Nomenclature
tions?
b. Identification
c. Systematics
d. Classification
121

Chapter 9
Environmental
Microbiology
Chapter Outline
9.1 Interrelationship with other Fields

9.2 Air Microbiology
9.3 Microbiology of Water
9.4 Water Pollution and Microbial
Microorganisms are found in every corner of the earth
Contamination from miles below the surface to boiling hot springs to Antartic
9.5 Sewage Treatment ice. Every ecosystem on earth contains microorganisms
thay occupy unique niches based on their specific metabolic
9.6 Recycling of Treated Sewage properties.
9.7 Composting
9.8 Biogas Production

b iotic and abiotic environments.
Learning Objectives
Microorganisms in the environment

After studying this chapter the student are diverse in origin and ubiquitous.
will be able, Environmental Microbiology involves
the study of the applied effects of
• To gain knowledge of various layers of
microorganisms on the environment and
atmosphere and micro fauna of air.
on human activity, health and welfare.
• To understand air pollution and air
It was in the 1970s that a new area of
borne diseases.
Microbiology emerged and developed into
• To learn water borne diseases and
the field of Environmental Microbiology.
water treatment procedures.
The initial focus was on water quality
• To know Eutrophication. pathogens in the environment in the
• To know composting techniques. context of public health safety. The
• To gain knowledge of biogas developing field of environmental
production. microbiology expanded to several other
areas of applied research. These include
microbial interactions with chemical
Environmental Microbiology is the field pollutants in the environment and the
of science that examines the r elationship use of microorganisms for resource
between microorganisms and their

production and resource recovery.
122

Chemical pollutants in soil and 9.1 Interrelationship with
ground water have pronounced effects on other Fields
human population. The cost of cleanup
Since environmental microorganisms
or remediation of the contaminated
affect so many aspects of life and are easily
sites is too high. The overall objective of
transported between environments,
this chapter is to define the important
the field of Environmental Microbiology
microbes involved in environmental
interfaces with a number of subspecialities.
microbiology, the nature of the different
It includes soil, aquatic, aeromicrobiology,
possible environments in which the
as well as bioremediation, Water
microbes are situated, the methodologies
quality, Occupational health, Infection
used to monitor the microbes and their
control, Food safety and Industrial
activities and finally the possible effects of
Microbiology.
the microbes on human activities.
Hazardous waste
Bioremediation
Aeromicrobiology Aquatic Microbiology
Biotechnology Water Quality
Environmental
Microbiology
Soil Diagnostic
Microbiology Microbiology
Industrial Microbiology Food safety
Occupational health/
infection control
Modern environmental microbiology discovery and identification of new

has a much wider scope. There are microbes and their products that may
many different fields which recognises have practical application for protection
the problems in various fileds of of environment and human health and
Environmental Microbiology. It includes commercial applications.
123

9.2 Air Microbiology Microorganisms are frequently found
in the lower portion of the troposphere,
Earth’s environment is endowed with
where they are dispersed by the air
atmosphere, hydrosphere, lithosphere and
currents. Most of the microbes present
biosphere. In the 1930s, F.C. Meier coined
in the troposphere are either spore
the term “Aerobiology”. Air is a natural
formers or microbes that are easily
resource and is fundamental to human life
dispersed in air. The stratosphere has a
as it makes breathing possible. Its universal
temperature range of -80°C to -10°C.
presence and requirement for the survival
The temperature in the stratosphere can
of human beings make air an important
reach a maximum of several thousand
environmental factor. Air contains
degrees. Microorganisms are not found
significant number of microorganisms. It
in the upper regions of the atmosphere
also acts as a medium for the transmission
because of the temperature extremes,
of microorganisms including bacteria,
scarcity of available oxygen, absence
viruses, fungi, yeast and protozoa. Air borne
of nutrients and moisture, and low
transmission is one of the important
atmospheric pressure. The relatively low
modes for the transmission of infectious
humidity in the atmosphere (especially
diseases. Aeromicrobiology is the study of
during daylight hours) and UV rays from
air borne microorganisms.
the sun, limit the types and number of
Infobits microorganisms that are able to survive
in this part of the biosphere.
Miquel (1883) and Carnally & Exosphere
colleagues (1887) carried out the Thermosphere
most systematic studies in airborne Mesosphere

>700 km Stratosphere
microorganisms. 80–700 km Troposphere
50–80 km
12–50 km
9.2.1 Layers of Atmosphere 0–12 km
Earth’s atmosphere is divided into five

major layers (Figure 9.1) they are:
• Exosphere : 700 to 10,000 km
Figure 9.1: Diagram showing layers of
• Thermosphere : 80 to 700 km
atmosphere
• Mesosphere : 50 to 80 km
• Stratosphere : 12 to 50 km 9.2.2 Composition of Air
• Troposphere : 0 to 12 km The air in our atmosphere is composed
The air in the troposphere, the of different gaseous molecules. The most
layer close to the earth is constantly in common gases are nitrogen (78%), CO2
motion and the temperature decreases (0.034%) oxygen (21%) argon (1%) and
with increasing altitude, reaching a other molecules in trace level are present
low of -57°C at the apex of this region. in the atmosphere.
124

Table 9.1: Composition of Air Outdoor Microflora
Gas Symbol Content The air in the exterior environment is
Nitrogen N2 78.084% called outdoor air and the microbes that
Oxygen O2 20.947% 99.99% reside there are called outdoor microflora.
Argon Ar 0.934% Example: Bacillus, Aspergillus.
Carbon CO2 0.033%
9.2.4 Sources of Microorganisms in Air
dioxide
Neon Ne 18.20 ppm Air is not a natural environment for
Helium He 5.20 ppm microorganisms as it doesn’t contain
enough moisture and nutrients to support
Krypton Kr 1.10 ppm
their growth and reproduction. Soil
Sulphur SO2 1.00 ppm
microorganisms when disturbed by the
dioxide
blow of wind, gets liberated into air and
Methane CH4 2.00 ppm
remain suspended there for a long period.
Hydrogen H2 0.50 ppm
Man-made actions like digging,
Nitrogen N2O 0.50 ppm
ploughing the soil may also release
oxide soil borne microbes into the air.
Xenon Xe 0.09 ppm Microorganisms found in water may also
Ozone O3 0.07 ppm enter air by wind action or tidal action
Nitrogen NO2 0.02 ppm in the form of droplets or aerosols. Air
dioxide currents may bring the microorganism
Iodine I2 0.01 ppm from plant or animal surfaces into air.
Commensal as well as pathogenic flora
9.2.3 Microflora of Air of the human beings may enter the air by
Human beings and animals are continuously activities like coughing, sneezing, talking and
inhaling the microbes present in the air that laughing. The microorganisms are discharged
cause various infectious diseases. Most of out in different forms of particles which are
the respiratory tract infections are acquired grouped on the basis of their relative size
by inhaling the air pathogen. The microflora and moisture content. These are aerosols,
of air is studied under two categories such as droplets, droplet nuclei and infectious dust.
indoor and outdoor microflora. Aerosols
Indoor microflora An aerosols are mixture of small liquid or
The air found inside the closed solid particles dispresed in air. Aerosols can
environment (building/ room) is referred be natural or anthropogenic. Example: Dust
as indoor air and the microbes present in and smoke.
this region is called indoor microflora. Droplets
Example: Staphylococcus, Bacillus, Droplets are formed by sneezing, coughing
Penicillium. and talking which consists of saliva and
125

mucus (Figure 9.2). Droplets are relatively droplet nuclei. These are 1-4µm in size.
large, about 10µm or more in size, and They can remain in air for longer period
they tend to settle rapidly in still air. When and transmit various infectious airborne
inhaled these droplets are trapped on the diseases.
moist surfaces of the respiratory tract.
Infectious Dust
Thus, the droplets containing pathogenic
microorganisms may be a source of Large aerosol droplets settle out rapidly
infectious disease. from air on to various surfaces and get dried.
Nasal and throat discharges from patient
can also contaminate surfaces and become
dry. Microorganisms can survive for longer
period in dust. This creates a significant
hazard, especially in hospital areas.
9.2.5 Air Borne Diseases

Many microbial diseases are transmitted
through the air (Table 9.2). The incidence
of diseases caused by airborne transmission
Figure 9.2: Droplets released during
sneezing can be reduced by covering one’s nose and
mouth during coughing or sneezing and by
Droplet nuclei the use of face masks.
Small droplets in a warm, dry atmosphere
tend to evaporate rapidly and become
Table 9.2: Important airborne human diseases and causative agents (pathogens)
Human Diseases Pathogens
Bacterial diseases
Pulmonary tuberculosis Mycobacterium tuberculosis
Pneumonia Klebsiella pneumoniae
Streptococcal respiratory infections Streptococcus pyogenes
Fungal diseases
Aspergillosis Aspergillus fumigatus
Cryptococcosis Cryptococcus neoformans
Viral diseases
Influenza Influenza Virus
Common cold Picorna Virus
Protozoal diseases
Pneumocystosis Pneumocystis carinii
126

Nosocomial infection
Hospital acquired infection are also
known as a nosocomial infection. It is
acquired in a hospital or other health
care facility. Infection is spread to
the susceptible patient in the clinical
setting by various means, one of them
being air droplets. The infection can
originate from another infected patient,
staff, or in some cases, the source of the
infection cannot be determined. The
most common pathogens that cause
nosocomial infections are Staphylococcus
aureus, Pseudomonas aeruginosa, Figure 9.3: Settle plate technique
and Escherichia coli. One of the common showing bacterial and fungal growth
nosocomial infections is respiratory
pneumonia. Choice of the medium depends
upon the kind of microorganisms to
9.2.6 Enumeration of Microorganisms be enumerated. For bacterial isolation
in Air Nutrient agar and for fungal isolation
There are several methods adopted Sabourauds dextrose agar (SDA) can be
to enumerate microorganisms in air. used.
The most important methods are solid During sampling it is better to keep the
impingement and liquid impingement, plates about one meter above the ground
filtration, sedimentation, centrifugation level. Then the plates are uncovered in the
electrostatic precipitation. Many tools selected position for the required period
have been designed for the collection of time. Immediately after exposure for the
of air samples. Choosing an appropriate given period of time the plates are closed
sampling device is based on many factors, with the lids. Then the plates are incubated
such as availability, cost, volume of air to for 24 hrs at 37°C for aerobic bacteria and
be sampled, sampling efficiency and the for 2 days at room temperature (27°C) for
environmental conditions under which fungi. Enumeration results are expressed
sampling will be conducted. One of the as numbers of viable organisms per unit
methods is Settle plate technique, where area of air colony forming unit (CFU/
the microorganism carrying particles mm3).
are allowed to settle onto the medium
(solid impingement) for a given period HOTS
of time and incubated at the optimum
temperature (Figure 9.3). It works under Can microorganisms grow in clouds?
the principle of gravitational force.
127

9.3 Microbiology of Water 9.3.2 Estuaries
Aquatic microbiology is the study A partly enclosed coastal body of water
of microorganisms and microbial in which fresh water is mixed with seawater
communities in water environments. is called an estuary. As a consequence
Aquatic environments occupy more than of this geographical location, estuaries
70% of the earth’s surface. Water is essential are of salinity levels that range from less
for life and one of the most important than 0.5 to 2.5 grams of dissolved salts
natural resource. Thus, protection and per 100 grams of water at their mouths,
preservation of aquatic environments are where the water flows into the marine
vital for the continuation of life. There are environment. Estuaries are ecologically
two kinds of water found on earth: sensitive environments and serve as
habitats for fish and wide variety of marine
1. Salt water (97%)
life. There is an increasing concern about
2. Fresh water (3%) the quality of life in estuaries seriously
damaged by human impact, including
9.3.1 Salt Water Microflora
over development and pollution from
Salt water contains a significant level of industrial and waste water discharges. The
dissolved salts. The major bodies of salt bacterial population in estuaries consists of
water are oceans, seas, estuaries, and Pseudomonas, Flavobacterium, and Vibrio,
certain salt water lakes. Average salinity as well as enteric organisms. The quantities
of ocean is 3.5 grams per 100 grams and types of organisms vary, and depend
of water. The pH of salt water remains on the tide, rainfall, salinity, depth and
relatively constant at a range of 8.3 to 8.5. temperature. Most of the bacteria found
The temperature of seawater fluctuates in water runoff come from animal and
depending on location, season and depth.
The hot, sulphur-rich, acidic pool of yellow stone national park (U.S.)
is home to many Archaebacteria. The colour differences in the pool result
from the different communities of microbes that are able to thrive at extreme
water temperatures. Pyrolobus fumarii is a unique Archaebacteria, which
is hyperthermophilic that can grow at the temperature of 113°C. Some Archaebacteria live
in thousands of miles deep in ocean near superheated volcanic vents.
128

Salt
Fresh Brackish
Riv
er
Estu
ary
Ocean
Figure 9.4: Estuary

fowl fecal matter deposited on the ground.
Sometimes overflow from sewage systems Barophiles are
contributes to these higher number of bacteria that thrive
bacteria in the water. Microbiota are deep in the ocean
the primary producers in the aquatic and live at pressures
ecosystem since they play a major of 400-700 atmosphere but die at
role in food chain in water. Drifting 1 atmosphere. A strain MT-41,
microbial life of aquatic environment a bacteria is isolated from the marine
is called plankton. It is composed of sediments in the Mariana Trench near
phytoplankton and zooplankton. The the Phillippines where the depth exceeds
bottom region of the water harbours 10 kilometres. This strain has optimum
largest number of microorganisms growth at a pressure of 709 bar.
called as benthic microorganisms.
Microorganisms have been found at all 9.3.3 Freshwater Microflora
depths and at all latitudes within the The study of fresh water ecosystem is
ocean. Microorganisms are abundant near referred to as Limnology. The major
shore regions, where organic pollution bodies of fresh water are rivers, streams,
as well as microbial pollution from the swamps, marshes and lakes. The fresh
surrounding environment occurs. water ecosystem is divided into lentic
Algae are common in these e nviron- (still water) and lotic (flowing water)
ments. Because of their dependence on ecosystems. Lentic ecosystem is divided
light as a source of energy, algae occur into zones based on light penetration and
primarily in the upper strata of the temperature. They are littoral, limnetic
oceans and seas. Although they constitute and profundal zones (Figure 9.5).
a vital part of the food chain in the Most lakes are surrounded by rooted
marine environments, they also can be a vegetation in a large littoral zone along
nuisance and threat to other forms of life. the shore. Light penetrates the shallow
Nutrient levels in such environments can littoral and open-water limnetic zone but
significantly increase as a result of sewage is unable to reach the profundal zone in
plant discharges. Under such conditions the deep portions of many lakes.
algal blooms are common.
129

As lake vegetation and animal life impart an earthy odour and taste to river
decompose, their organic matter provides water. Rivers also receive high concentration
a source of nutrients. Lakes that have of bacteria and agricultural chemicals
very high concentrations of nutrients through surface runoff water from adjoining
(particularly nitrogen and phosphorus) soil during heavy rains and irrigation. In
are termed eutrophic and have low oxygen many countries, rivers are heavily polluted
concentrations because of extensive with sewage bacteria, especially Escherichia
microbial decomposition of organic matter. coli, Enterobacter fecalis, Proteus vulgaris
In comparison, the lakes that receive small and other intestinal bacteria.
amounts of nutrients and are nutrient
Infobits
sparse are oligotrophic. Caulobacter grows
well in oligotrophic lakes. Red Tides
Red tide is a common name for a
The common microorganisms
phenomenon known as an algal
found in fresh water are Pseudomonas, bloom which is caused by a species of
Flavobacterium, Serratia, Dinoflagellates (Gymnodinium) and the
Chromobacterium, Micrococcus, bloom takes on a red colour due to the
Aeromonas and Alcaligenes. presence of photosynthetic pigments
As these waters reach the surface or with the production of toxins. The most
conspicuous effects of red tides are
inland bodies of water, they become
associated with mortalities of marine
contaminated with different types of species
microorganisms. Rivers flowing water
(lotic) in close contact with the soil may
contain large numbers of soil bacteria
(Bacillus, Actinomyces and Streptomyces),
Fungi (Polyphagus, Penicillium and
Aspergillus), and algae (Microcystis and
Nostoc). These microorganisms frequently
Littoral Zone
Limnectic Zone
Profundal Zone
Benthic Zone
Figure 9.5: Light penetration zones of a freshwater lake

130

9.3.4 Eutrophication • Blue green algae like Microcystis,
The addition of excess quantities of Anabaena, Gonyaulax and other
nutrients to aquatic ecosystems termed Dinoflagellates produce toxins which
eutrophication often cause damage on the are poisonous to aquatic lives such as
communities involved. Eutrophication fish.
is an enrichment of water by nutrients, • Algal toxin may also contaminate the
especially nitrogen and phosphorus which drinking water supply.
are maintained at high levels that causes • Plankton blooms of green alga create
structural changes to the eco system. problems of O2 supply in the water.
The sudden influx of abundant nutrients • Musty tastes and odors in drinking
encourages a heavy surface growth of water are the other effects of
cyanobacteria and algae called a bloom Eutrophication.
(Figure 9.6). This heavy matt of biomass
• Excessive growth of aquatic weeds
effectively shuts off the oxygen supply
which impair fishing, swimming,
to the lakes below. The lack of oxygen
boating, shell fish production.
greatly disturbs the ecological balance of
the community. It causes massive death • Irrigation canals may become clogged.
of strict aerobes. Fish invertebrates and
Control measures
anaerobic or facultative microbes will
Five different methods have been suggested
survive.
to control eutrophication they are,
Effects of eutrophication
1. Ecological management (control the
• Eutrophication can have serious, long flow of nutrients into natural wastes)
term effect. The most notable effect of
2. Advanced treatments Example:
eutrophication is algal blooms.
Phosphate removal is effected by
• When a bloom occurs, the river, lake precipitation with lime.
or ocean becomes covered with algae,
3. Chemical algicides Example: copper
which is usually bright green.
sulphate
Figure 9.6: Algal blooms in a polluted eutrophic lake

131

4. Biological algicides Example: Bacteria taste, harmful chemicals, turbidity and
5. Destratification Example: physical/ microorganisms is called potable water,
mixing / forced aeration. which is safe to drink and can use for
food preparation without risk of health
9.4 Water Pollution and Microbial problems.
Contamination Biological oxygen demand (BOD)
Water is polluted by both natural as well as Biological oxygen demand
man-made activities. Polluted water is one is one of the common
which consists of undesirable substances parameter to monitor
rendering it unfit for drinking and domestic water quality and purity.
use. BOD is the amount of
dissolved oxygen needed
Sources of water pollutants by aerobic organisms to break down organic
1. Industrial waste material present in a given water sample at
2. Sewage waste certain temperature over a specific period
3. Mining activities of time. The amount of decomposable
4. Marine dumping organic material in sewage is measured by
5. Accidental oil leakage the biochemical oxygen demand, or BOD.
6. Burning of fossil fuels The BOD of the water sample is determined
7. Chemical fertilizers and pesticides by aerating it, measuring the amount
8. Radio-active waste of oxygen in sample before incubation,
The most prevalent biological placing the sample in a sealed container
contaminants in water are microbes, (BOD bottle) and incubating the container
particularly bacteria and viruses. Most of the for five days at 20°C. During this five day
bacteria carried by storm runoff originate period, microorganisms in the water grow
from animal fecal matter. Studies have and oxidize any organic materials in it. After
shown that during storms, the water that incubation period, the BOD of the water can
drains off the land and into sewage systems be determined by measuring the quantity of
also carries large quantity of bacteria and residual oxygen in the container. BOD of
chemicals. The chemicals include pesticides drinking water should be below 3ppm or
applied to lawns, chemical wastes near 3mg/litre.
industrial plants, and organic matter
deposited o the ground by different sources. Indicator Microorganisms
In addition to the chemical and biological Indicator organism are frequently used
contaminants, physical properties also affect to monitor bacterial contamination of
the quality of biological life in water. Among water. These indicator organisms provide
these are pH, temperature, dissolved oxygen a representative index of the water
concentration and salinity. contamination by pathogenic microbes.
The indicator organisms generally used in
Potable Water water quality monitoring are those that are
Clean water free from odour, disagreeable associated with the gastrointestinal tract
132

Infobits
Microbes at work:
One of the major environmental problems today is hydrocarbon contamination
resulting from the activities related to the Petrochemical Iindustry. Accidental
releases of petroleum products are of particular concern in the environment.
Hydrocarbon components have been known to belong to the family of carcinogens
and neurotoxic organic pollutants. Petroleum-based products are the major
source of energy for industry and daily life. Leaks and accidental spills occur
regularly during the exploration, production, refining, transport, and storage of
petroleum and petroleum products. There are the two main approaches to oil spill
bioremediation: (a) Bioaugmentation, in which known oil-degrading bacteria are
added to supplement the existing microbial population, and (b) Biostimulation,
in which the growth of indigenous oil degraders is stimulated by the addition of
nutrients or other growth-limiting cosubstrates. Bacteria like Pseudomonas putida
and Alcanivorax borkumens are the most active agents in petroleum degradation,
and they work as primary degraders of spilled oil in environment. Bioremediation
is a potential source for clean and green environment.
The Marine Bacterium Alcanivorax feeds on oil
Sea Water
Alcanivorax secretes natural
emulsifiers which help to break
up oil droplets
Oil Droplet
Biofim of Alcanivorax
at oil water interface
1000 nanometres
1/1000 of a millimetre)
133

and fecal matter. The most common group Filteration (MF) technique. The number
of indicator organisms used in water quality of coliforms per 100ml of water sample is
monitoring are the coliforms, bacteria that estimated to find the quality of water and its
are Gram negative, aerobic or facultative suitability for drinking purposes. In addition
anaerobic, non-spore forming rods that to coliforms, coli phages, Clostridia and
ferment lactose with gas production within human enteric viruses are also monitored in
48 hours at 35°C. Examples of coliforms drinking water.
are Escherichia coli, Enterobacter aerogenes
Waterborne diseases
and Klebsiella pneumonia. Two analytical
procedures were followed to check the Waterborne diseases are posing a serious
presence of coliforms in water. They are Most threat to health (Table 9.3).
Probable Number (MPN) and Membrane
Table 9.3: Waterborne diseases

Waterborne diseases Causative agent Symptoms
Bacterial diseases
Enteric fever Salmonella typhi Fever & enlargement of spleen
Cholera Vibrio cholerae Vomiting & watery diarrhea
Leptospirosis (Weils Leptospira interrogans High fever, red eyes, muscle pain and
Disease) vomiting
Viral diseases
Infectious Hepatitis Hepatitis A Jaundice, vomiting & abdominal pain
Gastroenteritis Rotavirus Diarrhoea, vomiting
Poliomyelitis Coxsackie Virus Head ache, neck stiffness, flaccid
paralysis.
Protozoan diseases
Giardiasis Giardia lamblia Chronic diarrhoea, abdominal cramp,
fatigue & weight loss
Amoebiasis Entamoeba histolytica Stomach pain, bloody stools, fever
Meningoencephalitis Naegleria fowleri Ulceration, watery, bloody diarrhoea
Treamatode disease
Schistosomiasis Schistosoma Drowsy, confusion, head ache, stiff
neck, Diarrhoea
9.5 Sewage Treatment disposal into natural bodies of water. In

broad terms, water is said to be polluted
Wastewater treatments also called sewage
when it contains enough impurities to
treatment which removes the impurities
make it unfit for a particular use, such as
from wastewater, or sewage, before
134

drinking, swimming, or fishing. Water • To reduce the spread of pathogenic
pollution is caused primarily by the microorganisms
drainage of contaminated wastewater • To avoid health hazards while
into surface water or groundwater. The swimming and boating in the water.
wastewater treatment is a major element • To prevent the development of
of water pollution control. objectionable colours and tastes
Sewage The predominant method of wastewater
disposal in large cities and towns is discharge
into a body of surface water. Suburban
and rural areas rely more on subsurface
disposal. In either case, wastewater must
Water (99.9%) Solid (0.1%) be purified or treated to some degree in
order to protect both public health and
water quality. Suspended particulates and
biodegradable organics must be removed
to varying extents. Pathogenic bacteria
must be destroyed. It may also be necessary
Organic Inorganic to remove nitrates, phosphates (plant
(70%) (30%) nutrients) to neutralize or remove
industrial wastes and toxic chemicals. The
Goal of Sewage Treatment degree to which wastewater must be treated
varies, depending on local environmental
• To convert waste water into a reusable
conditions and governmental standards.
resources.
HOTS
List the major oil spills that occurred recently in India and other countries.
Is it practically possible to clean up these oil spills using bacteria?
135

Sewage Treatment Methods
Sewage treatment defined as an artificial
process in which sewage is subjected to
remove / alter its constituents to render
it less offensive. There are three levels of
wastewater treatment (Figure 9.7): primary,
secondary, and tertiary (or advanced).
1. Primary treatment
2. Secondary treatment
3. Tertiary / Final treatment
Primary treatment
Primary treatment removes about 60 percent
of total suspended solids and about
35 percent of BOD; dissolved impurities
are not removed. It is the physical method
which remove large floating and suspended
solids from sewage water. Example: papers, Figure 9.8: Bar Screening in Sewage
leaves, bottles, rocks, pieces of metal or Treatment
wood. These objects are removed by passing
the sewage through screens (Figure 9.8)
and grit chambers. The screened water are removed with a skimmer. The liquid
is then sent to settling tanks or basin, wastewater remaining in the settling tank or
where the suspended solids are allowed to basin is then ready for secondary treatment.
settle as primary sludge. Materials such as The fluid from primary treatment is called
oil or grease, which float on the surface, primary effluent.
Preliminary Primary Secondary stage Tertiary

stage stage (Activated sludge process) stage
Grit Primary Secondary Tertiary Disinfection

Bar screens chamber clarifier Aeration tank filter
clarifier zone
Hospital
influent
Final efluent
(Raw sawage)
Air for disposal
Recycle sludge
Sludge
Primary sludge digestion Secondary sludge
tank
Sludge disposal
Figure 9.7: Schematic diagram of waste water treatment

136

Secondary treatment Oxidation ponds (Figure 9.10) are
Secondary treatment removes more than used in some communities to handle small
85 percent of both suspended solids and loads of sewage. Small communities and
BOD. Secondary treatment involves isolated areas frequently use oxidation
the oxidation of the primary effluent by ponds for treatment of waste water.
microorganisms. The common types Sewage is channeled into an initial pond
processes used are: where the sludge settles out. The liquid
portion of the sewage is then pumped
1. Trickling filter process
into an adjacent series of ponds where
2. Activated Sludge process aeration allows bacterial growth and
3. Oxidation ponds degradation of organic matter. These
A trickling filter consists of a large tank secondary ponds often are seeded with
or basin filled with a bed of crushed stone, algae which provide oxygen for the
gravel, slag or other porous material. growth of aerobic, heterotrophic bacteria.
Sewage is sprayed in a fine mist over the
Tertiary treatment
rocks. As the sewage trickles through the
bed, organic matter clings to the rocks, Tertiary processes can remove 99 percent
where it is digested by heterotrophic of all the impurities from sewage.
microorganisms (Figure 9.9). The Although effluents from secondary
microorganisms are contained within treatment have a low BOD, they may
a biofilm which are produced by slime contain eutrophication inducing salts
forming bacteria such as Zooglea and the (Phosphorus and Nitrogen compounds),
organic matter, is oxidized to gases and organic and inorganic suspended solids,
inorganic products. and poorly biodegradable organic
materials. Advanced or tertiary treatment
In the activated sludge process sewage
process are designed to reduce or eliminate
is mixed with a slime forming bacteria
these materials depends more on physical
(Zooglea) in a large aeration tank. As the
and chemical processes than biological
mixture is aerated, large flocs, or clumps,
processes. For phosphorus elimination the
form. These clumps contain not only
phosphates are converted to poorly soluble
the original slime forming bacteria but
aluminum, calcium or iron compounds
also large population of heterotrophs,
and removed by precipitation. Nitrogen
which oxidize organic matter within
in sewage effluent is removed primarily
these clumps. In this system wastewater
through nitrification by microorganisms.
is continuously pumped into the tank
The extent of nitrification during
and the treated water is removed into a
tertiary treatment depends on adequate
holding tank and the flocs are allowed
treatment plant designed and the proper
to settle. The settled floc material is
removal of sludge so that these bacteria
recycled into the tank as an inoculums to
are grown under optimal conditions.
continue the process. The remaining floc
Otherwise, large amounts of nitrogenous
is further treated or removed for burial or
compounds may escape tertiary treatment
incineration.
and released in the effluent. Suspended
137

Sprinkler
Filter
Feed pipe Air

Filter support Outlet
Collection
Figure 9.9: Trickling Filter in sewage treatment
solids are eliminated in sewage through The residue that accumulates in sewage
filtration or sedimentation. Poorly treatment plants is called sludge.
biodegradable substances can be removed
Sludge Digestion
by the use of specialized microorganisms
capable of using them as substrates. Treatment of sewage sludge may include
Chlorine is frequently added to tertiary a combination of thickening, digestion,
treated effluent to kill any remaining and dewatering processes. Among these
microorganism. digestion is mediated by microbes. Sludge
digestion is a biological process in which
9.6 Recycling of Treated Sewage organic solids are decomposed into stable
substances. Digestion reduces the total mass
Water recycling is reusing treated of solids, destroys pathogens, and makes it
wastewater for beneficial purposes such easier to dewater or dry the sludge. Digested
as agricultural and landscape irrigation, sludge is inoffensive, having the appearance
industrial processes, toilet flushing, and and characteristics of a rich potting soil.
replenishing a ground water basin. Recycled
water can satisfy most water demands, as Most large sewage treatment plants use a
long as it is adequately treated to ensure two-stage digestion system in which organics
water quality appropriate for the use. are metabolized by bacteria anaerobically.
138

Sun Carbon
Oxygen dioxide
Wind action
Aerobic zone
Algae O2 Bacteria CO2 + NH3 + PO4
Anaerobic zone
Organics Organic acids, etc. CO2 + NH3 + CH4
Figure 9.10: Oxidation Ponds
Sludge digestion may also take place Digested sewage sludge is usually
aerobically The sludge is vigorously dewatered before disposal. Sludge-drying
aerated in an open tank for about 20 days. beds provide the simplest method of
Methane gas is not formed in this process. dewatering. A digested sludge slurry is
spread on an open bed of sand and allowed
Septic tank is a small scale anaerobic treatment process. It is commonly

used in rural areas. It is simple, inexpensive and satisfactory if properly
operated. The septic tank consists of an underground sedimentation
container into which sewage from a home enter and is retained for a
short time. The organic matter in the sewage settles to the bottom of the tank where
it is covered by a thin organic film that excludes oxygen. Anaerobic bacteria in the
sediment digests the organic matter into simpler chemical compounds and gases.
The gases are then discharged through a vent in the tank. Liquids in the tank rise
and overflow through an outlet pipe and are distribute in the surrounding soil. As
the water trickles through the soil any remaining organic matter is decomposed by
aerobic prokaryotes. Septic tank should not be located near water supplies because
not all bacterial pathogens are removed by this treatment. Undigested solids in the
bottom of the septic tanks must be periodically removed.
139

to remain until dry. Drying takes place by Municipal use, Irrigation and
a combination of evaporation and gravity Agricultural use.
drainage through the sand.
Infobits
Sludge Disposal There is a huge business opportunity
The final destination of treated sewage in finding ways to use these waste
dumps for productive purposes - energy,
sludge usually is the land. Dewatered organic fertilizer. This requires methods
sludge can be buried underground in of dealing with old waste that has been
a sanitary landfill. It also may be spread on accumulating over the years as opposed to
agricultural land in order to make use of new/fresh waste.
its value as a soil conditioner and fertilizer.
Since sludge may contain toxic industrial 9.7 Composting
chemicals, it is not spread on land where
Compost is organic matter that has been
crops are grown for human consumption.
decomposed and recycled as a fertilizer and
When a suitable site for land disposal is soil amendment. It is a mass of rotted organic
not available, as in urban areas, sludge may matter made from waste. Example: garbage,
be incinerated. Incineration completely paper, sugarcane trash, paddy straw, aquatic
evaporates the moisture and converts the weeds, other agricultural waste.
organic solids into inert ash. The ash must be
Composting is a natural process in which
disposed of, but the reduced volume makes
aerobic and anaerobic microorganisms
disposal more economical. Air pollution
decomposes organic matter into valuable
control is a very important consideration
manure called as compost. The primary
when sewage sludge is incinerated.
objective of composting is to convert an
Appropriate air-cleaning devices such as
unstable material into stable end product
scrubbers and filters must be used.
(Figure 9.11).
Benefits of Sewage Treatment Organic wastes
• Water recycling has proven to be Water hyacinth, sewage
effective and successful in creating a sludge etc.
new and reliable water supply, while Microorganisms

(Bacteria, fungi
Aeration
(oxygen)
not compromising on public health. etc,) Composting
H2O (vapour)
process
• Water recycling can help us find ways CO2 Heat
to decrease the diversion of water from

sensitive ecosystems Finished compost
(Humus)
• Water users can supplement their
Figure 9.11: Composting process
demands by using recycled water.
• Decreases wastewater discharges The humification of organic material
• Reduces and prevents water pollution occurs in three stages
• Recycled water can be further used in 1. Mesophilic stage - Mesophilic is the
Thermal power plant (for cooling), initial stage of decomposition, lasting
140

for about a week, during which sugars Compost bed types
and other simple carbohydrates are 1. Pit method
rapidly metabolized. This is
2. Heap method
an exothermic process and may cause
an increase in temperature by 40°C. Pit method
Example: Bacillus subtilis The compost pits dug in soil with
2. Thermophilic stage - Thermophilic is dimension of 3.5m × 2.5m × 1.5m
the second stage, lasting for about two (L×B×H). The pits are filled layer by layer
weeks, during which the temperature may using green plants and animal excreta.
rise to about 50 to 75°C. Such a drastic The layering is repeated until the pit is
increase in temperature is accompanied filled. Finally a layer of mud is plastered
by the decomposition of cellulose and on the top of the pit (Figure 9.12).
other resistant materials. It is important
that the material be thoroughly mixed
and kept aerated during this stage.
Example: Bacillus stearothermophilus
3. Curing stage - The temperature
decreases during this final stage and
the material being composted is
recolonized by mesophillic organisms,
Figure 9.12: Pit method
which often produce plant-growth
stimulating compounds. Heap method
The humification of organic material In regions with heavy rainfall, the compost
is characterized by an increase in may be prepared in heaps above the ground
concentration of humic acids approximately level and protected by a shed. The pile is
from 4 to 12 percent, and decreases during made with dimension of 2m × 2m × 1.5m
the composting process. (L×B×H) (Figure 9.13).
What should you

compost?
When selecting
materials for your
compost pile avoid the following:
• Wastes that attract
pests
• Diseased / insect ridden plants
• Non-biodegradable things
Figure 9.13: Heap method
Methods of compost preparation
1. Indore method
2. Bangalore method
141

Indore method (L×B×H)
This method was developed at Indore, • In the Bangalore method of composting,
India. In this method organic wastes dry waste material of 25cm thick is
are spread in the cattle shed to serve as spread in a pit and a thick suspension
bedding. Trenches are dug with dimension of cow dung in water is sprinkled over
of 10ft × 6ft × 2ft. for moistening.
Dry wastes with cattle dung and soil • A thin layer of dry waste is laid over
are added in ratio of 4: 2: 1 up to 2 inches the moistened layer
layer in composting pit. A moisture • The pit is filled alternatively with
level of about 40-50% is ideal for good dry layer of material and cow dung
composting. Odour and insect problems suspension till it rises 0.5m above the
can be controlled by covering the piles ground level and plastered with wet
with a layer of soil or wood chips. mud and left undisturbed for about
The heap is left undisturbed for about 8 4-6 months or till required.
to 9 months. Turning the pile for every 15 • This method saves labour cost because
days is important for coplete composting there is no need of turning & regular
because pile needs a periodic influx of O2. sprinkling of water.
Plant residues, weeds, sugarcane leaves,
Benefits of compost
grass, wood ashes, animal dung, and water
urine soaked mud can also be used as raw • Compost improves the quality of soil
materials for this type of composting. hence called as a soil conditioner.
• Compost contains a variety of the basic
Bangalore method
nutrients required for healthy growth
• This method was developed at of the plant.
Bangalore, India. It is recommended • Nitrogen, phosphorous, potassium and
as a satisfactory method for disposal certain micronutrients viz, manganese,
of town wastes and night soil. copper, iron and zinc are found in
• The compost pits dug in soil with compost.
dimension of 4.5m × 2.5m × 90cm
Two types of microbes which help in composting process are
Aerobes Anaerobes
which decompose organic matter which decompose organic matter

matter in the presence of oxygen in the absence of oxygen
Example: Bacillus subtilis Example: Clostridium thermocellum
142

Infobits
Role of microbes in vermicomposting:

Recycling organic wastes through Vermiculture Biotechnology (VBT) is being considered
an economically viable solution. Earthworms are regarded as natural bioreactors which
proliferate along with other microorganisms and provide required conditions for the
biodegradation of wastes. Vermicomposting involves bio oxidation and stabilization of
organic material through the interactions between earthworms and microorganisms.
Worms like to feed on slowly decomposing organic materials like fruit and vegetable
scraps. Worms produce castings that contain beneficial microbes and nutrients, which
makes a great soil amendment. Worms are very
efficient at breaking down food scraps and can eat
over half their body weight in organic matter every
day. Vermicasting, also called vermicomposting, is the
processing of organic wastes through earthworms . It
is a natural, odourless, aerobic process, much different
from traditional composting. Earthworms ingest
waste, then excrete casts – dark, odourless, nutrient-
and organically rich, soil mud granules that make an
excellent soil conditioner.
• The composted product is safe and easy • Compost material is principally used for
to handle, and does not induce nitrogen the reclamation of drastically disturbed.
deficiency in recipient plants by Example: mined soil, landscaping and
nitrogen stabilization in the compost. agriculture.
• It suppresses disease infestation by • Compost finds unrestricted application
partial sterilization and detoxifies in parks and gardens for ornamental
pollutants. plants, in land reclamation and
highway beautification projects.
143

9.8 Biogas Production Leftover foods fruit & vegetable wastes
and cow dung can be subjected to anaerobic
Worldwide energy consumption and
digestion for energy production in a variety
demand are growing up since past 50 years.
of ways.
With the growth of population, demand
for energy is also increasing leading to Production of Biogas
an uneven supply and distribution of Biogas production is carried out in an airtight
resources. Therefore, the requirement of cylindrical tank called biogas digester Cow
sustainable and eco friendly energy in India dung is mixed with equal volume of water and
to satisfy the energy demand is inevitable. made into slurry and fed through the inlet
Along with the source of sustainable green of the biogas unit. The digestion proceeds at
energy, biogas production is an alternative 37°C with sufficent amount of nitrogen and
way to produce clean energy through solid phosphorus. The production of biogas sets
waste management. around 40-50 days, under anaerobic conditions.
Biogas is a type of renewable energy that Production of biogas accomplished in
can be produced from decomposition of 3 stages namely Hydrolysis, Acetogenesis and
animal and plant waste. It is composed of 50– Methanogenesis
75% methane, 25–50% carbon dioxide, 0–10%
Steps
nitrogen, 0–3% hydrogen sulphide, 0–1%
hydrogen and traces of other gases. The term Hydrolytic fermentative stage
“anaerobic” suggests that the process occurs In this step, several microbes secrete
in the absence of free oxygen and produces different enzymes, which cleave the complex
CH4 through decomposition of waste in nature macromolecules into simpler forms.
and reduces environmental pollution. Organisms that are active in a biogas process
Biogas generating technology is a during the hydrolysis of polysaccharides
favorable dual purpose technology at include various bacterial groups such
present since used as fuel and fertiliser. as Bacillus, Clostridium, Cellulomonas.
Cooking
Inlet Lighting
Gas tank
Manure + Fertilizer
Gobar Soil
Soil
Out let
Scum
Compost tank
Figure 9.14(a): Biogas Figure 9.14(b): Biogas Digester

production schematic
diagram
144

Acetogenic stage
Deenabandhu model
Through various fermentation reactions,
the products from hydrolysis are converted It is a biogas production
mainly into various organic acids (acetic model popular in India
acid, propionic acid, butyric acid, succinic which means “Friend of the helpless”
acid, lactic acid), alcohols, ammonia
(from amino acids), carbon dioxide and Small scale biogas unit
hydrogen. Facultative anaerobes and The biogas production is carried out in
hydrogen producing bacteria Example: an air tight cylindrical tank called biogas
Acetovibrio cellulosolvens, Bacteroid digester (Figure 9.15).
cellulosolvens are involved.
Applications
Particulate organic matter
1. Biogas used as fuel
(carbohydrate, protein, lipid
2. Used to generate electricity
Hydrolysis 3. Biogas is used to run any type of heat
engine in order to generate electrical
Amino acid, sugar, alcohol,
fatty acid and mechanical power.
4. Producing high quality fertilizer.
Acidogenesis
5. Reducing water and air pollution.
Intermediary products (volatile fatty acids,
acetate, propionate, ethanol, lactate)
Summary
Acetogenesis
Environmental microbiology is the field of
Acetate H2, CO2
science that examines the relationship between
Methanogenes microorganism and their biotic and abiotic
(rate limiting step)
Acetoclastic Hydrogenotrophic environments.
Identification of new microbes and
Biogas
(mainly methane) their products have practical application on
protecting the environment as well as human
health. Areomicrobiology is the study of
Methanogenic stage airborne microorganisms and is one of the
In this step, obligate anaerobic methane important modes for the transmission of
producing bacteria produce Methane gas as infectious diseases. The air in our atmosphere
the major end product along with Carbon is composed of different gaseous molecules.
dioxide, Hydrogen and traces of other gases. The air present both interior and exterior
Methanogenesis has six major pathways, of the environment is called indoor air
each converting a different substrate into and outdoor air. The microorganisms are
Methane gas. The six major substrates used discharged out in different forms which are
are Carbon dioxide, Formic acid, Acetic grouped on the basis of their relative size
acid, Methanol, Methylamine, and Dimethyl and moisture content. They are aerosols,
sulphate. The methanogenic bacteria include droplet, droplet nuclei and infectious dust.
Methanococcus voltae and Methanobacterium Hospital – acquired infection are also known
formicum (Figure 9.14 a, b).
145

as nosocomial infection. Solid and liquid BOD is the amount of dissolved oxygen
impingement, filtration, sedimentation, needed by aerobic organisms to breakdown
centrifugation, electrostatic, precipitation are organic material present in a given water sample
used to enumerate microorganisms in air. at certain temperature over a specific period of
The Aquatic Microbiology is the study of time. Indicator organisms are frequently used
microorgansims and microbial communities to monitor bacterial contamination of water.
in the water environment. Eutrophication is Waste water treatment are called sewage
an enrichment of water by nutrients, especially treatment which removes the impurities
nitrogen and phosphorus, which makes the from waste water and sewage. Trickling
overgrowth of the “algal bloom”. Apart from filter, activated sludge, oxidation ponds
microbes and chemicals, pH, temperature, are generally used. Compost is a natural
dissolved oxygen concentration and salinity are process in which aerobic and anaerobic
the physical properties that affect the quality of microorganisms decomposes organic matter
biological life. in to valuable manure called as compost.
BIO REMEDIATION
146

Evalution 8. In which process of treating sewage,99
percent of all the impurities from the
Multiple choice questions sewage are removed.
1. In 1930, the term a. Primary treatment process
aerobiology was coined b. Secondary treatment process
by . c. Tertiary treatment process
a. F.C.Meier b. Miquel d. None of the above
c. Carnelly and colleagues 9. Primary treatment is a method
d. None of the above a. Physical b. Chemical
2. The gas molecules which are more in c. Biological d. All of the above
atmosphere. 10. Activated sludge process is an example
a. Nitrogen b. Oxygen for treatment
c. Carbon dioxide d. Neon a. Physical b. Chemical
3. are water droplets c. Biological d. Composting
containing several types of 11. Chlorination is an example for
microorganisms released in to the air treatment
from the water sources. a. Physical b. Chemical
a. Droplet nuclei c. Biological
b. Infectious dust d. None of the above
c. Droplet d. Aerosols 12. is an open tank
4. Hospital acquired infections are where algal forms are allowed to grow
otherwise called as a. Trickling filter
a. Nosocomial infection b. Oxidation pond
b. Gastro intestinal infections c. Sludge digester
c. Ocular infection d. None of the above
d. All the above 13. Trickling filter is an example for
6. Is the amount of treatment
dissolved oxygen needed by aerobic a. Physical b. Chemical
organisms to breakdown organic c. Biological d. None of the above
materials?
14. Which one of the following is a good
a. BOD b. COD source for making compost?
c. DOB d. DOC a. Plastic b. Aluminum foil
7. is called as c. Vegetable peel d. Polythene
Indicator organisms
15. Algal boom in pond water is called
a. Escherichia coli
a. Eutrophication
b. Staphylococcus aureus
b. Acclimatization
c. Pseudomonas aeruginosa
c. Algalization
d. Green manuring
147

16. The most common toxic algal bloom 23. Which compost method is employed
among the following in the regions with heavy rainfall?
a. Euglena b. Microcystis a. Heap method
c. Paramaecium d. Hydra b. Pit method
17. Organism inhabiting the bottom c. Indore method
sediment of aquatic environments d. Banglore method
constitute community Answer the following
a. Pelagic b. Benthic 1. What are properties favour survival of
c. Abyssopelagic d. Episammon microorganisms in the atmosphere?
18. Study of flora and fauna of fresh water 2. Define Nosocomial infections
3. What is droplet nuclei?
a. Ornithology b. Zoology
4. What is the purpose of bacteriological
c. Paleontology d. Limnology analysis of water?
19. A partly enclosed coastal body of 5. What is potable water?
water in which river water is mixed 6. What is sewage?
with sea water 7. Define sludge.
a. Lake b. Estuary 8. Explain the sewage treatment
c. Creek d. Bay processes.
9. Is compost a fertilizer?
20. Chemical agent used for disinfecting 10. Describe the process of composting?
water 11. What is Biogas?
a. Glycols b. Chlorine 12. What role can biogas play in supplying
c. Hydrogen peroxide our energy needs?
d. None of the above 13. Draw the light penetration zones of a
fresh water lake.
21. The main component of natural gas 14. What is Eutrophication?
is 15. Write a note on trickling filter.
a. CO2 b. Carbon monoxide 16. Discuss the air borne diseases.
c. O2 d. Methane 17. Write in detail about settle plate
22. Biogas is a mixture of technique.
18. Write in detail about biogas
a. Methane, Nitrogen, Hydrogen
production processes.
b. Methane, Nitrogen, Oxygen
19. Discuss the Indore method of
c. O2, CO2, N2
composting in detail.
d. None of these 20. Discuss the sludge digestion methods.
21. Explain the benefits of waste water
treatment?
Student Activity
1. Set up a small scale anaerobic digester for anaerobic digestion using cow dung / fruits &
vegetable waste.
2. Instruct the students to bring algal bloom sample from their residential area.
3. Visit nearby sewage treatment plant.
148

Chapter 10 Soil crust microbes
Millipede
Spider
Microfungi
on leaf surface
Soil Microbiology Bacteria Earthworm

Ground
Beetle
Chapter Outline Mite

Fungal
Springtail hyphae
Microfungi in soil
in soil
10.1 Soil in General Nematode
Protozoa
10.2 Pioneers of Soil Microbiology Fungus

feeding on
nematode
10.3 Components of Soil
10.4 Soil Microorganisms
10.5 Microbial Interactions Soil is inhabited by a living microscopic population
which is responsible for the numerous reactions taking place
10.6 Rhizosphere in the soil. The soil microorganisms affects the life economy
of man in many ways.
10.7 Phyllospere
10.8 Spermosphere
Learning Objectives
10.1 Soil in General
After studying this chapter the student Soil is the outer covering of the earth.
will be able, It consists of loosely arranged layer of
• To know the composition of soil. materials composed of inorganic and
• To understand the importance of organic constituents. Soil provides the
soil microorganisms in soil fertility. physical support needed for the anchorage
of root system and serves as the reservoir
• To learn about the beneficial and
of air, water and nutrients that are essential
harmful interaction between soil
for plant growth.
microorganisms.
10.1.1 Formation of Soil
Knowledge of Soil Microbiology is
essential to understand the agricultural The processes involved in the formation of
soil are slow, gradual and continuous. The
and environmental science. Without soil
sum total of environmental effects on rocks
microorganisms, life as we know could
collectively known as the weathering of
not exist on earth. Organic matter would rocks. Weathering of rocks is a continuous
accumulate in the form of undecomposed phenomenon and add more and more
substances. Why should we study soil soil to the surface of the earth. There are
Microbiology? If we understand what is different types of parent materials of rocks
happening in soil, we get a better idea of available for the formation of soil.
how other biological systems work on earth.
149

Figure 10.1: Soil Horizons
10.1.2 Soil Horizons of science dealing with study of soil

Each type of soil is characterized by the microorganisms and their activities in soil
presence of different horizons which can is known as 'Soil Microbiology'.
be seen in a soil profile (Figure 10.1).
10.2 Pioneers of Soil Microbiology
The formation of soil horizons depends
on climate, living organisms, parent rock Scientists studied the microorganisms
material, topography and time; all of from water, air and soil. They recognized
which control the weathering of rocks. the role of microorganisms in natural
processes. They realized the importance
10.1.3 Physical and Chemical of soil microorganisms in growth and
Properties of Soil development of plants. Soil Microbiology
emerged as a distinct branch of soil science
Physical properties of a soil type depends during first half of the 19th century.
on the size of particles, soil texture, soil
Sergei N. Winogradsky discovered the
temperature and soil pH.
autotrophic mode of life among bacteria
Chemical properties of soil includes and established the microbiological
three main components which provides transformation of Nitrogen and Sulphur.
nutrients for plant growth. The three He isolated nitrifying bacteria for the first
components are the organic matter, the time and demonstrated the role of these
derivatives of parent rock materials and bacteria in nitrification (1890). Further he
the clay fraction. demonstrated that free-living Clostridium
The fertility of soil depends not only pasteurianum could fix atmospheric
on its chemical composition, but also on Nitrogen (1893). He developed the
the qualitative and quantitative nature of Winogradsky column (Figure 10.2),
microorganisms inhabiting it. The branch a self contained ecosystem for studying the
150

Cyanobacteria
and algae Air
Aerobic zone
Nonsulfur
Micro-
photosynthetic Liquid aerophilic zone
bacteria
(e.g., Rhodomicrobium)
(Less anaerobic)
Purple
photosynthetic Anaerobic zone
bacteria (More anaerobic)
(e.g., Chromatium)
Green photosynthetic Mud mixed with
bacteria (e.g., sulfate and carbonate
Chlorobium) salts and cellulose or
other organics
Figure 10.2: Winogradsky column
Sulphur cycle. Therefore, he is considered • Organic matter

as the 'Father of Soil Microbiology'. • Living organisms
M. W. Beijerinck (1888) isolated root One gram of soil contains approximately
nodule bacteria in pure culture from one million microorganisms. The soil
nodules in legumes and named them as has organic matter, soil solution and soil
Bacillus radicola. Thus, he is considered air. All these components are affected by
as the 'Father of Microbial ecology'. the activities of microorganisms. Soil is
Beijerinck and Winogradsky (1890) a constantly changing medium. The soil
developed the enrichment culture solution in agricultural soil has ions like K+,
technique for isolation of soil organisms, Na++, Mg++, Ca++, Fe+, S-, NO3, SO4, PO4 and
proved independently that transformation others.
of nitrogen in nature is largely due to These ions are very essential in culture
the activities of various groups of soil media. In a fertile soil, these elements
microorganisms (1891). Therefore, in mineral form are supplemented by
they are considered as 'Pioneers in Soil organic compound, derived from the
Bacteriology'. decomposition of animal and plant
residues. Thus the soil is an excellent natural
10.3 Components of Soil medium for growth of microorganisms.
The soil is composed of five major
10.4 Soil Microorganisms
components
Soil contain five major groups of
• Mineral matter microorganisms. They are Bacteria,
• Water Actinomycetes, Fungi, Algae and Protozoa
• Air (Table 10.1).
151

Table 10.1: Soil Microorganisms with This is because all kinds of organic refuse
example are disposed on the soil
Soil Microorganisms Examples Many of the soil bacteria perform useful
functions like decomposition of organic
Bacteria Agrobacterium
matter, conversion of soil constituents into
Bacillus
useful materials, production of antibiotics
Clostridium
in the soil and biogeochemical cycling of
Pseudomonas
elements like Carbon, Nitrogen, Phosphorus,
Actinomycetes Actinomyces Iron, Sulphur and Manganese. The bacterial
Nocardia population of the soil exceed the population
Streptomyces of all other groups of microorganisms in
Fungi Aspergillus both number and variety.
Fusarium
Soil Actinomycetes
Alternaria
Cladosporum The actinomycetes population is present
as many as millions per gram of soil.
Soil algae Anabaena
The most predominant genera present
Oscillatoria
in the soil are Nocardia, Streptomyces
Nostoc
and Micromonospora. Actinomycetes
Protozoa Colpoda are capable of degrading many complex
Nematodes organic substances and therefore
Pleurotricha play an important role in building
Heteromita soil fertility. One of the most notable
Bacteriophages T4 characteristics of the actinomycetes
Bacteriophages is their ability to produce antibiotics.
Examples: Streptomycin, neomycin,
One teaspoon of erythromycin and tetracycline.
productive soil can
Soil Fungi
contain between 100
million and 1 billion Next to bacterial population in soil,
bacteria. These living microorganisms fungi dominates in all kinds of soil. It
recycle organic material, promoting soil possess filamentous mycelium composed
fertility and supporting plant growth. of individual hyphae. All environmental
By practicing conservation tillage, factors which influence the distribution of
farmers can maintain biodiversity in bacteria and actinomycetes also influence
their soil. the fungal flora of soil. The quality and
quantity of organic matter present in the
soil have a direct influence on the fungal
Soil Bacteria
numbers in soil. Fungi are dominant in
Among the soil microorganisms, bacteria acidic soils because acidic environment is
are most dominant group of organisms. not supportive for the existence of either
All kinds of bacteria are found in soil. bacteria or actinomycetes.
152

adverse soil conditions. The protozoans
Soil microbes create prefer certain species of bacteria for their
humus: Humus is nutrition. Protozoa are abundant in the
the dark organic upper layer of the soil and their numbers
matter in soil. The are directly dependent on bacterial
humus is formed when dead plant population.
and animal matter are decayed by soil
microorganisms. Humus has many HOTS
nutrients that improve the fertility of
soil, Nitrogen being the most important. “Without fungi even death will be
Humus helps soil to retain moisture, incomplete” - Pasteur
and encourages the formation of soil Justify the statement.
structure. Soil organisms promote plant
growth. 10.4.1 Factors Influencing Microbial
Population in Soil
Soil Algae The major factors that
influence the microbial
Soil algae are ubiquitous in nature wherever
community in soil are
moisture and sunlight are available. They
are visible to the unaided eye in the form • Moisture
of green scum on the surface of soils. • pH
Numerically, they are not as many as Fungi, • Temperature
Bacteria or Actinomycetes. Some of the • Gases
common algae in Indian soil are Chlorella, • Organic and inorganic fertilizer
Chalmydomonas, Chlorochytrium, • Organic matter of soil
Chlorococcum and Oedogonium. • Types of vegetation and growth stages
Blue green algae, or Cyanophyceae, • Ploughing
are responsible for Nitrogen fixation. • Season
The amount of Nitrogen they fix • Depth of soil
depends more on physiological and
environmental factors rather than the Infobits
organism’s abilities. These factors include
intensity of sunlight, concentration of Some soil microbes produce a variety
of substances that promote plant
inorganic and organic Nitrogen sources
growth, including Auxins, Gibberellins
and ambient temperature and stability.
and antibiotics.
Soil Protozoa
Soil protozoa are unicellular. They are 10.5 Microbial Interactions
characterized by a cyst in their life cycle Microorganisms in soil interact with
which can help the species to withstand themselves and lead to beneficial and
153

harmful relationships (Flowchart 10.1). Symbiosis (mutualism)
Some of the interaction and Mutualism is an example of symbiotic
interrelationship have been discussed in relationship in which each organisms
this connection in Table 10.2. benefits from the association. One type
of mutualistic association is involving the
Microbial interactions
exchange of nutrients, between two species,
a phenomenon called syntrophism. Many
Beneficial Harmful microorganisms synthesise vitamins and
interactions interactions amino acids in excess of their nutritional
requirements. Other have a requirement
for one or more of these nutrients.
Symbiosis Parasitism
Symbiosis is an obligatory relationship
Commensalism Amensalism
between two populations that benefit
Protocooperation Competition
both the population. Both populations
Flowchart 10.1: Microbial interactions live together for mutual benefit.
10.5.1 Beneficial Interactions A B

The beneficial interactions such as
symbiosis (mutualism) and commensalism
are found to operate among the soil
inhabitants.
Table 10.2: Types of microbial interaction in soil
Interaction Microorganisms A Microorganisms B
Neutralism No effect No effect
Commensalism + No effect
Amensalism No effect -
Mutualism
Synergism
+ +
Protoco-operation
Symbiosis
Competition - -
Parasitism + -
Predation + -
+ = positive effect
– = negative effect
154

The relationship between algae and producing organisms benefit vitamin and
fungi that result in the formation of growth factors requiring organisms.
lichen is a classical example of mutualistic
intermicrobial relationship (Figure 10.3). 10.5.2 Harmful Microbial Interactions
Lichens are composed of primary Harmful microbial interaction is otherwise
producer, the phycosymbiont (algae) and described as negative interaction or
a consumer the mycosymbiont (fungus) antagonistic interaction. Any inhibitory
Lichen morphology effect of an organism created by any
means to the other organism is known
Reproductive unit
Algal cells Fungal hyphae as harmful interaction or antagonistic
interaction and the phenomenon of this
activity is called antagonism
Ammensalism
Algal layer
Ammensalism is the phenonmenon
where one microbial species is affected
by other species, where as other species is
Fungal hyphae unaffected by first one. Ammensalism is
Figure 10.3: Lichen Morphology accomplished by secretions of inhibitory
substances such as antibiotics. Certain
Commensalism organisms may be of great practical
In a commercial relationship between importance, since they often produce
two microbial population, one population antibiotics or other inhibitory substances,
is benefited and other population which affect the normal growth of other
remains unaffected. Commensalism is an organisms. Antagonistic relationships are
unidirectional relationship between two quite common in nature. For example:
population. The unaffected population Pseudomonas aeruginosa is antagonistic
does not benefit by the action of second towards Aspergillus terreus (Figure 10.4).
population. For the receiving population,
the benefit provided may be essential. In
commensalism, the unaffected population
modifies the habitat in such a way that
another population is benefited.
Benefits
A B
Figure 10.4: Microbial antagonism
Benefits
Parasitism
For example: A population of facultative
This is a relationship in which one of the
anaerobes utilizes oxygen and creates a
population benefits from the other and
habitat suitable for the growth of anaerobes.
the host is usually harmed. Parasitism
In soil, vitamin and growth factor
155

is one of the most complex microbial 10.6 Rhizosphere
interactions. The line between parasitism
In 1904, L.Hiltner for the first time coined
and predation is difficult to define. The
the term “rhizosphere” to denote the area
parasites feed on the cells, tissues or fluids
of intense microbiological activity that
of another organisms the host, which is
extends several millimeters from the root
harmed in this process.
system of the growing plants.
Harms The region which is adjacent to the
A B root system is called rhizophere. The
Benefits microbial population on and around root
system is considerably higher than the root
The parasites depends on the host and free soil or non rhizophere soil. This may
lives in intimate physical and metabolic be due the availability of nutrients from
contact with the host. All types of plants plants root in the form of root nodules,
and animals are susceptible to attack by secretion, lysates, mucigel and sloughed
microbial parasites. off cells (Figure 10.5).
Rhizoplame
Rhizosphere
5 mm
Stele
(xylem↑, phloem↓) Root hair
Epidermis
Mucigel (plant &

Cortex bacterial mucilage)
Endodermis
Root cap
Plant mucilage
Sloughed root
cap cell
Figure 10.5: Root hair and Rhizosphere
The rhizospere region can be divided Rhizophere Effect

into two zones. The rhizophere is a zone of increased
• Exorhizosphere microbial community as well as microbial
• Endorhizophere activities influenced by the root itself.
However the root surface is termed as Greater rhizosphere effect is seen
“rhizoplane”. with bacteria (R: S values ranging from
156

Pathogenic interactions: Mutualistic interactions:
Increased disease response, PGP traits, pathogen
decreased biomass/nutrient protection, induced systemic
content resistance
Endophytic
microbiome
Plant exudates
Microbiome
exudates
Potential rhizoplane and
endophytic microbiome
Bulk soil
microbiome
Figure 10.6: Effect of Rhizosphere in plant growth
10 to 20 or sometimes more) than with

Infobits actinomycetes or fungi (Figure 10.6).
From the agronomic point of view,
Bioleaching:
the abundance of Nitrogen fixing and
Soil microorganisms are very closely Phosphate solublising bacteria in the
involved as catalytic agents in rhizosphere of crop plants assumes a
many geological processes. These natural significance.
include mineral formation, mineral
It has been reported that amino acid
degradation, sedimentation and
requiring bacteria exist in the rhizosphere
geochemical cycling. In recent
in large numbers than in the root free
years, a new discipline of mineral
soil. The rhizosphere effect improves the
science namely bio-hydrometallurgy
physiological conditions of the plant and
or microbial mining (mining with
ultimately result in higher yield.
microbes) is rapidly growing. Broadly
speaking, bio-hydrometallurgy deals
with the application of biotechnology 10.7 Phyllosphere
in mining industry. In fact, The term “Phyllosphere” was coined
microorganisms can be successfully by the Dutch Microbiologist Ruinen.
used for the extraction of metals The leaf surface has been termed as
(Example: copper, zinc, cobalt, lead, Phylloplane and the zone on leaves
uranium) from low grade ores. Mining inhabited by the microorganisms as
with microbes is both economical and Phyllosphere (Figure 10.7). In forest
environmental friendly. vegetation, thick microbial epiphytic
157

Figure 10.7: Microscopic appearance of Phyllosphere Bacteria
Infobits
PGPB can promote plant growth. The bacteria include those that are free-living, those
that form specific symbiotic relationships with plants (Example: Rhizobia and Frankia),
bacterial endophytes that can colonize some or a portion of a plant’s interior tissues, and
cyanobacteria (blue-green algae). PGPB may promote plant growth directly usually by
either facilitating resource acquisition or modulating plant hormone levels, or indirectly
by decreasing the inhibitory effects of various pathogenic agents on plant growth and
development, that is, by acting as biocontrol bacteria. It is envisioned that in the not
too distant future, plant growth-promoting bacteria (PGPB) will begin to replace the
use of chemicals in Agriculture, Horticulture, Silviculture, and environmental cleanup
strategies.
associations exist on leaves. The Pseudobacterium, Phytomonas are also

dominant and useful microorganisms on encountered on the leaf surface. The
the leaf surfaces in the forest, vegetation age of plant, its leaf spread, morphology
happened to be Nitrogen fixing bacteria and maturity level and the atmospheric
like Beijerinckia and Azotobacter. factors greatly influence the phyllosphere
Apart from Nitrogen fixing bacteria, microflora.
other genera such as Pseudomonas,
158

ATION
E NER BIOTA
G CRO
XT I M AE
NE AIN M IC R
GR R
O BIO
O
BI T
C A
ANAEROBIC MICROBIOTA
ERE
PH IC
OB T A
OS
AEROBIO
RM
R
MIC
SPE
A
M NAE
IC
RO R O B I C
BIO
TA
Figure 10.8: Spermosphere
10.8 Spermosphere There are five major components in the

soil, that includes mineral matter, water,
The region which is adjacent to the
air organic matter and living organisms.
seed surface is termed as spermosphere
The soil environment is unique in
(Figure 10.8). Healthy seeds carry specific
several ways. It consists of bacteria,
bacterial flora in respect to number and
fungi, actinomycetes, algae and protozoa.
species. There are several reports in the
Several factors influences the moisture,
literature on the quantity and quality of
pH, temperature, organic and inorganic
microorganisms carried by the seeds of
matter of the soil.
different plants species both externally and
internally. When the seed is sown in soil, Microorganisms in soil interact
certain interactions between the seed borne themselves and lead to both beneficial
microflora and the soil microorganisms and harmful interactions. Beneficial
take place under the influence of chemicals interaction includes symbiosis and
excluded by the germinating seed. commensalism. Harmful microbial
interaction includes parasitism. The
Summary region adjacent to the root system is
called rhizosphere. Bacteria predominate
Soil is the outer most covering of the in rhizophere. Soil and their growth is
earth. Soil consists of living and non living influenced by nutritional substances
components that contribute its fertility. released from plant tissues.
159

160

The leaf surface has been termed 6. Leaf surface has been termed as
as phylloplane and the zone on leaves .
inhabited by the microorganisms is a. Rhizosplane b. Spermoplane
phyllosphere. The region, which is c. Phylloplane d. All the above
adjacent to the seed surface is termed as
7. Spermosphere is
spermosphere.
a. Leaf and microorganisms
Evaluation b. Root and microorganisms
Multiple choice questions c. Seed and microorganisms
d. All the above
1. Example for soil algae
a. Anabaena
b. Oscillatoria
1. What is soil?
c. Nostoc
2. Give examples for the soil bacteria?
d. All the above
3. What are the types of microbial
2. Lichens are example for
interaction?
a. Symbiosis
4. What is harmful microbial interac-
b. Parasitism
tion?
c. Commensalism
5. Define rhizophere.
d. All the above
6. Define rhizoplane.
3. The relationship between and
that result in the 7. Define phyllosphere.
formation of lichen 8. Define spermospere.
a. Bacteria and virus 9. Explain parasitism.
b. Algae and bacteria 10. Explain commensalism.
c. Algae and fungi 11. Give examples for the soil Fungi &
d. Virus and fungi Actinomycetes.
4. Harmful interaction is otherwise 12. What are the components of soil?
called as 13. Mention the different types of soil
a. Mutualism microorganism with help of chart.
b. Antagonism 14. Explain – factors influencing
c. Commensalism microbial population in soil.
d. Symbiosis 15. Explain – microbial interaction.
5. first coined the term 16. Write about symbiosis or mutualism.
rhizophere 17. Describe rhizosphere.
a. L. Hiltner 18. Explain rhizophere effect.
b. Ruinen
c. Pasteur
d. Koch
161

Chapter 11
Agricultural Microbiology
Chapter Outline
11.1 Biogeochemical Cycles

11.2 Biofertilizers
11.3 Biopesticides
The Anabaena azollae-Azolla association is a symbiotic

relationship between a blue green alga and a rice plant. Azolla is
a small, fast growing floating water fern. In the cavities
of leaves of azolla, Anabaena azollae a blue green algae
(a cyanobacterium) fixes nitrogen from the air.
Learning Objectives and Nitrogen are microbe mediated and

play a very significant role in replenishing
After studying this chapter the student the nutrient supply in soil. Certain
will be able, microorganisms like bacteria, fungi
• To understand the various biogeo and viruses are economically important
chemical cycle since they cause plant diseases and are
responsible for severe crop losses. The
• To know the nitrogen fixation
study that deals with plant diseases is
process
called plant pathology.
• To infer about the biofertilizer There are microorganisms capable
• To learn the role of biopesticides in of producing plant hormones thereby
agriculture enhancing the plant growth. Some micro
organisms can be used as biopesticides
as they have ability to kill insects that
Agricultural microbiology is a branch of damage plants.
microbiology which deals with the study
of microorganisms that are involved in 11.1 Biogeochemical Cycles
various agricultural processes taking place Biogeochemical cycling is defined as the
in soil, plants and agro industries. movement and conversions of materials
Agriculturally important processes like by biochemical activities throughout
the Biological nitrogen fixation, nutrient air, water and soil. All living organisms
cycling of Carbon, Sulphur, Phosphorus participate in biogeochemical cycling but
162

microorganisms play a major role. This is Atmospheric CO2, dissolved carbon
because of their high enzymatic activity in oceans and freshwater, organic matter
and their ubiquity. are actively cycled carbon reservoirs.
Most macro elemental components of Sediments and fossil fuels like coal,
living organisms like Carbon, Nitrogen, petroleum and natural gas are slowly
Sulphur, Oxygen, Phosphorus and cycled carbon reservoirs. Carbon is
Hydrogen are cycled most intensely and cycled between these reservoirs by the
other elements like Copper, Chromium, biochemical activities of micro organisms
Iron, Zinc, Nickel are cycled less intensely. and other living things (Figure 11.1).
The different stages or processes
11.1.1 Carbon Cycle involved in carbon cycle are
Carbon is a macro element present in all 1. Photosynthesis
living cells. In microorganisms, they are
2. Decomposition
present in all macromolecules like cell
wall, cytoplasmic membrane, proteins and 3. Methanogenesis
nucleic acids.
1. Photosynthesis
Reservoirs of Carbon: It is a process where atmospheric CO 2 is
Reservoirs are the storage places of converted to organic carbon (CH2O)n.
nutrients that are present in nature. They This is carried out by higher plants,
store nutrients in large amounts for longer photosynthetic bacteria, cyanobacteria
periods of time. and algae using radiant energy from
Organic compounds
Death in dead organisms
Death
Feeding
Organic compounds
Organic compounds Organic compounds in fossil fuels
in green plants in consumers
Respiration Decay and
Respiration cecompositicn-
returns CO2 to
the atmosphere CO2 released
as microbes
Respiration respire
CO2 in the air and
Photosynthesis dissolved in water,
removes CO2 from the particularly oceans Burning
environment
Figure 11.1: A simplified diagram of Carbon cycle is as follows
163

the sun. This can be explained by the
equation, Infobits
CO2 + H2O  (CH2O)n + O2 Fistulated cow:

Fistulated cows are very useful in
where (CH2O)n represents the organic studying rumen microorganisms and
form of carbon (Example: Carbohydrates) ruminant nutrition and are used to
which gets incorporated into the treat indigestion in cows. Fistula is
photosynthetic organisms. This organic a sampling port that allows access to
carbon serves as food for herbivores and the rumen. Rumen is the first and
in turn for c arnivores. the largest chamber of the stomach of
2. Decomposition cows and other ruminant animals.
The organic matter fixed as a result of

photosynthesis is eventually degraded by
microorganisms to CO2 during processes
like respiration and decomposition. When
aerobic and anaerobic organisms respire,
CO2 is released into the atmosphere. Much
of the CO2 is released when dead organisms
decompose in the soil predominantly
by the activities of soil microorganisms.
Burning of fossil fuels also release CO2 is needed for the formation of proteins,
into the atmosphere. amino acids, nucleotides and is present in
a number of oxidation states inside the cell.
3. Methanogenesis Nitrogen is cycled between atmosphere,
It is an anaerobic process where CO2 gets organic compounds in living things, soil
converted to CH4 (methane) by strict and sediments.
anaerobes like methanogens (Example: The processes that are involved in
Methanobacterium). Methanogens are Nitrogen cycle are
a group of Archaebacteria found in
anaerobic environments like swamps, 1. Nitrogen fixation
marshes, rumen of ruminants, paddy 2. Nitrification
fields and gut of termites. 3. Ammonification
4. Denitrification
H2 + HCO3- + H+  CH4 + 3H2O
Nitrogen fixation
Methane is converted back to carbondioxide Nitrogen is present as N2 (N N) in
by a process called Methylotrophy. air (78% N2). The triple bonded state of
nitrogen makes it very stable and nitrogen
11.1.2 Nitrogen Cycle in its gaseous state cannot be assimilated
The element Nitrogen (N) is a key by plants or animals for their metabolism.
constituent in microbial cell. Nitrogen Only few groups of prokaryotes are
164

capable of breaking the triple bond and Ammonification
use it for building up their proteins and The production of ammonia during
aminoacids. The process of reduction of the decomposition of organic nitrogen
gaseous nitrogen (N2) to ammonia (NH4) compounds, by micro organisms after
is called Nitrogen fixation. This process the death of plants and animals is called
is carried out by a group of prokaryotes ammonification (Figure 11.2). Much
called diazotrophs. of the ammonia released by aerobic
decomposition in soil is taken up rapidly
N2+8H  2 NH3 + H2 by plants and micro organisms and is
converted to amino acids.
Cyanobacteria, Rhizobium and Frankia are
some of the examples of diazotrophs that Bacteria like Bacillus, Clostridium,
can fix atmospheric nitrogen. The fixed Pseudomonas and fungi like Aspergillus,
ammonia gets incorporated into proteins Mucor and Penicillium are few examples
and amino acids, thus building up organic of micro organisms that can ammonify.
nitrogen. Nitrification
The oxidation of ammonia (NH3) to nitrate
assimilation
NH4 Proteins, amino acids (NO3) is called nitrification. It is carried
Nitrogen in atmosphere (N2)
Plants
Assimilation
Denitrifying
bacteria
Nitrogen-fixing Nitrates
bacteria in root (NO3–)
nodules of Decomposers
legumes (aerobic and anaerobic
bacteria and fungi)
Nitrifying
bacteria
Ammonification Nitrification
Ammonium (NH4+) Nitrites (NO2–)
Nitrogen-fixing soil bacteria Nitrifying bacteria
Figure 11.2: Simplified diagram of nitrogen cycle

165

out by nitrifying bacteria. It is a two step symbiotic association with other plants or
process where ammonia is first converted micro organisms and fix N2.
to nitrite (NO2) and then to nitrate (NO3). Organisms capable of BNF are
2NH3 + 3O2  2HNO2 + 2H2O Free living

Aerobic Azotobacter
The above given oxidation reaction is Cyanobacteria
the first step that produces nitrite. This
Anaerobic Clostridium
reaction provides energy and is carried
out by Nitrosomonas and Nitrosococcus. Symbiotic Rhizobium
In the second step, the nitrite is Frankia.

oxidized to nitrate BNF by Rhizobium in leguminous plants
Leguminous plants belong to the family
2HNO2 + O2  HNO3
Leguminaceae and bear seeds in pods.
This reaction is carried out by Example: Black gram, Green peas,
Nitrobacter. Soyabean, Subabul. The bacteria belonging
to the genus Rhizobium which can exist in
Nitrates are readily assimilated by
free living state in soil but can enter into
plants but are very water soluble and
symbiosis with legume plants and carry
rapidly leached from soil.
out nitrogen fixation.
Denitrification Process of BNF
The reduction of NO3 from soils by
-
It consists of the following steps
denitrifying bacteria to gaseous nitrogen
1. I nfection of legume roots by Rhizobium
is called denitrification. In this process,
carried out by bacteria like Pseudomonas, 2. Formation of root nodules
Thiobacillus denitrificans, organic 3. Reduction
of N N to NH4 in root
compounds serve as hydrogen donors and nodules
nitrates serve as electron acceptor. 1. Infection of legume roots by Rhizobium
Rhizobium recognises and attaches to the
NO3  NO2  N2O  N2
root hairs of legume plant. It invades the
Biological Nitrogen Fixation root hairs and secretion of certain nod
factors result in root hair curling typically
One of the most significant biological
called Shepherd’s crook symptom which
process taking places on the Earth is
leads to the formation of infection thread.
biological nitrogen fixation (BNF). This
Infection thread is a cellulosic tube like
fixation of atmospheric nitrogen carried
structure through which Rhizobium moves
out by few prokaryotes is cost efficient
into the cortex from root hairs.
because industrial production of ammonia
by Haber’s Bosch process is very expensive. 2. Formation of root nodules
Organisms carry out BNF in a free The invaded plant cells are stimulated to
living state in soil or they can establish divide repeatedly thus forming a mass
166

of tissue on the roots which are called
root nodules (Figure 11.3). Root nodules
are fleshy light pink colored globose
structures seen on the roots. The bacteria
inside the root nodules transform into
swollen, mishappened forms. These are
called bacteroids. The bacteroids are
capable of nitrogen fixation (Figure 11.4)
3. Reduction of N N to ammonia in root

nodules
This biochemical process is catalysed by
an enzyme called Nitrogenase present in
bacteroids and happens under diminished
O2 levels. The O2 levels in the nodules are
controlled by an oxygen binding protein
called leghemoglobin. This is a red, iron
containing protein which can keep the Figure 11.3: Root nodules in the
nodule environment free of oxygen. leguminous plant root
Nitrogenase
The enzyme nitrogenase is a complex 11.1.3 Phosphorus Cycle
enzyme consisting of 2 enzymes, The element phosphorus (P) is an essential
dinitrogenase reductase and dinitrogenase. macro element in all living organisms. They
Electrons from organic compounds like are found in nucleic acids and phosphate
pyruvate are passed on to dinitrogenase esters. It is an essential component of ATP
reductase first and then to dinitrogenase and other high energy phosphates and
which in turn passes them to N N thus phospholipids.
reducing it to NH4. This reduction needs 16
ATP, ferredoxin and cytochromes. Reservoirs of Phosphorus
1. Phosphate
rock like apatite, a large
N2 + 8e- + 8H+  2NH3 + H2 inert reservoir
N N + 4H HN=NH + 2H 2. Marine
and aquatic sediments
H2N-NH2 + 2H 2NH3 3. Dissolved
phosphates in soils and
waters
4. Organic
phosphates in dead and living
HOTS
organisms.
Will nitrogen fixation occur in the Phosphorous transformations mostly

presence of air? What will be the fate happen as inter conversion of inorganic to
of nitrogenase enzyme in aerobic organic phosphate and insoluble form to
condition? soluble phosphates.
167

Cortical cells Root hair
Rhizobium bacteria
Root hairs release a substance

that attracts Rhizobium.
Infection
thread
Rhizobium proliferates and causes an

infection thread to form
Cortical cells begin to divide,
Infection thread grows

into the cortex of the root.
Root haoir
Infection thread releases bacterial cells,

which become bacteroids (bacterial cells,
membrane disappears) in the root cells.
Cytokinins from bacteria cause plant
cortical cells to divide.
Root nodule
Root cap
Root nodule forms from rapidly dividing,

infected cortical cells. Bacteroids perform
symbiotic nitrogen fixation.
Bacteroids
Figure 11.4: Showing stages of formation of root nodules on legume plant
Infobits
Phosphate solubilizations
Most of phosphates occur in combination The ability of bacteria to solubilise
with Calcium, Iron, Magnesium and phosphates can be tested in the
Aluminium (inorganic P) and thus are laboratory by streaking the bacterial
insoluble and unavailable to plants and culture in Pikovaskaya agar which
micro organisms. Some micro organisms contains tricalcium phosphate. Positive
solubilize those insoluble phosphates cultures show clear halo around
by producing organic acids. Example: growth.
Thiobacillus, Bacillus thus enabling the
plants to utilize it.
Phosphate assimilation
Plants and micro organisms can readily
assimilate soluble forms of inorganic
phosphates like H2PO4-, HPO4-2 and HPO4-3
and incorporate them as organic forms of
phosphates like ATP, nucleic acids.
168

Phosphate mineralisation 3. Sulphur reduction
Breakdown of organic phosphates to 4.
Organic sulphur compound
form soluble inorganic phosphates oxidation or reduction
is called mineralisation. Organisms 5. Desulfurylation
produce phosphatase enzymes and
catalyse mineralisation. The mineralised Sulphide/Sulphur oxidation
phosphates can be utilized by plants (H2S  S0  SO42-)
(Figure 11.5). It is carried out by prokaryotes under
aerobic and anaerobic conditions. Under
Phytic acid Inositol
Phytase aerobic conditions, H2S is spontaneously
oxidized at neutral pH to elemental
11.1.4 Sulphur Cycle sulphur. Elemental sulphur is oxidized to
Sulphur is present in sulphur containing suphates by chemolithotrophic bacteria
aminoacids. The sulphur cycle involves like Thiobacillus, Beggiatoa.
oxidation – reduction reaction between If light is available, H2S can be used as
Sulphate (SO4), Elemental S and H2S and electron donor to carry out photosynthesis
hence there is change in the valence states
of sulphur from -2 to +6.
HOTS
The basic steps involved in sulphur
cycle are
If there is no biogeochemical cycle in
1. Sulphide/ sulphur oxidation the ecosystem, What will happen to
2. Sulphate reduction the earth?
Uplifting
of rock Phosphates
in organic
Weathering compounds
of rock
Phosphates
in rock Animals
Plants
Runoff
Detritus
Phosphates
Phosphates
in soil
in solution
(inorganic)
Decomposition
Precipitated Detritivores
Rock in soil
(solid) phosphates
Figure 11.5: Phosphorus cycle

169

under anoxic conditions by phototrophic is called as assimilatory sulphate
sulphur bacteria like Chromatium and reduction. The H2S thus produced is
Chlorobium.

immediately incorporated into organic
compounds.
Sulphate reduction
When sulphate is present in habitats, Sulphur reduction (S0H2S):
different groups of microorganisms
The dissimilative sulphur reducing
are capable of carrying out sulphate
bacteria can reduce elemental sulphur
reduction.
to hydrogen sulphide. Example:
Beijerinck described the use of sulphate Desulfuromonas, an obligate anaerobe.
(SO42-) as a terminal electron acceptor Under aerobic conditions, organisms like
during anaerobic respiration to form Pseudomonas, Proteus and Salmonella are
sulphide (H2S). This process is called also capable of performing this reaction.
Dissimilatory Sulphate Reduction(DSR).
The anerobic bacteria capable of carrying Organic sulphur compounds
out DSR are Desulfovibrio, Desulfococcus, reduction/oxidation
Desulfotomaculum (Figure 11.6) This Organic sulphur compounds like dimethyl
reaction by sulphate reducers requires sulphide can be used as carbon and energy
organic carbon sources like pyruvate or source for many microorganisms.
lactate. H2S accumulated in such habitats
by the action of sulphate reducers is toxic to Desulfurylation
aerobic organisms. It is a process where organic sulphur
compounds are used up by microorganisms
The reduction of sulphate to H 2S, for
for energy to produce H2S.
building up aminoacids and proteins
Aerobic conditions Elemental

Beggiatoa, sulfur
Acid deposition H2SO3
Thiobacillus
S0 Beggiatoa,
SO2 Thiobacillus
Microbial
oxidation
Burning of
fossil fuels
Reduction by Des
ulfovibr
SO2– io
4
Assimilation S0 Desu
by plants and lfuro
mon
bacteria as
SH sulfhydryl H2S
groups of Decomposition
proteins by microbes
Purple and green (dissimilatory)
phototrophic bacteria
S0 Purple and green
phototrophic bacteria
Anaerobic confditions
(mostly soil and sediments)
Elemental sulfur
Figure 11.6: Sulphur cycle

170

11.2 Biofertilizers Based on the nutrients that they provide,
biofertilizers are of the following types
In India, the availability and affordability
of fossil fuel based chemical fertilizers Nitrogenous biofertilizers-
at the farm level have been ensured • Rhizobium,
only through imports and subsidies.
• Azotobacter,
Indiscriminate and imbalanced use of
• Azospirillum
chemical fertilizers, especially urea, along
with chemical pesticides and unavailability • Frankia
of organic manures has led to considerable Phosphate solubilisers
reduction in soil health. Biofertilizers can • Bacillus
act as a renewable supplement to chemical
• VAM
fertilizers and organic manures. They have
the capacity to produce natural resistance 11.2.1 Rhizobium
in plants against pests and soil borne
diseases, adding fertility to soil. Rhizobium – legume symbiosis is a well
studied plant microbe interaction and
Nitrogen fixation by leguminous and
Rhizobium is the most extensively used
other crops is reported to be 44 million metric
nitrogenous biofertilizer in India.
tons per annum. The appropriate strain of
Rhizobium can increase the crop yield up to Rhizobium is a gram negative, non-spore
10-35%. Also, residual N is beneficial for the forming aerobic bacillus inhabiting the
next crops grown in the same field. soil in a free living state. The colonies
of Rhizobium on YEMA (Yeast Extract
It has been estimated that 40-250 kg
Mannitol Agar) plate are gummy, pale white
N / hectare(ha) / year is fixed by different
in colour (Figure 11.7). They can establish
legume crops by the microbial activities of
symbiotic relationship with leguminous
Rhizobium.
plants and fix atmospheric nitrogen thereby
Definition greatly improving soil fertility.
Biofertilizers are preparations containing
beneficial micro organisms like N2 fixers,
PO4 solubilizers in a viable static state
intended for seed or soil application and
designed to improve soil fertility.
Advantages
1. They reduce the need for chemical
fertilizers.
2. They provide the plant with certain
vitamins, plant growth promoting
substances and increase the vigour of
the plant. Figure 11.7: Pale pink mucoid colonies
of Rhizobium on Yeast Extract Mannitol
3. It is cheap and cost effective. Agar plate
171

Mass production of Rhizobium Method of application of Rhizobium to
The flowchart explaining the mass plants
production of Rhizobium biofertilizer is Carrier based Rhizobium inoculants are
given below mixed with water to form slurry to which
the seeds of plants are added (Figure 11.8).
The coated seeds are dried in shade and
Efficient N2 fixing strain of Rhizobium
used for sowing.
are selected and initially grown in YEM
broth for 24 hours.
11.2.2 Phosphate Solubilizers
Several soil bacteria like Pseudomonas
and Bacillus possess the ability to convert
Inoculum level is increased from
insoluble mineral phosphates into soluble
250 ml to 1000 ml in large flasks
form by secreting organic acids thereby
making it available to plants.
For mass cultivation and inoculant
Cultures are then grown in large
preparation, the cultures are grown in
fermentor for large scale production
Pikovaskaya broth for 7-18 days and
mixed in suitable carrier like peat or
lignite. After curing for a week, the
Broth is blended with powdered carrier inoculants are packed and made ready
material (peat or lignite) for use in a similar manner as Rhizobium
inoculants.
Packing in polythene bags 200g per bag 11.2. 3 VAM

Mycorrhiza means fungus root. It
describes the symbiotic association
Curing at 25°C between plant and fungus. Vesicular
Arbuscular Mycorrhiza (VAM) is an
endomycorrhiza which is used as a fungal
Dispatch to farmers biofertilizer. They mobilize the soluble
Appressorium Epidermis
at entry point
Hypodermis
Intracellular Intercellular
hyphae Cortex hypha in air
channel
Arbuscules
Vesicle
Figure 11.8: Showing the colonization of VAM fungi in root cells of plants
172

phosphates in the root zone of plants
and satisfy the phosphorus nutrition of
plants.
Morphology
VAM is an example of endomycorrhiza
meaning, the storage organelles of
phosphates like vesicles and arbuscles are
seen intracellularly. Vesicle is a globose
structure and arbuscle is a tree like branching
structure present in the root cortical cells Figure 11.10: Microscopic view of
(Figure 11.9). VAM fungi are naturally most Anabaena azollae
prevalent in angiosperms. gymnosperms,
pteridophytes and bryophytes. fixation and photosynthesis. Most of the
filamentous forms have specialized large,
thick walled cells called heterocysts which
are sites of nitrogen fixation.
Example: Nostoc, Anabaena is
examples for filamentous BGA. Gleocapsa
is an example of unicellular BGA. Some
of the filamentous forms do not possess
heterocysts but still fix atmospheric
nitrogen. Since they need standing water
for their growth, BGA can effectively
colonize paddy fields and enrich the soil
Figure 11.9: The fresh water fern Azolla. with nitrogen.
Mass production Mass cultivation of BGA

Root based inoculum is used for preparing Applying BGA to paddy fields can reduce
VAM biofertilizer (Figure 11.10). The the amount of chemical nitrogenous
selected spores of VAM fungi are allowed fertilizer applied for the growth of paddy
to infect plants like onion, sorghum and crop. Therefore cultivation of BGA in large
other grasses. After 3-4 months, the roots quantities is necessary. Mass cultivation of
along with the soil are macerated or BGA has the following steps.
pelleted with an inert material and packed 1. Isolation of BGA
in polythene pouches which can be used 2. Mass cultivation of BGA
as biofertilizer.
Isolation of BGA
11.2.4 Cyanobacteria / Blue green BGA can be isolated from soil or paddy
Algae fields. Appropriate dilutions from serially
Blue green algae are single celled or diluted algal sample are inoculated in
filamentous prokaryote capable of nitrogen the liquid flasks containing algal media
173

like BG-11 or Pringsheim’s media. After Anabaena azollae (Figure 11.10). The fern
several weeks of incubation at 28°C, and the cyanobacteria exhibit symbiotic
the individual colonies are picked up, relationship in which Anabaena provides
identified and stored. This can be used as the fern with fixed nitrogen and fern
starter culture for mass cultivation. Mass provides niche for the cyanobacteria
culture can be done in 2 ways. free from competition from other
microorganisms.
Mass cultivation of BGA
Azolla can be used as a nitrogenous
1. Open air shallow culture:
biofertilizer for paddy crop. When applied
into the paddy fields, Azolla provides
Pits are prepared (6΄ × 3΄ × 9΄)
nitrogen nutrition to standing rice crop
and can reduce the need for synthetic
fertilizers.
Filled with soil and 200g of
superphosphate and water is added Infobits
Mycorrhiza and orchid germination
Starter culture is sprinkled over water In the early stages of their life
cycle, all terrestrial orchids are
non photosynthetic, totally lacking
1 week chlorophyll and relying on carbon(C)
acquired from a fungal symbiont
(Mycorrhiza) for growth until the
production of the first green leaves
Algal scum is formed on the surface
above the ground, a nutritional
strategy termed mycoheterotrophy.
Around 200 species of orchids
Water is allowed to dry and the dried
remain achlorophyllous throughout
up algal flakes are collected and stored
their lifetimes. Species such as
in poly bags
Galeola, Gastrodia, Corallorhiza,
Rhizanthella and many others
The dried algal flakes around
continue to gain carbon from
10kg/ha can be applied in paddy fields
mycorrhizal fungi.
after transplantation.
11.2.5 Azolla
Azolla is a floating freshwater fern. The
plant has a branched stem, deeply bilobed
leaves which are arranged alternately on
the stem and each leaf has a dorsal and
ventral lobe (Figure 11.9). The dorsal
lobe houses the cyanobacterial symbiont
174

Mass multiplication of Azolla detrimental to ecosystem if the usage is
prolonged and pests may develop resistance
Small plots (50-100 sq.m) or concrete to the pesticides.
tanks with standing water are The term biopesticides refers to
inoculated with Azolla compounds that are used to manage
agricultural pests by means of specific
biological effects. It refers to products
containing biocontrol agents like natural
pH is adjusted to 8.0 with lime. substances such as plants, certain minerals,
animals, micro organisms including their
genes or metabolites.
They are an important part of
Integrated Pest Management (IPM)
Superphosphate is applied as a nutrient
strategy in controlling the pest.
and carbofuran is applied to deter insects
Advantages
They are less toxic to humans and
After 20 days, Azolla biomass can be environment and they do not leave
harvested and used as biofertilizer for harmful residues.
rice plantings They affect only the target pest.
They cause long term suppression of
Method of application of Azolla in rice pest populations since they persist in the
fields environment.
Azolla is grown on the flooded rice fields Microbial biopesticides are of three kinds
prior to planting for 2-3weeks. Then water 1. Bacterial biopesticide
is drained and Azolla is incorporated into
2. Fungal biopesticide
the soil followed by rice transplantation
within a week’s time. 3. Viral biopesticide
11.3.1 Bacterial Biopesticide

HOTS
Bacteria like Bacillus thuringiensis,
Bacillus papillae and Bacillus lentimorbus
Why biofertilizer are preferred to
have the potential to kill certain insect
chemical fertilizer?
pests and are entomopathogenic.
Bacillus thuringiensis
11.3 Biopesticides
It is a gram positive, spore forming, rod
Pests are insects that damage crop plants shaped soil bacterium. During sporulation,
and stored products. They feed on leaves the bacterium produces insecticidal
and roots or suck the sap of the plants proteins as parasporal crystals. These are
causing severe crop losses. Chemical called delta endotoxin also called as Cry
pesticides sprayed on plants can be proteins. Cry proteins are specifically
175

toxic to insects belonging to Lepidoptera, Various species of Bt are able to work
Coleoptera and other few insect orders. against cotton boll worm, cabbage worm
Mode of action of Bt and gypsy moths.
The Bt cells sprayed on the leaves have

to be ingested by the larval forms of the Infobits
insects in order to exert its action. This is
Photograph of a cotton plant showing
because the Bt toxin gets activated in the
opened and unopened bolls. BT
insect gut at a specific pH.
cotton is a genetically modified cotton
Process plant (GM crop) which has the gene
for the crystal toxin integrated in its
Insect ingests the parasporal crystals genome. Crystal toxin is expressed
containing Cry protein
in plant parts which reduce the need
for spraying pesticides. BT cotton
is the only GM crop approved for
The crystals dissolve in the alkaline commercial cultivation in India.
environment of the insect gut
11.3.2 Fungal Biopesticides

The solubilised inactive delta endotoxin are These entomopathogenic fungi attack
cleaved rendering them active insects and cause diseases in insect body
which lead to insect death. Two prominent
fungi used as mycopesticide are
The activated toxin binds to specific • Beauveria bassiana which causes white
receptor of the midgut epithelium muscardine disease
• Metarhizium anisopliae which causes
green muscardine disease
This leads to the insertion of toxin thereby
developing pores in the epithelium Mode of action of Beauveria bassiana
Beauveria bassiana, a filamentous fungus
belongs to class Deuteromycetes also called
Cell lysis and disruption of epithelium releases imperfect fungi. It can be successfully used
the cell contents leading to insect death. against Colarado potato beetle, (Figure 11.11)
Codling moth and American boll worm.
This fungus invades the haemocoel of
Symptoms insects through spores. Once the spores
• Larvae stops feeding attach to the cuticle, it germinates and
• Larvae becomes sluggish and static the hyphae penetrates the insect cuticle
• Water oozes out from the body (cuticle is the outer membrane of insects)
Penetration is aided by formation of
• Larvae dies and falls off the leaf
appresorium and penetration peg. The fungi
176

secrete chitinases, lipases and proteases • Nuclear Polyhedrosis viruses (NPVs)
which can dissolve the cuticle. The hyphae • Granulosis viruses (GVs)
enter the haemolymph and proliferate • Non occluded viruses
and colonise the entire insect and release
blastospores. Insect death occurs due to Mode of action of NPV
nutrient depletion of the haemolymph or by The virus enters the insect
toxaemia by secretion of toxic metabolites. body via ingestion by
insects and infects the
midgut cells by membrane
fusion. The NPV uncoat
within the nucleus of cells and pass through
the intestinal epithelium (Figure 11.12)
and establish a systemic infection of the
haemocoel.
Symptoms
Figure 11.11: Picture of insect infected Discoloration (larvae turns brown or
with Beauveria bassiana yellow)
• Decomposition or softening of larvae
Biopesticides • Lethargy
registered in India: • Infected larvae hang upside down twigs
1. Bacillus thuringien-
• Larvae become swollen with fluid
sis var. israelensis
containing virus and eventually die
2. Bacillus thuringiensis var. kurstaki turning black in color.
3. Bacillus thuringiensis var. galleriae
4. Bacillus sphaericus Mass production of NPV
5. Trichoderma viridae NPV are mass produced in laboratory
6. Trichoderma harzianum using suitable larval hosts. The fifth stage
7. Pseudomonas fluorescens larvae are fed with food infected with
NPV. After 4-5 days, the dead larvae are
11.3.3 Viral Biopesticides collected and macerated. The liquid is
centrifuged and the pellet containing the
Viral insecticides are pathogens that
viruses is suspended in sterile distilled
attack insects and other arthropods. Viral
water. This viral suspension can be used
pesticides are used to control Lepidopteran
for spraying in the fields.
larvae like Helicoverpa, Spodoptera sp
on Cotton, Corn, Sorghum, tomatoes.
1Summary
Baculoviruses are the commonly used viral
biopesticide. They are extremely small and Carbondioxide fixation and Biological
Nitrogen Fixation are the most significant
are composed of double stranded DNA. The
biological processes taking place on planet
genus Baculoviruses contains 3 subgroups. Earth. Methanogenesis is an anaerobic
177

process converting CO2 to CH 4. It is carried Azolla and Anabaena share symbiotic
out by methanogens like Methanobacterium relationship in which Anabaena provides
sp. Phosphorous transformations mostly fixed atmospheric nitrogen to Azolla.
happen as inter conversion of inorganic to
organic phosphate and insoluble form to Vesicles and arbuscles in VAM fungi are
soluble phosphates.Purple and green sulphur the storage organelles of polyphosphates.Blue
bacteria store sulphur as granules as a result Green algae are prokaryotes that can perform
of which they appear yellow in colour. both photosynthesis and nitrogen fixation.
Biofertilizers are preparations Biopesticides refer to compounds that
containing beneficial micro organisms like are used to manage agricultural pests
N2 fixers, PO4 solubilizers in a viable static by means of specific biological effects.
state intended for seed or soil application Bacillus thuringiensis produces crystal
and designed to improve soil fertility. toxin which is detrimental to insects.
178

Evaluation d. VAM
Multiple choice 7. The other name for Blue Green Algae is
questions a. Green algae
b. Brown algae
1. The conversion of
atmospheric nitrogen c. Cyanobacteria
to ammonia by prokaryotes is called d. Blue algae
a. Biological Nitrogen Fixation 8. The selective media for Rhizobium
b. Nitrification YEMA contains the sugar
c. Ammonification a. Maltose
d. Denitrification b. Mannitol
2. The oxidation of sulphide is carried c. Glucose
out by d. Lactose
a. Thiobacillus
9. Solubilisation of inorganic phosphates
b. Purple bacteria
by Bacillus is brought about by the
c. Beggiatoa a. Production of enzymes
d. Both a and b
b. Production of organic acids
3. Phosphate solubilisation by bacteria
is mediated by the production of c. Production of alkali
a. Organic acids d. Mineralisation
b. Phosphatases 10. The mass cultivation of VAM is
c. Phosphoric acid preferably done in
d. Phytases a. Sorghum roots
4. The process of production of CH4 b. Rice roots
from CO2 is called c. Potato roots
a. CO2 fixation d. Cotton roots
b. Methylotrophy 11. is an example of
c. Methanogenesis entomopathogenic fungi
d. Photosynthesis a. Verticillium
5. The reduction of sulphate for building b. Beauveria bassiana
up aminoacids and proteins is called
c. Metarhizium anisopliae
a. Desulfurylation
b. Assimilatory sulphate reduction d. All of the above
c. Dissimilatory sulphate reduction 12. The toxic effect of Bacillus
d. Sulphur reduction thuringiensis is due to
6. An example of nitrogenous a. Cry protein
biofertilizer b. Delta endotoxin
a. Bacillus c. Parasporal crystals
b. Pseudomonas d. All of the above
c. Rhizobium
179

13. The action by mycopesticide depends 13. Explain the mass production of
on the phosphate solubilisers.
a. Ingestion of fungi by insects 14. What is Azolla-Anabaena symbiosis?
b. Penetration of cuticle by fungi 15. What is crystal toxin? Write short
c. Ingestion of leaves infected with notes on BT cotton.
fungi 16. Explain the process of root nodule
d. Ingestion of spores by fungi. formation with appropriate diagram.
14. Crown gall is caused by Agrobacterium 17. Give in detail the reactions involved in
on phosphorus/carbon/sulphur/nitrogen
a. Monocots cycle.
b. Dicots only
18. Explain the mass production of VAM.
c. Monocots and dicots
d. Ferns 19. Give detailed account on Bacillus
thuringiensis.
15. Smut is a disease caused by
a. Puccinia sp
b. Ustilago sp Student Activity
c. Verticillium
• Sow two groundnut seeds in a
plastic cup/earthern pot. After
one month, pull out the plant
and observe the root system for
1. What is the role of purple and green nodules.
bacteria in sulphur cycle?
• Collect pictures of all organisms
2. What is nitrogenase? Give its function. involved in sulphur cycle and
3. Define biofertilizers. prepare a collage showing its role.
4. What is VAM? • Prepare a chart work showing the
5. Define biopesticides. biogeochemical cycles of carbon,
6. What is a smut? nitrogen and phosphorus
7. What is NPV? • Collect pictures of various diseases
of plants and prepare a chart.
8. List out Bacterial diseases of plants.
• Prepare a model on the mode of
9. What is the end result of decompo-
action of BT.
sition of organic matter in carbon
cycle? Give the role of microorganisms • Collect diseased parts of plants
with examples. and identify the symptoms of the
disease.
10. What is the function of leghemo-globin?
11. Give the method of application of
Azolla in paddy fields.
12. Give the salient features of
Rhizobium.
180

Chapter 12
Medical Microbiology
Chapter Outline
12.1 Microbial Infections of the Human

Body
12.2 Skin and Wound Infections
12.3 Respiratory Tract Infections
12.4 Gastrointestinal Tract Infections
12.5 Ocular Infections
12.6 Urinary Tract Infections
12.7 Reproductive Tract Infections Medical Microbiology or Clinical Microbiology plays
an important role by providing the necessary diagnostic
12.8 Infections of the testing, means of epidemiological detection, and future
Nervous System innovation required in an era of emerging and reemerging
infectious diseases.
12.9 Systemic
Infections
Learning Objectives 12.1 Microbial Infections of the

Human Body
After studying this chapter the student Medical microbiology is the branch
will be able, of microbiology which deals with
• To describe the importance of prevention, diagnosis and treatment of
medical microbiology. infectious diseases. There are four kinds
• To understand the types and sources of microorganisms that cause infectious
of infections. diseases. They are bacteria, fungi, parasites
and viruses. Any disease that spreads
• To know the types of infectious
from one host to another, either directly
diseases and virulence factors of the
or indirectly is said to be a communicable
pathogen.
disease. Chicken pox, measles, genital
• To tell the etiological agents of skin
herpes, typhoid fever and tuberculosis are
wound respiratory, gastro intestinal,
examples of such diseases, that are easily
ocular, urinary, reproductive, ner-
spread from one person to another.
vous system and systemic infections.
A non communicable disease does
• To know the causative agents of
not spread from one host to another.
varoius human diseases and their
For example, Clostridium tetani, a soil
portal of entry.
181

inhabitant, produces Tetanus when it is some of the virus producing respiratory
introduced into a wound or an abrasion. infections.
Tetanus is thus an infectious disease, but c. Ingestion
not communicable.
Intestinal infections are generally
Infectious disease occurs when the acquired by the ingestion of food or
infecting microorganism causes damage drinks contaminated by pathogens.
to the host. The term infection refers to Infection transmitted by ingestion may
the establishment of the microorganisms be waterborne (cholera), food borne
in the tissues resulting in injury or (typhoid) or fecal-oral route (dysentery).
harmful effect to the host. Infection is a
d. Inoculation
pathological condition due to the growth
of microorganisms in a host. To initiate an Pathogens, in some instances, may be
infection, a pathogenic microbe enters the inoculated directly into the tissues of the
tissues of the body by a characterization host. Tetanus spores implanted in the
route, the portal of entry. depth of wounds, rabies virus deposited
subcutaneously by dog bites, inoculation
12.1.1 Routes of Infections through unsterile syringes and surgical
equipments are examples that enter
There are various ways in which through direct inoculation.
microorganisms enters into the host are
explained below. e. Congenital
Some pathogens are able to cross the
a. Contact
placental barrier and infect the fetus in
Infection may be acquired by contact uterus. Bacteria like Treponema pallidum,
which may be direct or indirect. Sexually viruses like Rubella, Cytomegalovirus
transmitted diseases such as syphilis parasite like Toxoplasma gondi are some of
and gonorrhea spread by direct contact. the organisms that enter through placenta
Indirect contact may be through the and cause disease in the newborn.
agency of inanimate objects such as
clothing, pencils or toys which may be 12.1.2 Types of Infections
contaminated by a pathogen from one
person to another. Pencils shared by Infections may be classified in various
school children may act as fomites in ways. Initial infection with a parasite
the transmission of diphtheria, and face in a host is called a primary infection.
towels in trachoma. Subsequent infections by the same parasite
in the host are termed reinfections. When
b. Inhalation
a new parasite sets up an infection in
Respiratory infections such as influenza a host whose resistance is lowered by
and tuberculosis are transmitted by a preexisting infectious disease, this is
inhalation of the pathogen in droplet and termed secondary infection.
droplet nuclei that are shed by the patients
When in a patient already suffering
during sneezing, speaking or coughing.
Common cold virus, Adenovirus is from a disease, a new infection is setup
182

from another host or other external 1. When the skin is breached normal
sources it is termed cross infection. Cross flora enters the tissues.
infections occurring in hospitals are 2. When the urethral organisms ascend,
called nosocomial infections. Iatrogenic they cause urinary tract infection
infection refers to physician induced 3. When a patient is treated with
infections resulting from investigative, antibiotics, normal flora is eliminated
therapeutic or other procedures. and replaced by potential pathogens
Depending on whether the source 4. When the intestine is perforated,
of infection is from the host’s own body normal flora enter the previously
or from external sources, infections are sterile body parts
classified as endogenous or exogenous, 5. Similarly when the pH of the vagina
respectively. increases potential pathogens occupy
Endogenous infection the space.
Endogenous infections are acquired from However normal flora helps host against
the host himself from the normal flora of pathogen and benefits the host in many
the body. ways
Microorganisms are present in certain • Normal flora of skin produces fatty

areas of the body in all human beings. They acids which inhibit other species
are called normal flora. The common areas • Intestinal bacteria secrete antibacterial
are Nose, Mouth, Teeth, Throat, Intestine, substances (bacteriocins, colicins) and
Urethra, Vagina and Skin (Figure 12.1). many metabolic products that prevent
other species to survive.
Bacterial flora in a normal person in the community
Upper respiratory tract

Staphylococcus sp.
Streptococcus sp.
– Streptococcus pneumoniae
– Viridans streptococcus
Haemophilus sp.
Anaerobes
Skin
Staphylococcus sp.
Coryneform bacteria or
‘‘Diptheroids’’
Propionibacterium sp.
Gastrointestinal tract
Anaerobes
Enterococcus sp.
Enterobacteriaceae
– Escherichia coli
– Klebsiella sp.
Streptococcus sp.
Lactobacillus sp.
Genital tract
Lactobacillus sp.
Streptococcus sp.
– Streptococcus agalactiae
Figure 12.1: Microorganisms present as normal flora

(There are many organisms in a site. Only few are listed)
183

• Because of their large numbers other Transmission may be mechanical or
species do not have space in the biological. Mechanical transmission is the
intestine passive transport of the pathogens on the
• Acidic environment created by vaginal insects feet or other body parts. Example:
Lactobacilli suppresses growth of other Houseflies can transfer the pathogens of
bacteria. Typhoid fever and Bacillary dysentery
HOTS Infobits
The story of typhoid Mary

How do normal flora help host against
The classic example of role of carriers
pathogenic microorganisms?
in disease transmission is the story of
Mary Mallon.
Exogenous sources of infections Mary Mallon was an Irish
Human beings: The commonest source immigrant who worked as a cook
of infections in human are from other in New York in the early twentieth
human beings. The parasite may originate century. Over seven years, from
from a patient or a carrier. A patient is 1900 to 1907, Mallon worked for
a person who harbours the pathogenic number of different households.
microorganism and suffers from ill effect Unknowingly spreading illness to
because of it. A healthy carrier is the the people who lived in each one.
one who harbours the pathogens but has Later George Soper, tracked Mallon
never suffered from the disease caused by linked 22 cases of typhoid fever
the pathogen. A convalescent carrier is through her. He discovered that
one who has recovered from the disease Mallon was a carrier for typhoid but
and continues to harbor the pathogen in was immune to it herself. Although
his body (Figure 12.2). active carriers had been recognized
before, this was the first time that
Animals: Many pathogens are able to
an asymptomatic carrier of infected
infect both human beings and animals.
had been identified. Epidemiologists
Infectious disease transmitted from
were able to trace 51 cases of typhoid
animals to human beings are called
fever and three deaths directly to
zoonoses. Zoonotic diseases may be
Mallon, who is remembered as
bacterial (Example: plague from rats) or
“Typhoid Mary”. She was forced to
viral (Example: rabies from dogs).
prison and then released under the
Insects: Blood sucking insects may transmit conditions that she could no longer
pathogens to human beings. The diseases so be a cook. She assumed a false name
caused are called arthopod borne diseases. and began cooking again and of
Insects such as mosquitoes, ticks, mites, course, infecting numerous people.
flies, fleas and lice that transmit infections She was again prisoned where she
are called vectors. died 26 years later of pneumonia.
184

Figure 12.2: The steps involved when a microbe causes disease in a host
(shigellosis) from feces of infected people Localised infections: An infection

to food. Such vectors are called mechanical that is restricted to a specific location or
vectors. region within the body of the host is called
Biological transmission is an active localised infection.
process and is more complex. The pathogens Generalised infections: An infection
multiply in the body of the vectors often that has spread to several regions or areas
undergoing part of a developmental cycle in the body of the host. This involves
in it. Such vectors are termed biological the spread of the infecting agent from
vectors. Example: Aedes aegypti mosquito the site of entry through tissue spaces or
transmitting dengue, Anopheles mosquito channels, along the lymphatics or through
transmitting malaria. the bloodstream.
Soil: Some pathogens are able to survive Circulation of bacteria in the blood is
in the soil for very long periods. Spores known as Bacteremia. Septicemia is the
of tetatus bacilli may remain viable in the condition where bacteria circulate and
soil for several decades and serve as the multiply in the blood, form toxic products
source of infection. and cause high fever. Pyemia is a condition
where pyogenic bacteria produce
Water: Water may act as the source of septicemia with multiple abscesses in the
infection due to contamination with internal organs such as the spleen, liver
pathogenic microorganisms. Example: and kidney.
Cholera causing Vibrio cholerae.
Occurrence of a disease
Food: Contaminated food materials may To understand the full scope of a disease,
act as source of infection. The presence we should know about its occurrence.
of pathogens in food may be due to Epidemiology involves in the study of the
external contamination. Example: Food frequency and distribution of disease and
contaminated by Staphylococcus. other health related factors in defined
populations. The incidence of a disease
12.1.3 Types of Infectious Diseases
is the number of people in a population
Infectious diseases may be localised or who develop a disease during a particular
generalised. time period. The prevalence of a disease
185

is the total number of existing cases with
respect to the entire population. Facts about Fever:
Depending on the spread of infectious Fever is as more healthful than harmful.

disease in the community, they may be An experiment with vertebrates shows
classified into different types. that fever increases the rate of antibody
synthesis. Increased temperatures
• Endemic diseases are those which are stimulate the activities
constantly present in a particular area. of T cells and increase
Typhoid fever is endemic in most parts the effectiveness of
of India. interferon. Fever
• Epidemic disease is one that spreads appears to enhance
rapidly, involving many persons in phagocytosis. Fever almost never
an area at the same time. Example: occurs as a single response; it is usually
Epidemic of Dengue in 2017. accompanied by chills. The explanation
• A pandemic is an epidemic that spreads lies in the natural physiological
through many areas of the world interaction between the thermostat in
involving very large numbers of persons the hypothalamus and the temperature
within a short period. Example: H1N1 of the blood. For example: If the
Influenza outbreak in 2009. Ebola thermostat has been set (by pyrogen) at
outbreak in 2014-2016 in West Africa 102°F but the blood temperature is 99°F,
was the largest in history and first ever the muscles are stimulated to contract
epidemic, affecting multiple countries. involuntary (shivering) as a means of
• If a particular disease occurs only producing heat. In addition, the vessels
occasionally, it is called a sporadic in the skin constrict, creating a sensation
disease. The most commonly of cold and the piloerector muscles in
occurring sporadic diseases in India the skin develops ‘goose bumps’.
are Diphtheria and Hepatitis A and E.
Severity or duration of a disease • A latent disease is one in which the
causative agent remains inactive for
Another useful way of defining the scope
a time but then becomes active to
of a disease is in terms of its severity or
produce symptoms of the disease.
duration.
• An acute disease is one that develops 12.1.4 Interaction between Microbes
rapidly but lasts for a short time. and Host
• A chronic disease develops more Pathogen is a microorganism which causes
slowly, and the body’s reactions may disease.
be less severe, but the disease is likely Pathogenecity is the ability of a
to be continual or recurrent for long pathogen to produce disease.
periods.
Virulence is the degree of pathogenecity
• A disease that is intermediate between
of a microorganism. Virulence is not
acute and chronic is described as a
generally attributable to a single property
subacute disease.
186

but depends on several parameters related to intestine, Haemotoxin lyses red blood
the organism, the host and their interaction. cells, and Nephrotoxins damages the
Microorganisms first enter the body, kidneys.
survive, multiply and elaborate many A toxin molecule secreted by a living
factors and produce the disease. bacterial cell into the infected tissue is an
Adhesion: The initial event in the exotoxin. A toxin that is not actively secreted
pathogenesis of many infections is the but is shed from the outer membrane is an
attachment of the bacteria to body endotoxin. the difference between exotoxin
surfaces. Adhesions may occur as and endotoxin were given in Table 12.1.
organized structures, such as fimbriae and Production of enzymes
pili. Adhesions serve as virulence factors.
Some enzymes like proteases, DNAases
Capsule: It is an envelope or slime and phospholipases are produced and they
layer surrounding the cell wall of certain help in destruction of the cell structure
microorganisms. Capsule plays important and to hydrolyse host tissues.
roles in immune evasion as it inhibit’s
phagocytosis, as well as protecting the Antigenic variation
bacteria while outside the host. Microorganisms evade the host immune
responses by changing their surface
Toxins: Toxins are specific chemical
antigens. Antigenic drift and antigenic
products of microbes, plants and some
shift are common in influenza viruses.
animals that are poisonous to other
The distinction between the commensal
organisms. Toxigenicty is the power to
and the organism associated with disease.
produce toxins.
A toxin is named according to specific 12.1.5 Diagnostic Cycle
target of action: Neurotoxin acts on the Specific diagnosis is important for better
nervous system. Enterotoxin acts on the patient care, use of appropriate antibiotics
Table 12.1: Differences between endotoxin and exotoxin

Exotoxins Endotoxins
Heat labile proteins, secreted by certain Heat stable polysaccharide proteins, lipid
species of bacteria and diffuse readily into complex which form an integral part of the
the surrounding medium cellwall of Gram negative bacteria
Proteins with a strong specificity to a target A Lipopolysaccharide (LPS), which is part
cell and extremely powerful sometimes of the outermembrane of gram negative
deadly cell walls
Highly immunogenic Less immunogenic
Toxoids can be made by treating toxins Toxoids cannot be made
with formalin
Produced mainly by Gram positive bacteria Produced by Gram negative bacteria
but also by some Gram negative bacteria
187

and to initiate appropriate preventive believed to be present in the clinical sample.
measures. The diagnostic cycle begins Quality of patient specimens and their
when the clinician takes a microbiological transport to the laboratory is important.
sample and ends when a clinician
receives the laboratory report and uses Infections and samples used
the information to manage the condition Respiratory tract infections: Nasal and
(Figure 12.3). bronchial washings, throat and nasal
swabs, sputum.
The steps in diagnostic cycle are
Eye infections: Conjunctival swab or
1. Clinical request and provision of scraping.
clinical information.
Wound infections: Pus, skin scraping,
2. Collection and transport of appropriate wound swap.
specimens.
Gastrointestinal infections: Stool, rec-
3. Laboratory analysis.
tal swabs.
4. Interpretation of microbiology report
Genital infections: Vesicle fluid or
and use of the information.
swab.
Specimen Collection and transport:
Urinary tract infections: Urine.
It is important to collect the specimen
appropriately and protect it from Blood borne infections: Blood.
contamination. Transport media are used Nervous system infections: Cerebro-
that are compatible with the organism spinalfluid (CSF).
Figure 12.3: The steps in diagnostic cycle

188

Figure 12.4: Different approaches of diagnosis
Laboratory diagnosis of infectious agents can enter through skin breaks that are
Direct diagnosis: It is the not readily apparent, and the larval
demonstration of the presence of an forms of a few parasites can penetrate
infectious agent, antigen or nucleic acids the intact skin. The skin has up to seven
Indirect diagnosis: It is the layers (Figure 12.5) of ectodermal tissue
demonstration of presence of antibodies and guards the underlying tissues viz;
to a particular infectious agent, cytopathic muscles, bones, ligaments and internal
effects, haemagglutination, inclusion organs. Nearly all human skin is covered
bodies and neutralization. with hair follicles. Because it interfaces
with the environment, skin plays an
The different approaches for diagnosis
important role in protecting the body
or identification of infectious agents are
against pathogens and excessive water
shown in Figure 12.4.
loss. Its other functions are insulation,
temperature regulation, sensation,
12.2 Skin and Wound Infections
synthesis of vitamin D, and the
The skin, which covers and protects the protection of vitamin B folates. Severely
body, is the body’s first line of defense damaged skin will try to heal by forming
against pathogens. As a physical barrier, scar tissue. This is often discolored and
it is almost impossible for the pathogens depigmented.
to penetrate it. However, microorganisms
189

12.2.1 Normal Microbiota of the Skin may also cause infection. The most common
The skin’s normal microbiota contains normal flora of the skin are: Staphylococci,
relatively large numbers of Gram positive and various Streptococci, Sarcina sp,
bacteria, such as Staphylococci and anaerobic Diphtheroids, Gram negative
Micrococci. Bacteria in the skin tends to rods and others.
be grouped into small clumps. Vigorous Infected area
Pus
washing can reduce their numbers but (white
will not eliminate them. Microorganisms Skin Blood celis)
remaining in hair follicles and sweat glands
after washing will soon reestablish the
normal populations. Areas of the body
with high moisture, such as armpits and Bacteria
between the legs, have higher populations
of microorganisms. They metabolize Figure 12.6: Bacterial infection
secretions from the sweat glands and are the on the skin
main contributors to body odour.
Post operative infections
Also part of the skin’s normal microbiota
Gasgangrene organisms like Clostridium
are Gram positive pleomorphic rods called
perfringens, Staphylococcus aureus and
diphtheroids. Some diphtheroids, such
Clostridium tetani may cause post operative
as Propionibacterium acnes, are typically
infections.
anaerobic and inhabit hair follicles. These
bacteria produce propionic acid, which HOTS
helps maintain the low pH of skin, generally
between 3 and 5
1. What are the possible infecting
12.2.2 Wound Infection agent you could pick up when you
Wound can be defined as any interruption are injured while playing on the
of continuity of external or internal surfaces ground? List them and name the
caused by violence diseases that they could cause.
2. What are the possible infectious
Wounds may occur following: surgery,
agent that can infect you when you
trauma or injections
are injured by a rusted nail?
Wound infections may occur mainly
after surgical procedures
Route of entry
Wound sepsis is the result of cross
infection from human sources and from Wounds may occur following surgery,
other outside sources. trauma or injections. Wound infections
may occur mainly after surgical procedures.
Bacteria associated with wound Wound sepsis is the result of cross infection
infections from human sources and from other outside
Many bacteria are associated with wound sources. Infections of skin are listed in
infection (Figure 12.6). The normal flora Table 12.2.
190

Mechanisms of damage
1. Organisms enter through the skin,
multiply there and produce the disease
in the skin.
For example, impetigo, abscess and
cellulitis (Figure 12.7) are caused by
Staphylococcus aureus and Streptococcus
pyogenes.
Figure 12.8: Ulcer formation
2. Organisms multiply in the skin and

produce disease in internal organs. For
example some Group A Streptococci
Figure 12.7: Cellulitis
multiply in the skin and produce disease
As soon as the organisms enter the skin known as Acute Glomerulonephritis
they multiply and produce various toxins causing damage to the kidneys. Some
that kill the cells and produce cellulitis. times Corynebacterium diphtheriae may
Further damage leads to necrosis and multiply in the skin and affect the heart
ulcer formation (Figure 12.8). due to the toxin
Table 12.2: Bacterial Infections of the skin

Disease Pathogen Signs and Symptoms Transmission
Cellulitis Streptococcus Localised inflammation Through cut or abrasion
pyogenes of dermis and
hypodermis; skin red,
warm, and painful to
the touch
Erysipelas Streptococcus Inflamed, swollen patch Through cut or abrasion
pyogenes of skin, often on face;
may be suppurative
Impetigo Staphylococcus Vesicles, pustules, and Highly contagious,
aureus, Streptococcus sometimes bullae around especially via contact
pyogenes nose and mouth
Wound infections Pseudomonas Formation of biofilm in Exposure of wound
aeruginosa, others or on wound to microbes in
environment; poor
wound hygiene
191

3. Sometimes organism may multiply in Lower respiratory tract includes
the skin and produce the toxin which trachea, bronchi, bronchioles, lungs and
affect the Central Nervous System alveoli (Figure 12.9).
(CNS) and the effects seen. In the case of
Clostridium tetani infection, convulsions
and paralysis occur due to the production
of a powerful toxin.
12.3 Respiratory Tract Infections

With every breath, we inhale several
microorganisms and therefore the
respiratory system is a major portal of
entry for pathogens. In fact, respiratory
system infections are the most common
type of infections and among the most
damaging. Some pathogens that enter via
respiratory route can infect other parts of
the body, such as skin incase of measles,
mumps and rubella.
Figure 12.9: Structure of human
The upper respiratory system has respiratory tract
several anatomical defenses against
airborne pathogens. Coarse hairs in the 12.3.2 Normal Defenses against Infections
nose, filter large dust particles from the air. Respiratory tract infection are divided
The nose is lined with a mucous membrane into upper respiratory tract (URT) tract
that contains numerous mucous secreting infection and lower respiratory tract (LRT)
cells and cilia. The upper portion of the infection. Infection of the respiratory tract
throat also contains a ciliated mucous are listed in the Table 12.3.
membrane. The mucous moistens inhaled
air and traps dust and microorganisms. URT: Infections are Sinusitis, Pharyn-
The cilia help to remove these particles gitis Laryngitis and Epiglotitis
by moving them towards the mouth for LRT: Infections are Trachiitis, Tracheo
elimination. bronchitis, Bronchitis, Alveolitis and
Pneumonia.
12.3.1 Structure of Respiratory Tract
The structure of respiratory tract is divided
into two main parts viz: upper respiratory
tract (URT) and lower respiratory tract (LRT).
Upper respiratory tract includes
mouth, nose, nasal cavity, sinuses, throat
or pharynx, epiglottis and larynx.
192

Upper respiratory system
Diseases Pathogen Symptoms
Bacterial diseases
Epigottitis Haemophilus influenzae Inflammation of the epiglottis
Streptococcal Streptococci, especially Inflamed mucous membranes of the
pharyngitis (strep Streptococcus pyogenes throat;
throat)
Diphtheria Corynebacterium diphtheriae Bacterial exotoxin interferes with
protein synthesis; damages heart,
kidney, and other organs; membrane
forms in throat; cutaneous form also
occurs;
Otitis media Several agents, especially Accumulations of pus in middle ear
Staphylococcus aureus, build up painful pressure on eardrum
Streptococcus pneumonia and
Haemophilus influenza
Viral diseases
Common cold Rhino virus Familiar symptoms of coughing,
sneezing, running nose.
Lower respiratory system
Bacterial diseases
Pertussis (whooping Bordetella pertussis Cilia in upper respiratory tract
cough) inactivated, mucus accumulates, spasms
of intense coughing to clear mucus;
Tuberculosis Mycobacterium tuberculosis Tubercle bacilli entering lungs
survive phagocytosis, reproduce in
macrophages; tubercles formed to
isolate pathogen; defenses eventually
fail, and infection becomes systemic;
Viral diseases
Respiratory syncytial Respiratory syncytial virus A serious respiratory disease of infants;
virus (RSV)
Fungal diseases
Blastomycosis Blastomyces dermatitidis Abscesses; extensive tissue damage;
Bacterial pneumonia
Pneumococcal Streptococcus pneumonia Infected alveoli of lung fill with fluids;
pneumonia interferes with oxygen uptake
Haemophilus Haemophilus influenzae Symptoms resemble pneumococcal
influenzae pneumonia pneumonia
193

12.4 Gastrointestinal Tract Infections Difference between infection and
intoxication
Human systems function by the energy
produced from the digested food Microbial diseases of digestive system
molecules. The food is swallowed through are typically transmitted by a fecal oral
mouth and digested in the gastro intestinal route. Most such diseases result from the
tract. The food we consumed should be ingestion of food or water contaminated
free of contaminations. The contaminated with pathogenic microorganisms or their
food causes gastrointestinal infections. toxins. These pathogens usually enter the
food or water supply after being shed in
Through contaminated food and water
the feces of people or animals infected
the pathogens are ingested and they enter
with them.
the GIT. In the small intestine they initiate
an infection. Many times the pathogens
• E ach milliliter
that cause intestinal infections multiply
of saliva can
in the GIT and produce their pathogenic
contain millions
effect in the intestine itself. Example:
of bacteria
Shigellosis, Cholera.
• Stomach/small intestine has very
The gastrointestinal tract (GIT) or few microorganisms because of
alimentary canal includes the mouth, hydrochloric acid present in the
pharynx, throat, oesophagus (food Tube stomach.
lead to the stomach), stomach, small and • L arge intestine harbours microbial
large intestine. It also includes accessory population exceeding 100 billion
structures salivary glands, liver, gall of bacteria per gram of feces (40%
bladder and pancreas lying outside the fecal masses contain microbial cell
GIT. Secretions of these organs enhance the material)
digestion of food molecules (Figure 12.11). • Large intestine microbial population
Gastro intestinal tract mainly contain anaerobes and
Salivary Mouth facultative anaerobes.
glands
After ingestion of pathogenic
microorganisms, localization and
Liver multiplication of organisms takes place in the
Duodenum
Stomach GIT and is called infection. Microorganisms
Pancreas may penetrate into intestinal mucosa and
Gall bladder
grow there or they may penetrate to other
organs. Gastroenteritis is usually classified
Small Large
intestine intestine as either infection or intoxication. Food
Rectum borne diseases can arise from either
Anus infection or intoxication. In both cases,
Figure 12.11: The structure of bacterial toxins are typically responsible for
Gastro intestinal tract producing disease signs and symptoms. In a
food infection the microbial agent ingested
194

colonise in the gut and then produces toxins characterized by nausea, vomiting,
that damage host cells. diarrhea, abdominal discomfort.
In case of food intoxication the toxins
produced by bacteria in the food are Stomach is acidic
ingested. Infection and intoxication differ because of the pres-
in their onset of symptoms. Infections are ence of hydrochlo-
characterized by a delay in the appearance ric acid. So in this
of gastrointestinal disturbance until the acidic condition organisms gen-
pathogen increases in number or affects erally not survive except one bac-
invaded tissue. Infection is correlated terium Helicobacter pylori. This
with onset of fever, one of the basic body’s bacterium is the leading cause of
general responses to an infective organism. stomach ulcers. This bacterium has
In case of intoxication, the symptoms are maximum evidence of correlation
characterized by sudden appearance of with the development of stomach
gastrointestinal disturbances like cramping, and intestinal cancer.
nausea, vomiting or diarrhoea. Stomach
HOTS
What is likely to happen to a child who

drinks contaminated water? H. pylori
12.4.1 Microbial Flora of

Gastrointestinal Tract
The stomach and gastrointestinal tract are
Diarrhea: Condition in which feces,
not sterile and are colonized by the organisms
that perform functions beneficial to the are discharged from the bowels frequently
host, including the manufacture of essential and in a liquid form.
vitamins. Escherichia coli found in the
intestine help the body to produce vitamin K Botulism is a special
and Bifodobacteria can synthesizes vitamins case of intoxication
such as vitamin B12, folate, and riboflavin. because, the ingestion
Humans cannot produce these vitamins. of the preformed toxin
The normal flora changes according to the affects the nervous system rather than
diet, age, cultural conditions and the use of GIT.
antibiotics (Table 12.4). Infant Botulism is the infectious
form of Botulism which results when
12.4.2 Terms used in GIT Infections
spores of Clostridium botulinum
Gastroenteritis: Inflammation of lining of swallowed colonise in the intestine.
stomach and intestine. It is a syndrome Botulism spores can be found in honey.
195

Table 12.4: Normal flora of human gastrointestinal tract
At human birth Stomach and intestine are sterile
Breast fed babies Lactobacillus bifidus
Bottled milk fed babies Enteric bacteria, Lactobacillus bifidus, Enterococci,
Clostridium sp
Small intestine Lactobacilli, Enterococcus faecalis, Escherichia coli
Large intestine Anaerobic bacteria, Streptococci, Bacteroides,
Bifidobacteriumbifidum
Dysentery: Inflammatory disorder of the GIT associated with pus and blood in feces.
Gastritis: Inflammation of the stomach lining that results in swelling.
Enteritis: Inflammation of the intestinal mucosa
Colitis: Inflammation of the colon
Hepatitis: Inflammation of the liver
Enterocolitis: Inflamation involving the mucosa of both large and small intestine.
Peritonitis: Inflammation of peritoneum (it is the serous membrane that forms the
lining of the abdominal cavity). Infections of digestive system are listed in Table 12.5.
Table 12.5: Diseases of the digestive system
Infection Pathogen Symptoms
Bacterial Diseases
Staphylococcal food Staphylococcus aureus Nausea, vomiting, and diarrhea
poisoning
Shigellosis (bacillary Shigella sp- Tissue damage and dysentery
dysentery)
Salmonellosis salmonella enterica Nausea and diarrhea
Typhoid fever Salmonella typhi High fever, significant mortality
Cholera Vibrio Cholerae Diarrhea with large water loss
Yersinia gastroenteritis Yersinia enterocolitica Abdominal pain and diarrhea,
usually mild; may be confused with
appendicitis
Viral Diseases
Mumps Mumps virus Painful swelling of parotid glands
Paramyxoviridae
Viral gastroenteritis Rotavirus Vomiting, diarrhea for 1 week
Fungal Diseases
Ergot poisoning Claviceps purpurea Restricted blood flow to limbs;
hallucinogenic
Aflatoxin poisoning Aspergillus flavus Liver cirrhosis; liver cancer
196

12.5 Ocular Infections protect the external surfaces of the eye,
A number of microorganisms cause both mechanically and biologically.
infection when introduced into the Infection of Eyelid
mucosa of the eye. In general, bacterial Most common cause of eyelid infection is
eye infections can lead to inflammation, Staphylococcus aureus.
irritation, and discharge, but they vary in Infection involves lid margins and
severity. Some are typically short-lived, and cause blepharitis.
others are chronic and lead to permanent When the eyelid glands or follicles
eye damage. Prevention requires limiting are affected stye (sticky eye) is seen
the exposure to contagious pathogens. (Figure 12.14).
When infections do occur, prompt
Conjunctivitis (inflammation of
treatment with antibiotics can often limit
conjunctiva) Conjunctivitis or pink eye
or prevent permanent damage.
can be caused by many different kinds of
The external surfaces of the eye viz. the viruses and bacteria.
conjunctiva and cornea are susceptible to
Infected eyelid and conjunctiva
infection. These are exposed to external
world and are easily accessible to infective Eyelid margin
agents. Particularly the conjunctiva is Infected eyelid
susceptible because it is covered with eyelid

Pus accumulation
that provides warm, moist and enclosed
environment in which contaminating
organisms can quickly establish a focus
Figure 12.14: Infected eyelid and
of infection. However, eyelid and tears
conjunctiva
Table 12.6: Bacterial infections of the eye
Disease Pathogen Signs and Symptoms Transmission
Acute bacterial Haemophilus Inflammation of Exposure to
conjunctivitis influenza conjunctiva with secretions from
purulent discharge infected individuals
Bacterial keratitis Staphylococcus Redness and irritation Exposure to
epidermidis, of eye, blurred vision, pathogens on
Pseudomonas sensitivity to light; contaminated
aeruginosa progressive corneal contact lenses
scarring, which can
lead to blindness
Neonatal Chlamydia Inflammation of Neonate exposed to
conjunctivitis trachomatis, conjunctiva, purulent pathogens in birth
Neisseria discharge, scarring canal of mother
gonorrhoeae and perforation of with chlamydia or
cornea; may lead to gonorrhea
blindness
197

Trachoma The urinary system consists of two kidneys,
Trachoma, or granular conjunctivitis, is a two ureters, a single urinary bladder and a
common cause of preventable blindness that single urethra. Wastes are removed from the
is rare in the United States but widespread blood as it circulates through the kidneys
in developing countries, especially in Africa (Figure 12.15).
and Asia. The condition is caused by the
same species that causes neonatal inclusion
conjunctivitis in infants, Chlamydia Pyelonephritis
UPPER TRACT
Kidney
(Kidney infection)
trachomatis. Chlamydia trachomatis can
be transmitted easily through fomites such
Ureter Ureteritis
as contaminated towels, bed linens, and (Ureter infection)
clothing and also by direct contact with

infected individuals. Chlamydia trachomatis
LOWER TRACT
can also spread by flies that transfer infected Bladder
Cystitis
(Bladder infection)
mucous containing Chlamydia trachomatis Urethritis
Urethra
from one human to another. Infections of (Urethra infection)
eye are listed in Table 12.6. Figure 12.15: Structure of lower and
12.6 Urinary Tract Infections upper urinary tract infection
The urinary system is composed of organs
that regulate the chemical composition Infections of the kidney, ureter and
and the volume of the blood excrete mostly bladder constitute Urinary Tract Infections
nitrogenous wastes products and water. (UTI). When infection occur in the kidney
and ureter it is called upper urinary tract
infections and bladder downwards is called
Infobits
lower urinary tract infections. Urinary tract
Many of the bacteria which cause infection is common in females than males.
UTI’s have developed resistance to The urinary system normally contains few
antibiotics. Research has turned to microbes but it is subjected to opportunistic
probiotic (Lactobacillus) strain which infections that can be quite troublesome.
stimulates immune function, lowers Almost all such infections are caused by
acidity levels in the urinary tract, bacteria although occasional infections
by pathogens such as parasites, protozoa
and discourages the growth of UTI
and fungi also occured. Microorganisms
causing organisms.
invloved in UTI are listed in Table 12.7.
Table 12.7: Microorganisms involved in UTI

Microorganisms Examples
Bacteria (most common) Escherichi coli, Klebsiella, Enterobacter, Proteus
Viruses Adenovirus, Mumps
Fungi Trichomonas vaginalis, Schistosoma haematobium
Parasites Candida
198

12.6.1 Predisposing Factors for UTI involved in other infections like wound
Urinary tract infection is common in infection peritonitis) UTI is common in
females than in males. The urethra in (a) married women (b) elderly men with
females are shorter and wider and is prostate enlargement.
less effective in preventing the bacteria Pathogenesis of cystitis in woman
entering the bladder. Sexual intercourse
Bladder infections can result from the
is a predisposing factor in females. High
downward migration of organisms from
incidence is seen in pregnant women due
an infected kidney. But majority arise by
to hormonal changes and impairment
ascent of pathogens from the rectum and
of urine flow due to pressure on urinary
vagina to the urethra meatus and bladder,
tract.
leading to cystitis. If left untreated, the
infection can further ascend to involve the
Obesity increases kidneys (pyelonephritis) (Figure 12.16).
the risk of UTI’s The rectum and vagina function as
in men. A 2013 the reservoir of bacteria for sporadic
study examineed infections
how obesity affected the chance of
In men, the longer urethra is believed
developing UTI and it was found that
to protect against ascending infections.
obese men were twice more likely to
develop the UTI than obese women. When Escherichia.coli (and other
Gram Negative rods) causes UTI, usually
12.6.2 Urinary Tract Infection caused the number of organisms in freshly passed
by Escherichia coli urine is more than 100,000 organisms/ml.
Escherichia coli is the predominant cause This is called “significant bacteriuria”.
of UTI. Counts less than this is associated with
contaminants from urethra or externalia.
It is a normal flora of the gut and can
Infection of urinary tract are listed in
cause extra intestinal infections (UTI,
Table 12.8.
Wound infection.) UTI (it can also be
Table 12.8: Microbial Diseases of the Urinary System

Disease Pathogen Symptoms
Bacterial Diseases of the Escherichia coli, Difficulty or pain in
Urinary system Staphylococcus urination
Cystitis (Urinary bladder saprophyticus
infection)
Pyelonephritis (Kidney Primarily Escherichia coli Fever; back or flank pain
infection)
Leptospirosis (Kidney Leptospira interrogans Headaches, muscular aches,
infection) fever; kidney failure a
possible complication
199

Stages of a urinary tract infection
Acute kidney Bacteria continues to cascade

5 on to the kindneys, leading to
injury
acute kidney injury. Kidneys
Infection of the renal parenchyma

4 Pyelonephritis causes an inflammatory
response called pyelonephritis.
Bacteria ascends towards the Ureters

3 Ascension kidneys via the ureters.
Pathogen penetrates bladder

Uroepithelium Bladder
2 and bacteria replicates, poten.
penetration
tially forming biofilms.
Urethra
Pathogen colonizes the
urethra and ascends towards
1 Colonization
the bladder.
Figure 12.16: Various stages of a urinary tract infection
12.7 Reproductive Tract Infections organisms normally present in the genital

tract of healthy women. Example: Bacterial
Reproductive tract
Vaginosis or Vulvo Vaginal Candidiasis.
infections are caused
by organisms normally 3. Iatrogenic Infections
present in the
These infections are associated with
reproductive or genital
improperly performed medical procedures
tract or introduced
such as unsafe abortion or poor delivery
from the outside during sexual contact
practices. The endogenous organisms
or medical procedures. It occur both
in the vagina or sexually transmitted
in men and women. Based on mode of
organisms in the cervix may be transferred
infection reproductive tract infections
during a transcervical procedure into the
are classified into three types:
upper reproductive tract and cause serious
1. Sexually Transmitted Disease infections of the uterus, fallopian tubes,
and other pelvic organs.
It is caused through means of sexual
contact. Examples: Chlamydia, In men reproductive tract infections
Gonnorhea, Chancroid, and Acquired transmitted by sexual contact are much
Immuno Deficiency Syndrome (AIDS). more common than by endogenous or
iatrogenic reproductive infections. In women
2. Endogenous Infections
reproductive infections spread through non
These are caused by the overgrowth of sexual routes are usually more common.
200

12.7.1 Mode of Transmission flora during this period contain normal
skin microorganisms. The vaginal pH
Reproductive tract infections are caused
is mild alkaline. The normal vaginal
by pathogenic bacteria, parasite, virus. It
flora often includes Listeria, anaerobic
is mainly caused by pathogens entering
Streptococci, Mycoplasma, Gardnerella
into the body through the mucous mem-
vaginalis, Neisseria, Spirochetes, Candida,
branes during unprotected vaginal, oral,
Staphylococcus epidermidis.
anal intercourse with an infected part-
ner. In developing countries bacterial
infections like Gonorrhoea, Chlamydia, 12.7.3 Pathogenesis
Syphilis, Bacterial Vaginosis, Lympho-
After the entry of pathogenic organisms,
granuloma Venereum, Trichomoniasis,
with sufficient incubation time, symptoms
Chancroid, and viral infections caused
are clearly manifested in the affected
by Human Papilloma Virus, Hepatitis B
individual. The most common symptoms
Virus, Herpes Simplex Virus, Human Im-
include unusual vaginal discharge, penile
munodeficiency Virus are very common.
discharge, pelvic pain, itching, abnormal
or heavy vaginal bleeding, rashes, warts,
12.7.2 Normal Flora of Reproductive lesions, burning or pain during urination.
Tract However most of the infections are
Mycobacterium smegmatis, a harmless asymptomatic, which act as a effective
commensal found in the smegma of the control of reproductive tract infections.
genitalia of both men and women. In Diseases of reproductive system are listed
nomal men aerobic and anaerobic bacteria, in Table 12.9.
lactobacilli, alpha haemolytic Streptococci,
Chalmydia trachomatis and Ureaplasma
urealyticum may also be present.
The adult female genital tract has a
very complex microflora. The character
of the population changes with the
variation of the menstrual cycle. Mostly
the predominant bacteria are acid tolerant Infobits
Lactobacilli. Glycogen is accumulated in
the vaginal wall due to ovarian hormonal Tamilnadu has AIDS testing centres
activity. The breakdown of glycogen at all district head quarters with more
by the lactic acid bacteria (Doderlien’s than 55 Anti Retroviral Therapy(ART)
bacillus) leads to the formation of acidic centres and 750 (ICTC)-Integrated
pH (4.4-4.5). This acidic nature prevents (voluntary) and confidential
the vagina from bacterial vaginosis counselling and testing centres under
the national AIDS control programme
and yeast infections. However before
at district level government hospitals
puberty and after menopause there is
and medical colleges across the state.
no glycogen formation. The normal
201

Table 12.9: Microbial diseases of the reproductive system
Disease Pathogen Symptoms

Bacterial Diseases
Gonorrhea Neisseria Painful urination, discharge of pus in
gonorrhoeae males, abnormal vaginal discharge in
females
Nongonococcal Chlamydia Painful urination and watery discharge,
urethritis (NGU) trachomatis or other Chronic abdominal pain in females
bacteria, including
Mycoplasma hominis
and Urea plasma
urealyticum
Syphilis Treponema pallidum Initial sore at site of infection, later skin
rashes and mild fever; final stages may be
severe lesions, damage to cardiovascular
and nervous systems.
Lymphogranuloma Chlamydia Swelling in lymph nodes in groin
venereum (LGV) trachomatis
Viral Diseases
Genital Herpes Herpes simplex virus Painful vesicles in genital area
type 2; HSV type 1
Genital warts Human papilloma Warts in genital area
viruses
AIDS Human loss of appetite, weight loss,
Immunodeficiency persistent cough, attack on T cells
virus (HIV) (immunocompromise), easily prone to
fungal and other bacterial pathogens as
secondary opportunistic infections.
Fungal Diseases
Candidiasis Candida albicans Severe vaginal itching, yeasty odor, yellow
discharge
Protozoan Diseases
Trichomoniasis Trichomonas Vaginal itching, greenish yellow discharge
vaginalis
202

12.8 Infections of the Nervous System spinal cord. It controls most functions
Some of the most devastating infectious of the body and mind. The peripheral
diseases are those that affect the nervous nervous system (PNS) consists of all
system, especially the brain and the spinal the nerves that branch off from the
cord. Damage to these areas can lead to brain and spinal cord. These peripheral
deafness, blindness, learning disabilities, nerves are the lines of communication
paralysis and death. Microbial infections of between the CNS, the various parts of
CNS are infrequent but often have serious the body and the external environment
consequences. In pre antibiotic times, they (Figure 12.19).
were almost always fatal. An infection of Brain and spinal cord are covered
CNS can be life threatening condition, by three layers of membranes called
especially for children with weakened meninges. These layers are the outermost
immune system. These infections need dura mater, the middle arachnoid mater,
quick diagnosis and immediate treatment and the innermost pia mater. Between the
by an infectious disease specialist. Bacteria, pia mater and arachnoid membranes is a
Fungi and viruses are the most common space called the subarachnoid space, in
causes of CNS infections. which there is cerebrospinal fluid (CSF)
12.8.1 Structure of Nervous System circulating.
The human nervous system is organized 12.8.2 Barriers of CNS

into two divisions: The Central Nervous Dyes such as Trypan blue injected into
System (CNS) and Peripheral Nervous the systemic circulation stain virtually
System (PNS). The Central Nervous all tissues, with the exception of the
System (CNS) consists of brain and brain and spinal cord. This blood brain
Scalp
Skull bone
Dura mater
Venous blood
Arachnoid mater
Subarachnoid
space (contains
Venous cerebrospinal
blood fluid)
Pia mater
Brain (cerebrum)
Cerebrospinal fluid
Pia mater
Arachnoid
Meninges
mater
Ventricles
Dura mater
Subarachnoid
space of brain
Figure 12.19: Structure of central nervous system

203

barrier excludes most macromolecules, configuration of cerebral capillaries, the
microorganisms, immunocompetent cells choroid plexus and arachnoid cells. It
and antibodies. Even pathogens that are acts as a natural barrier that prevents
circulating in the bloodstream usually the invasion of microorganisms into
cannot enter the brain and spinal cord the brain. If this is breached organisms
because of blood brain barrier. Certain enter the brain. The blood CSF barrier
capillaries permit some substances to (Figure 12.22) (also calle brain CSF
pass from the blood into the brain but barrier) consists of endothelium with
restricts others. These capillaries are less fenestrations, and tightly joined choroid
permeable than others within the body plexus epithelial cells. It acts as a natural
and are therefore more selective in passing barrier that prevents the invasion of
materials (Figure 12.20). The blood brain microorganisms into the meninges.
barrier (Figure 12.21) is due to the cellular
Capillary (general) Capillary (brain)
cell Astrocyte
forming
capillary
wall
Tight junction
Water-lined Substance Pore (no pores)
Substance
pore in blood passage
in blood
Carrier-
mediated
Lipid-soluble Lipid-soluble transport
substances substances
Figure 12.20: Capillaries of brain
Tight junction
Blood
Endothelium
Basement membrane
Astrocyte
Brain
Figure 12.21: Blood brain barrier
Fenestration
Blood
Endothelium
Basement membrane
Chorid cells
Tight junction
Cerebrospinal fluid
Figure 12.22: Blood CSF barrier

204

12.8.3 Routes through which viruses that produce more widespread
microorganisms enter nervous system intracellular infections.
• Skull or bone fractures • Brain abcess is a focus of purulent
• Medical procedures infection and is usually due to bacteria.
Brain abscesses develop from either a
• Peripheral nerves contiguous focus of infection (such as
• Blood or lymph the ears, the sinuses, or the teeth) or
hematogenous spread from a distant
12.8.4 Clinical Manifestations of
focus (such as the lungs or heart,
Nervous System Infections
particularly with chronic purulent
Some of the symptoms of nervous System pulmonary disease, subacute bacterial
infections are headache, fever, stiff neck, endocarditis, or cyanotic congenital
focal signs, seizures, confusion, weakness, heart disease). In many cases the
hallucinations, stupor, coma, abnormal source is undetectable.
behavior and sleep disorder
Etiological agents of Meningitis
12.8.5 Infections of Nervous System
This can be caused by a wide range of
• Meningitis is an inflammation of microorganisms and can be classified as
the meninges (membrane covering pyogenic and non pyogenic meningitis.
the brain). Meningitis is a diffuse In pyogenic meningitis infiltration of
infection caused by a variety of pus cells (neutrophils) will be seen. In
different agents. Non pyogenic or aseptic meningitis
• Encephalitis is defined as inflammation infiltration of lymphocytes may be seen.
of the brain. Unlike an abscess, which Diseases of nervous system are listed in
is a localised area of bacterial or fungal Table 12.10.
growth, Encephalitis is usually due to
Drugs cannot cross the blood brain barrier unless they are lipid
soluble. Glucose and many amino acids are not lipid soluble, but they
can cross the barrier through special transport systems. The lipid
soluble antibiotic Chloramphenicol enters the brain readily. Penicillin
is only slightly lipid soluble, but, if it is taken in very large doses, enough may cross
the barrier to be effective. Inflammations of the brain tend to alter the blood brain
barrier in such a way as to allow antibiotics to cross that would not be able to cross if
there were no infection.
Antibodies found in the normal CNS are derived from the serum and are
present at low levels compared to serum levels. There are a few phagocytic cells and
complement is also largely excluded. CSF is especially vulnerable because it lacks
many of the defenses found in the blood, such as phagocytic cells. It is not easy for
the microorganisms to enter CNS but it hampers their clearance once it is penetrated.
205

Table 12.10: Microbial diseases of the Nervous system
Diseases Pathogen Portal of Entry Method of Transmission
Bacterial Diseases
Haemophilus Haemophilus. Respiratory Endogenous infection;
influenzae meningitis Influenzae tract aerosols
Meningococcal Neisseria Respiratory Aerosols
meningitis meningitidis tract
Pneumococcal Streptococcus Respiratory Aerosols
meningitis pneumoniae tract
Tetanus Clostridium tetani Skin Puncture wound
Botulism Clostridium Mouth Food borne intoxication
botulinum
Viral Diseases
Poliomyelitis Poliovirus Mouth Ingesting contaminated
water (fecal oral route)
Rabies Lyssavirus, includ- Skin Animal bite
ing rabies virus
Fungal Diseases
Cryptococcosis Cryptococcus Respiratory Inhaling soil contaminate
neoformans route with spores
Protozoan Diseases
African Trypanosoma Skin Tsetse fly
trypanosomiasis brucei
Rhodesiense,
Trypanosoma
brucei gambiense
12.9 Systemic Infections result from such common exposure is

transient and remains below the threshold
An infection that is in the bloodstream is
of detection. In severe cases, bacteremia
called a systemic infection. Systemic diseases
can lead to septicemia with dangerous
such as flu and typhoid affect the entire
complication such as Toxemia sepsis and
body. Bacteria can enter the circulatory and
Septic shock. In these situations, it is often
lymphatic systems through acute infections
the immune response to the infection that
or breaches of the skin barrier or mucosa.
result in the clinical signs and symptoms
Breaches may occur through fairly common
rather than microbes themselves.
occurrences, such as insect bites or small
wounds. Even the act of tooth brushing, Summary
which can cause small ruptures in the gums
In the branch of medical microbiology we
may introduce bacteria in to the circulatory
discussed about prevention, diagnosis and
system. In most cases, the Bacteremia
206

treatment of infectious diseases. Infections defence against infections. Conjunctivitis
are acquired through contact, inhalation, and Trachoma are the common eye diseases.
ingestion, inoculation and congenital. Sources Proper diagnosis and treatment should be
of infections are endogenous and exogenous suggested.
in origin. Normal flora are organisms present
Uninary tract infections are more
in certain areas of the body. Infectious
common in females than in males. There are
diseases may be generalised or localised.
many predisposing factors making female
Based on the occurrence of infectious diseases
prone to the infections. The predominant
the infection may be epidemic, endemic, or
causative agents in urinary tract infection is
sporadic. There are various virulence factors
Escherichia.coli. The number of organisms
which are responsibility for the pathogenicity.
in freshly passed urine is more than 100,000
Skin is the first line of defence against organisms/ml. It is called significant
pathogen. Normal uninterrupted skin bacteriuria.
provides protection against “invasion by
The infections spread through
bacteria”. Many exogenous and endogenous
reproductive tract by direct contact
factors are responsible for wound infections.
is called sexually transmitted disease.
The mechanism of damage may be in the
Mostly these infections are asymptomatic
skin or some cases its spreads to the internal
in women.
organs and CNS system.
Nervous system infection affect
Respiratory system of both lower and
brain and spinal cord. They are of two
upper is the major path for entry of pathogens.
types meningitis and encephalatis. An
The infections of upper respiratory tract are
infection that is in the blood stream
sinusitis, pharyngitis, laryngitis and epiglottitis.
is called systemic infections. Systemic
The infection of lower respiratory tract are
diseases like flu and typhoid affect the
trachitis, tracheobronchitis, bronchitis, and
entire body.
alveolitis.
Gastrointestinal tract infections are
infections of the digestive system. The food
borne infection and food intoxication are
the common cause of gastroenteritis. The
gut flora and natural defence mechanism
by defensins bacteriocins, globet cells, IgA
antibodies protect the individual against
pathogenic infection. Diarrhea, dysentery,
vomitting are the common symptoms of GIT.
Oral rehydration therapy, proper hygiene to be
manifested to reduce the risk of gastroenteritis.
The external structure and parts of the
eye are easily susceptible to infections. The
eyelids, tears, lysozyme, IgA are the natural
207

Modes of Infection
208

ICT CORNER
Respiratory Tract Infections
Know the myths

of cold
STEPS:
• Use the link or scan the QR code given below. “Cells Alive” home page will open.
You can select any topic you wish. For example click “understanding colds”
• “Understanding Colds” page will open. You can go through anatomy of the nose,
CAT scan view etc..
• At the top left of the page click on “Menu” and select “Treatments” and analyze.
• Also select “Special features” and go through the topic. Also you can select
how penicillin kills bacteria in the “Cells Alive” page, and know the action of
penicillin against bacteria.
Step1 Step2 Step3 Step4
URL:
209

Evaluation c. Between skull and dura mater
Multiple choice questions d. Sub dural space
6. antibody gives first line
1. Syphilis is
defense against respiratory tract
disease
infections.
a. Sexually
a. IgM
transmitted disease
b. IgA
b. Respiratory tract disease
c. IgD
c. Gastro tract disease
d. IgE
d. Urinary tract disease
7. The nose is lined with
2. is the person
membrane.
who harbours the pathogenic
a. Mucous
microorganisms and suffers from till
effect because of it? b. Epithelial
a. Carrier c. Secretion
b. Healthy carrier d. None of these
c. Patient 8. nature of stomach act as
a natural defense mechanism.
d. All the above
a. Acidic
3. Circulation of bacteria in the blood is
b. Neutral
known as
a. Septicimia c. Alkaline
b. Pyemia d. None of the above
c. Bacterimia 9. Traveller”s diarrhea is caused by
a. Escherichia coli
b. Staphylococcus aureus
4. From the skull down to the brain,
select the arrangement of layers of c. Vibrio cholerae
meninges from the following: d. All the above
a. Dura mater/Arachnoid mater/Pia 10. is the predominant cause
mater * of UTI?
b. Arachnoid mater/Dura mater/Pia a. Staphylocous aureus
mater b. Escherichia coli
c. Pia mater/Arachnoid mater/Dura c. Salmonella
mater d. Streptococcus pyogenes
d. Dura mater/Pia mater/Arachnoid 11. fungi involved in
mater urinary tract infection?
5. Cerebrospinal fluid (CSF) is present a. Klebsiella
in which of the following? b. Candida sp
a. Perivascular spaces c. Penicillium
b. Sub arachnoid space * d. Escherichia coli
210

12. During the breakdown of glycogen by lactobacilli in the vagina, makes vaginal pH
as .
a. Acidic
b. Neutral
c. Alkaline
1. Define congenital infection?
2. What is meant by nosocomial infection?
3. Define the term bacteremia, septicemia pyremia?
4. Explain mode of transfer of infection?
5. Define a wound.
6. What are the causes of wound?
7. Name two types of CNS infections.
8. Give the names of the etiologic agents of wound infection.
9. Describe microbial disease of upper respiratory tract infection?
10. State the difference between dysentery and diarrhoea?
11. What is the difference between food borne infection and intoxication?
12. Give the normal flora of the gastrointestinal tract of humans?
13. What is called significant bacteriuria?
14. Explain the predisposing factors for urinary tract infection?
15. Define iatrogenic infection.
16. Explain the role of lactobacilli in the prevention of bacterial vaginosis.
17. Give detailed study of various bacterial, fungal and viral infectious diseases of
reproductive tract infection.
211

Student Activity (1)
1. Get information from your parents/neighbor about types of diseases one gets due
to contamination. Example: If you drink contaminated water, you get diarrhoea.
No After certain activity Getting a disease Preventive method to

advocate
1 Contaminate Diarrhoea Don’t drink or Boil, cool
drinking water and drink
2
3
4
5
2. G
ive a list of organisms present as normal flora of the skin (include other than that
is given in the text book).
3. P
repare model of respiratory tract with innovations.
Prepare a list of URT infections with the etiologic agents and prevention.
Observe a chronic smoker. He coughs very often. List out the reasons for his cough.
collect information from nearby neighbors kids (10). How many of them are
immunized DTP vaccinated?
Where corporation
No Kid’s name DOB Immunized on or pvt
1
2
3
4. S tudent is asked to prepare a model of GIT with their innovations.

See for example:
What all the organisms that can be transmitted through the fly contaminated food.
Give a list.
5. ( 1) Write an assignment on Madras eye (conjunctivitis due to viruses)
(2) W rite Dos and Don’s when a dust particle comes into your eye.
6. 1) Draw the structure of urinary tract in a chart board using your innovation. Label
the parts (make a poster presentation material with flow of urine from kidney
to urethra).
7. P
repare a chart showing all sexually transmitted diseases.
Collect the disease photographs from the net.
8. Write a chart showing differences between pyogenic and aseptic meningitis.
212

Chapter 13
Immunology
Chapter Outline

13.2 Organs of the Immune System
13.3 Cells of Immune System
13.4 Immunity
13.5 Antigens
13.6 Antibodies
13.7 Antigen– Antibody Reactions
Non specific immunity or Innate immunity has four types
of defense barriers namely anatomical barriers, chemical
barriers, phagocytic barriers and inflammatory barriers.
Learning Objectives • To understand the properties of

antigen.
After studying this chapter the student
• To describe the basic structure
will be able,
and function of immunoglobulin
• To gain knowledge on the history of (antibodies).
immunology and Know the Nobel
• To explain the mechanism of antigen-
prize winners in immunology.
antibody interactions and their
• To know the structure and functions applications in clinical laboratory.
of primary and secondary lymphoid
organs.
• To know the cells of immune
system and understand the role of 13.1 Historical Background
granulocytes, mast cells, macrophages, Immunology is the study of immunity to
dendritic cells and lymphocytes. diseases. Immunology began as a branch
• To define immunity and Differentiate of Microbiology. Its origin is usually
between innate immunity and attributed to Edward Jenner who introduced
acquired immunity. variolation in 1796.
213

The success of vaccination enabled the stimulated the extension of Jenner’s strategy
World Health Organization to announce in of vaccination to other diseases.
1979 that small pox had been eradicated. Pasteur used attenuated culture and
Late in 19th century Robert Koch proved called it vaccine (Latin vacca, cow) in
that infectious diseases are caused by honour of Edward Jenner. Table 13.1 and
microorganisms. The discoveries of Koch Figure 13.1 list and shows the scientist who
contributed to the field of immunology.
Table 13.1: Scientists and their contributions to immunology

Year Name of the Contributions to immunology
Scientists
1796 Edward Jenner Discovered that cowpox or vaccinia,
induced protection against human
small pox
Discovery of humoral and cellular immunity
1890 Von Behring and Gave the first insights into the
Kitasto (von Behring mechanism of immunity
earned the Nobel Prize
in medicine in 1901)
1930’s Kabat Showed that gamma - globulin (now
immunoglobulin) a fraction of serum
exhibited the active component of
immunity
1883 Elie Metchnikoff He observed that certain white blood
cells, which he termed phagocytes,
were able to ingest microorganisms
and other foreign material
1903 Sir Almoth Wright Reported that antibodies could aid in

the process of phagocytosis. Wright
called these antibodies ‘opsonins’
1996 Claman, Chaperon and Discovered the presence and

Triplett cooperation of B cells and T cells
(Continued)
214

Year Name of the Contributions to immunology
Scientists
Specificity of immune response
Around 1900 Jules Bordet Demonstrated that nonpathogenic
substances, such as red blood cells
from other species, could also serve
as antigens
Karl Landsteiner Showed that injecting an animal
with almost any non-self, organic
chemical could induce production
of antibodies that would bind
specifically to the chemical
Molecular immunology
1959 Porter Separated fragments of
immunoglobulin
Edelman Heavy and light chains of antibodies
were separated by him
1965 Putnam, Hirschmann Discovered constant and variable
and Craig regions of immunoglobulin
1979 Kung et al. Described the first monoclonal
antibody identifying a T cell subset
1982-83 Allison et al and Isolated T cell receptor
Haskins et al.
Immunogenetics and Genetic Engineering
1936 Gorer Discovered the major
histocompatibility antigens
1968 McDevitt and Tyan Showed that immune response genes
were linked to the genes of the major
histocompatibility complex (MHC)
1974 Doherty and Reported that recognition of antigen
Zinkernagel by T cells was restricted by MHC
molecules
1978 Tonegawa et al. Demonstration of immunoglobulin
gene rearrangement
215

Infobits
Nobel Prizes for immunologic research
Year Recipient Country Research

1908 Elie Metchnikoff Russia Role of phagocytosis (Metchnikoff) and
Paul Ehrlich Germany antitoxins (Ehrlich) in immunity
1913 Charles Richet France Anaphylaxis
1919 Jules Bordet Belgium Complement-mediated bacteriolysis
1930 Karl Landsteiner United States Discovery of human blood groups
1972 Rodney R. Porter Great Britain Chemical structure of antibodies
Gerald M. United States
Edelman
1977 Rosalyn R. Yalow United States Development of radioimmunoassay
1980 George Snell United States Major histocompatibility complex
Jean Dausset France
Baruj Benacerraf United States
1984 Cesar Milstein Britain Technological advances in the development
George E. Kohler Germany of monoclonal antibodies
1991 E. Donnall United States Transplantation immunology
Thomas United States
Joseph Murray
2002 Sydney Brenner South Africa Genetic regulation of organ development
H. Robert Horvitz United States and cell death (apoptosis)
J. E. Sulston Great Britain
2008 Harald zurHausen Germany Role of HPV in causing cervical cancer
Françoise Barré- France (Hausen) and the discovery of HIV
Sinoussi Luc (Barré-Sinoussi and Montagnier)
France
Montagnier
2011 Jules Hoff man France Discovery of activating principles of innate
Bruce Beutler United States immunity (Hoff man and Beutler) and role
Ralph Steinman United States of dendritic cells in adaptive immunity
(Steinman)
216

Elie Metchnikoff karl Landsteiner Emil von Behring
Paul Ehrlich Robert Koch Niels K.Jerne
Jules J.B.V.Bordet Max Theiler Rodney R.Porter
Figure 13.1: Notable Scientists who contributed to the development of Immunology
13.2 Organs of the Immune System lymphoid organs serve as sites where
lymphocytes interact with antigen and
The immune system consists of structurally
undergo proliferation and differentiation
varied organs that are distributed
into antigen specific effector cells. The
throughout the body. Based on function,
spleen, lymph nodes and mucosal associated
the organs can be divided into primary and
lymphoid tissues (MALT) are secondary
secondary lymphoid organs (Figure 13.2).
lymphoid organs. These are discussed in
The primary lymphoid organs are
more detail below.
responsible for providing the appropriate
microenvironments for the development
13.2.1 Primary Lymphoid Organs
and maturation of antigen sensitive B and T
cells. The thymus is the primary lymphoid a. Thymus
organ for development of T cells and the The thymus is a highly organized
bone marrow is the primary lymphoid organ lymphoid organ located above the heart.
for development of B cells. The secondary The thymus consists of two lobes. Each
217

Medulla
Blood vessels
Cortex Tonsils
Thymus
(a) Thymus (site of T cell development)
Axillary lymph node
MALT (mucosal-associated
lymphoid tissue)
Breast, oral, respiratory,
gastrointestinal, and
genitourinary tract tissues
SALT (skin- Spleen
associated
lymphoid MALT (Peyer’s patches
tissue) in small intestine)
Inguinal lymph node
Bone marrow
Afferent lymphatic vessels
(b) Bone marrow

(site of B cell development)
A
Artery
Follicles Vein
V
Efferent lymph vessel
(c) Lymph node

(important site of T and B cell
interaction with antigens and
cells that present antigens)
Figure 13.2: The distribution of Lymphoid tissues in the body
lobe is surrounded by a capsule and is process known as thymic selection in which

divided into several lobules by strands of T cells that recognize host (self) antigens
connective tissue called trabeculae. Each are destroyed. The remaining 2% move into
lobule contains an outer cortex and an the medulla of the thymus, become mature
inner medulla. The cortex contains many T cells and subsequently enter the blood
dividing immature lymphocytes. The stream. These T cells recognize non host
medulla consists of reticular and epithelial (non self) antigens.
cells with fewer lymphocytes and isolated b. Bone marrow
Hassall’s corpuscles (Figure 13.3). The
In mammals, the bone marrow ( Figure 13.4)
primary function of the thymus is the
is the site of B cell maturation. Stromal cells
production of mature T cells. Precursor
within the bone marrow interact directly
cells from the bone marrow migrate into
with the B cells and secrete various cytokines
the outer cortex where they proliferate. As
that are required for B cell development. Like
they mature, about 98% die. This is due to a
218

Thymus
Capsule
Dead cell
Trabecula Thymocyte
Dividing thymocyte Nurse cell
Cortical epithelial
cell
Cortex
Medulla
Interdigitating
dendritic cell
Blood vessel
Medullary epithelial cell
Macrophage Hassall’s corpuscles
Figure 13.3: Diagrammatic Cross-section of a portion of the thymus
Spongy bone
Bone marrow
Epiphyseal
line Compact bone
Blood vessels
Figure 13.4: Structure of Bone marrow
thymic selection during T cell maturation, a The spleen is the most highly organized
selection process within the bone marrow secondary lymphoid organ. The spleen
eliminates non functioning B cells and those is a fist sized organ just behind the
bearing self reactive antigen receptors. In stomach. It collects and disposes of aged
birds, undifferentiated lymphocytes move red blood cells. Its organization is shown
from the bone marrow to the Bursa of schematically in Figure 13.5. The bulk of
Fabricius, where B cell mature; this is where the spleen is composed of red pulp which
B cells were first identified and how they is the site of red blood cell disposal. The
came to be known as “ B” (for bursa) cells. spleen is not supplied by lymphatic vessels.
The lymphocytes surround the arterioles
13.2.2 Secondary Lymphoid Organs entering the spleen, forming areas of white
a. Spleen pulp. The inner region of white pulp is
219

Capsule
Trabecula
Vascular Primary
sinusoid follicle
Marginal
zone White
Periarterial pulp
lymphoid
sheath
(PALS)
Red pulp Germinal center
Vein Artery
Figure 13.5: Structure of Spleen
divided into a Periarteriolar Lymphoid b. Lymph nodes

Sheath (PALS) containing mainly T cells. The lymph nodes are encapsulated round
The spleen filters the blood and traps blood structures located at the junction of
borne microorganisms and antigens. Once major lymphatic vessels. Lymph node is
trapped by splenic macrophages or dendritic morphologically divided into three regions:
cells, the pathogen is phagocytosed, killed the cortex, the paracortex and the medulla
and digested. (Figure 13.6). The outer most layer, the
Cortex
Paracortex
Medulla
Afferent
lymphatic
Germinal
vessels
centers
B lymphocytes Postcapillary
venule
Primary Capsule
lymphoid
follicle
Cross section post

Capsule
capillary venule
Germinal
centers
B lymphocytes
Efferent lymphatic
Lymphatic artery vessel
Lymphatic vein
Figure 13.6: Structure of Lymph node

220

cortex contains lymphocytes (mostly B class II MHC molecules by interdigitating
cells), macrophages and follicular dendritic dendritic cells in the paracortex, resulting
cells arranged in primary follicles. After in the activation of TH cells. Activated TH
antigenic challenge, the primary follicles cells release cytokines needed for B cell
enlarge into secondary follicles, each activation. Thus lymph nodes represent
containing a germinal centre. Beneath one environment where B cells differentiate
the cortex is the paracortex which is into memory cells and antibody – secreting
populated largely by T lymphocytes and plasma cells.
also interdigitating dendritic cells thought
to have migrated from tissues to the node. HOTS
These interdigitating dentritic cells express
high levels of class II MHC molecules, What happens when thymus is
which are necessary for presenting antigen removed from the human body?
to T helper (TH) cells. Lymph nodes are
specialized to trap antigen from regional c. MALT and SALT
tissue spaces. As antigen is carried into a The specialized lymphoid tissue in mucus
lymph node by the lymph, it is trapped, membranes is called mucosal associated
processed and presented together with lymphoid tissue (MALT). There are several
(a) (b)
Adenoids MALT
Tonsil
Thoracic duct
Thymus Left subclavian

vein
Right Lymph
lymphatic nodes
duct
Spleen
Right
subclavian
vein
Thoracic
duct
Peyer’s
Patches
Bone marrow
Large Villi
infestine Small intestine
Appendix
M cells
Lamina propria
Submucosa
Lymphocytes
High
endothelial Lymph
venule vessel
Tissue Lymphocytes Bcells

Tcells
lymphatics
Muscle layer
Germinal center
Peyer’s patch
Figure 13.7: Malt Mucosa Associated Lymphoid Tissue (MALT)
221

(a) The Peyer’s patch is a representative of the extensive MALT system that is found in
the intestine. (b) A stained tissue cross-section of Peyer’s patch lymphoid nodules in the
intestinal submucosa is schematically diagrammed in (c). The intestinal lamina propria
contains loose clusters of lymphoid cells and diffuse follicles.
(a) (b)
M cell Antigen Lumen
Antigen Intraepithelial
lymphocyte
Mucosal
epithelium IgA
TH cell M cell
Pocket IgA
Lamina Plasma
B cells propria cell
Organized
lymphoid
follicle
Macrophage
Figure 13.8: Structure of M cells and production of IgA:

(a) M cells, situated in mucous membranes, endocytose antigen from the lumen of the digestive,
respiratory, and urogenital tracts. The antigen is transported across the cell and released into
the large basolateral pocket. (b) Antigen transported across the epithelial layer by M cells at
an inductive site activates B cells in the underlying lymphoid follicles. The activated B cells
differentiate into IgA-producing plasma cells, which migrate along the lamina propria, the layer
under the mucosa. The outer mucosal epithelial layer contains intraepithelial lymphocytes, of
which many are T cells.
types of MALT. The system most studied is Antigen
the gut associated lymphoid tissue (GALT). Langerhans
GALT include the tonsils, adenoids, and cell

Keratinocytes Epidermis
appendix and specialized structures called

peyer’s patches (Figure 13.7) in the small Cytokines
Intraepidermal
lymphocyte
intestine, which collect antigen from the Inflammation
epithelial surfaces of the gastrointestinal Dermis

Lymphocytes
tract. In peyer’s patches, the antigen is Tissue Lymph vessel
collected by specialized epithelial cells called Lymphocytes
macrophage
M cells (Figure 13.8). The lymphocytes form

a follicle consisting of a large central dome of Lymph
B lymphocytes surrounded by small numbers Antigen-

presenting cell
containing Helper T cell
node
of T lymphocytes. Similar but more diffusely antigen
organized aggregates of lymphocytes protect

Cytokines released
the respiratory epithelium, where they are Antibody-
secreting cell
from T-cell influence
B-cell development
known as bronchial- associated lymphoid

tissue (BALT). B cell
Figure 13.9: SALT
222

Despite the skin’s defenses, at times Stem cells are cells that can differentiate into
pathogenic microorganisms gain access to other cell types. They are self renewing and
the tissue under the skin surface. Here, they they maintain their population level by cell
encounter a specialized set of cells called division. This chapter describes the formation
the skin associated lymphoid tissue (SALT) of blood cells and the properties of the
(Figure 13.9). The major function of SALT various cells of the immune system.
is to confine microbial invaders to the area
13.3.1 Hematopoiesis
immediately underlying the epidermis and
to prevent them from gaining access to Hematopoiesis is the formation and
the blood stream. One type of SALT is the development of blood cells of all types.
langerhans cell, a specialized myeloid cell In humans, hematopoiesis begins in the
that can phagocytose antigens. yolk sac in the first weeks of embryonic
development. As gestation continues, the
13.3 Cells of the Immune System site of hematopoiesis gradually shifts to
the bone marrow such that it becomes the
All blood cells arise from a type of cell
principle site at the time of birth.
called the hematopoietic stem cell(HSC).
Hematopoietic
stem cell
Self-
renewing
Dendritic cell Myeloid Lymphoid Natural killer

progenitor progenitor (NK) cell
Macrophage Monocyte
TH helper cell
Granulocyte T-cell
Neutrophil
monocyte progenitor progenitor
TC cytotoxic T cell
Eosinophil Eosinophil
progenitor
B-cell
progenitor B cell
Basophil Basophil progenitor
Dendritic cell
Platclets
Megakaryocyte
Erythrocyte
Erythroid progenitor
Figure 13.10: Hematopoiesis
223

As hematopoietic stem cells can give rise
Multilobed
to all of the different types of blood cells, Nucleus
Granules
they are often known as pluripotent stem
cells. (Figure 13.10)
Phagosome
The myeloid progenitor gives rise to Neutrophil
erythrocytes, neutrophils, eosinophils,

basophils, monocytes, mast cells and
platelets. The common lymphoid progenitor Glycogen
gives rise to B lymphocytes, T lymphocytes
and natural killer (NK) cells. Granule
13.3.2 Types of Leukocytes Basophil
The cells responsible for both innate

immunity and acquired immunity are the
leukocytes (Greek leukos, white and kytos Granule
cell). The average adult has approximately

7400 leukocytes (white blood cells) per Eosinophil
cubic millimeter of blood (Table 13.2). The
average value shifts substantially during Figure 13.11: Structure of granulocytes
an immune response. In defending the
host against pathogenic microorganisms, Table 13.2: Normal Adult Blood Count
leukocytes cooperate with each other first
Cell type Cells/ %
to recognize the pathogen as an invader
mm3 WBC
and then to destroy it. The different types of
leukocytes are now briefly described. Red blood cells 50,00,000 –
Platelets 2,50,000 –
a. Granulocytes Leukocytes 7,400 100
Granulocytes have irregularly shaped Neutrophil 4320 60
nuclei with two or five lobes. Their
Lymphocytes 2160 30
cytoplasm has granules that contain reactive
Monocytes 430 6
substances that kill microorganisms and
enhance inflammation. Three types of Eosinophils 215 3
granulocytes exist: basophils, eosinophils, Basophils 70 1
and neutrophils. Because of the many lobed b. Mast cells
(3-5) nuclei, neutrophils are also called
Mast cells are bone marrow derived cells that
polymorphonuclear neutrophils or PMNS
differentiate in the blood and connective
(Figure 13.11).
tissue.
HOTS c. Monocytes and Macrophages
Monocytes are mononuclear leukocytes.
Heard about stem cell treatment! They are produced in the bone marrow and
Why do we need stem cells bank? enter the blood, circulate for about eight
224

Lysosome
Nucleus
Phagosome
(a)
(b) (c)
Figure 13.12: (a) Structure of Monocytes (b) Phagocytosis by a Macrophage
(c) Dendritic Cell
hours, enlarge, migrate to the tissues and enhancement is termed opsonization.
mature into macrophages or dendritic cells Macrophages spread throughout the body
(Figure 13.12 a). and take up residence in specific tissues.
Macrophages are derived from monocytes Macrophages serve different functions in
and are classified as mononuclear phagocytic different tissues and are named according to
leukocytes. These microbial molecules are their tissue location.
examples of pathogen associated molecular • Alveolar macrophages in the lung
patterns (PAMPs) (Figure 13.12 c).
• Histiocytes in connective tissue
PAMPs enable macrophages to
• Kupffer cells in the liver
distinguish between potentially harmful
microbes and other host molecules. After the • Mesangial cells in the kidney
pathogen is recognized, the macrophages’, • Microglial cells in the brain
pattern recognition receptors (Example: Toll • Osteoclasts in bone
like receptors) bind the pathogen and
phagocytose it. Macrophages also have d. Dendritic cells
receptors for antibodies and complement Dendritic cells are not a single cell type.
proteins. Both antibody and complement They are a heterogeneous group of cells so
proteins can coat microorganisms named because of their Dendron (neuron)
and enhance their phagocytosis. This like appendages (Figure 13.12d). They arise
225

from various hematopoietic cell lineages. Memory B cells have a longer life span
Most dendritic cells are tissue bound, where than native cells. They express the same
they play an important role in bridging membrane bound antibody as their parent
innate immunity and acquired immunity. naive B cell. Plasma cells do not express
Dendritic cells can be classified by their membrane bound antibody. Plasma cells
location: secrete large quantities of antibodies.
Secreted antibodies are the major effector
• Langerhans cells found in the skin and
molecules of humoral immunity.
mucus membranes
ii) T Lymphocytes
• Interstitial dendritic cells which populate
most organs (heart, lungs, liver, kidney, T lymphocytes also arise in the bone
gastrointestinal tract) marrow. T cells then migrate to the thymus
to mature. During its maturation within
• Interdigitating cells present in T cell
thymus, the T cells express a unique a ntigen
areas of secondary lymphoid tissue and
binding molecule called the T cell receptor
the thymic medulla.
(Figure 13.13b) on its membrane. Unlike
• Circulating dendritic cells in the blood membrane bound antibodies on B cells,
and lymph. which can recognize antigen alone, T cell
receptor can recognize only antigen that
e. Lymphocytes
is bound to MHC molecules. There are
Lymphocytes are the major cells of the two major types of MHC molecules. Class
specific immunity. Lymphocytes can be I MHC molecules are expressed by all
divided into three populations: T cells, B nucleated cells. Class II MHC molecules are
cells, and NK (natural killer) cells. Clusters expressed only by antigen presenting cells.
of differentiation are group of monoclonal When a naive T cell encounters antigen
antibodies that identify the same cell combined with an MHC molecule on a cell
surface molecule. The cell surface molecule the T cell proliferates and differentiates into
is designated CD (cluster of differentiation memory T cell and various effector T cells.
followed by a number (CD1, CD2).
There are two subpopulations of T cells:
i) B Lymphocytes T helper (TH) and T cytotoxic (TC) cells.
B lymphocytes mature within the bone Although a third type of T cells called a T
marrow. When they leave bone marrow, each suppressor (TS) cell, has been postulated,
expresses a unique antigen binding receptor recent evidence suggests that it may not be
on its membrane. The B cell receptor is distinct from the TH and TC subpopulations.
a membrane bound antibody molecule T cells displaying CD4 function as TH cells
(Figure 13.13a). When a naive B cell, first whereas; those displaying CD8 function as
encounters the antigen that matches its TC cells (Figure 13.14).
membrane bound antibody, the binding of After a TH cell recognizes and interacts
the antigen to the antibody causes the cell to with an antigen-MHC class II molecule
divide rapidly. Its progeny differentiate into complex, the cell is activated. It becomes
memory B cells and effector B cells called an effector cell that secretes cytokines. The
plasma cells secreted cytokines activate B cells, TC cells,
226

B cell Receptor T cell Receptor
BCR TCR
recognition
Recognition
Light chain CD3 CD3
Heavy chain α β
ε δ γ ε
lgβ lgα
ITAM
ITAMa
Signaling ζ ζ
Signaling
Figure 13.13: (a) B cell receptor. (b) T cell receptor
(a) B cell (b) TH cell (c) TC cell
TCR CD4 TCR CD8
Antigen-
binding
receptor
(antibody)
Figure 13.14: Distinctive membrane molecules on lymphocytes
macrophages and various other cells that phagocytic granular lymphocytes that play
participate in the immune response. an important role in innate immunity. The
Under the influence of TH derived major NK cell function is to destroy cancer
cytokines, a TC cell that recognizes an cells and cells infected with microorganisms.
antigen-MHC class I molecule complex They recognize their targets in one of two
proliferates and differentiates into a ways. They can bind to antibodies that coat
cytotoxic T lymphocyte (CTL). Cells that infected or cancer cells. Thus the antibody
display foreign antigen complexed with a bridges the two cell types. This process is
class I MHC molecule are called altered self called antibody dependent cell mediated
cells. CTL destroy virus infected cells and cytotoxicity (ADCC) (Figure 13.15) The
tumor cells. second way that NK cells recognize infected
cells and cancer cells relies on the presence
iii) Natural killer (NK) Cells (Null cells) of specialized proteins on the surface of
NK cells are a small population of large, non
227

Antibodies Target cell infected
with virus
all nucleated host cells known as class II
Virus antigen
n MHC molecules. If a hosts cell loses this
+ MHC protein, as when some viruses or
cancers overtake the cell, the NK cells
(a)
kill it by releasing pore forming proteins
Antibody receptor
Target cell infected and cytotoxic enzymes called granzymes
with virus now coated
with antibody (Figure 13.16).
13.4 Immunity
NK cell
To establish an infection, an invading
(b)
microorganism must first overcome many
Target cell lysis surface barriers, such as skin, degradative
enzymes and mucus. These surface barriers
have either direct antimicrobial activity or
inhibit attachment of the microorganism
NK cell
to the host. Any microorganism that
(c)
penetrates these barriers encounters two
Figure 13.15: Antibody-Dependent levels of resistance: nonspecific resistance
Cell-Mediated Cytotoxicity mechanisms and the specific immune
response.
Natural
killer cell
Activating
ing Inhibiting
In
receptor receptor
Ubiquitous
MHC class I
molecule
molecule
Perforin
and
granzymes
Abnormal
cell lacking
Normal MHC class I
cell molecule
No attack Kill
(a) (b)
Figure 13.16: The system used by natural killer cells to recognize

normal cells and abnormal cells that lack the Major Histocompatibility
Complex Class I surface molecule
228

13.4.1 Types of Immunity part of the innate structure and function
The term immunity (Latin immunis, free of of each animal (such as skin, mucus and
burden) refers to the general ability of a host lysozyme). Innate immunity is the first
to resist infection or disease. There are two line of defence against any microorganism
interdependent components of the immune or foreign material encountered by the
response to invading microorganisms and vertebrate host. Innate immunity defends
foreign material. They are non-specific against foreign invaders equally and lacks
immune response or innate immunity or immunological memory.
natural immunity and specific immune II. Acquired immunity
response or acquired immunity or adaptive
Acquired immunity refers to the type
immunity.
of specific immunity that develops after
I. Innate immunity exposure to a suitable antigen (Figure 13.17).
Innate immunity refers to those general The effectiveness of acquired immunity
defence mechanisms that are inherited as increases on repeated exposure to foreign
Innate immunity
Physical barriers Cells Chemical barriers
Skin, mucous membranes pH, lipids, enzymes
Pattern recognition
molecules
PMN’s monocytes,
macrophages,
eosinophils,
NK cells
Cytokines Cytokines
Antibodies Cytokines
B cells T cells
Ag Specific receptors
Acquired immunology
Figure 13.17: The interrelationship between innate and acquired immunity
229

agents such as viruses, bacteria or toxins. ii) Mucous membranes
So acquired immunity has memory. The The mucous membranes of the eye
innate immunity and acquired immunity (conjunctiva), the respiratory, digestive and
work together to eliminate pathogenic urogenital systems withstand microbial
microorganisms and other foreign agents. invasion. One antibacterial substance in
Although innate systems predominate these secretions is lysozyme, an enzyme that
immediately upon initial exposure to lyses bacteria. Mucous secretions possess
foreign substances, multiple bridges occur the iron binding protein, lactoferrin.
between innate and acquired immune Lactoferrin sequesters iron from the plasma
system components. reducing the amount of iron available to
13.4.2 Mechanisms of Innate Immunity invading microbial pathogens and prevents
their ability to multiply. Mucous membranes
A potential microbial pathogen invading a produce lactoperoxidase, an enzyme that
human host immediately confronts a vast catalyzes the production of superoxide
array of nonspecific defence mechanisms. radicals, reactive oxygen intermediate that
Many direct factors (nutrition, physiology, is toxic to many microorganisms.
fever, age, genetics) and equally as many
indirect factors (personal hygiene, iii) Respiratory system
socioeconomic status, living conditions) Microbes smaller than 10μm pass through
influence all host microbe relationships. In the nasal cavity and are trapped by the
addition to these direct and indirect factors, mucociliary blanket and the trapped
a vertebrate host has the following four non microbes are transported by ciliary action
specific defence mechanisms. that moves them away from lungs. Coughing
and sneezing reflexes clear the respiratory
A. Physical barriers
system of microorganisms by expelling
B. Chemical mediators air forcefully from the lungs through the
C. Phagocytosis mouth and nose, respectively. Salivation also
D. Inflammation washes microorganisms from the mouth
and nasopharyngeal areas into the stomach.
A. Physical barriers
iv) Gastrointestinal tract
i) Skin
Most microorganisms that reach the
Intact skin contributes greatly to host
stomach are killed by gastric juice. (pH 2-3).
resistance. It forms a very effective
However, organisms embedded in food
mechanical barrier to microbial invasion.
particles are protected from gastric juice
Its outer layer consists of thick, closely
and reach the small intestine. The mucous
packed cells called keratinocytes, The skin is
membranes of the intestinal tract contain
slightly acidic (around pH 5-6) due to skin
paneth cells. These cells produce lysozyme
oil, secretion from sweat glands and organic
and cryptins (toxic for bacteria).
acids produced by commensal Staphylococci.
It also contains a high concentration of v) Genitourinary tract
sodium chloride and is subject to periodic Under normal circumstances, the kidneys,
drying. ureters and urinary bladder of mammals
230

are sterile. Urine within the urinary bladder B. Chemical mediators
is also sterile. In addition to removing • Antimicrobial peptides
microbes by flushing action, urine kills
They are low molecular weight proteins
some bacteria due to its low pH and the
that exhibit broad spectrum antimicrobial
presence of urea and other metabolic end
activity toward bacteria.
products (uric acid, hippuric acid, indican,
fatty acids, mucin, and enzymes). The acidic i) Cationic peptides
environment (pH 3-5) of the vagina is Cationic peptides are found in humans.
unfavorable to most microbes. There are three generic classes of cationic
peptides that have the ability to damage
vi) Eye
bacterial plasma membrane. Classes
The conjunctiva is specialized mucus of Cationic Peptides are Cathelicidins,
secreting epithelial membrane that lines Defensins and Histatin
the interior surface of each eyelid and the
exposed surface of the eye ball. It is kept ii) Bacteriocins
moist by the continuous flushing action Bacteriocins are produced by gram negative
of tears. Tears contain large amounts of and gram positive bacteria. For example,
lysozyme, lactoferrin, and antibody and Escherichia coli synthesize bacteriocins
thus provide chemical as well as physical called colicins. Colicins causes cell lysis.
protection (Figure 13.18).
Antimicrobial factors
in saliva (lysozymes, Lysozyme in
peroxidase, lactoferrin, tears and other
myeloperoxidase) secretions
Removal of
particles by
rapid passage
Commensals
of air over
turbinate
Mucus, cilia bones, hairs
Skin
physical barrier
fatty acids
Acid
commensals
Rapid pH Commensals,
change Paneth’s cells
Peristalsis
pH and
Flushing of
commensals
urinary tract
of vagina
Figure 13.18: Physical Barriers

231

• Cytokines Functions of complements
Cytokines are proteins made by cells that Some major functions of complements are:
affect the behavior of other cells. When • Opsonization and phagocytosis
released from mononuclear phagocytes, they
• Cell lysis
are called monokines. When released from
T lymphocytes they are called lymphokines. • Chemotaxis
When released from leukocytes they are • Activation of mast cells and basophils
called interleukins. Cytokines are required and enhancement of inflammation
for regulation of both the nonspecific and • Production of antibodies
specific immune responses. Interferons
(IFNS) are a group of cytokines produced • Immune clearance and inflamma-
by virus infected cells. Several classes tion by attracting macrophages and
of interferons are recognized. IFN γ is neutrophils.
synthesized by virus infected leukocytes, C. Phagocytosis
antigen stimulated T cells and natural killer i. Phagocytosis is the ingestion by
cells. IFN α / β is derived from virus infected phagocytic cells of invading foreign
fibroblasts. Interferons prevent viral particles such as bacteria. After
replication and assembly, thereby limiting ingestion, the foreign particle
viral infection. is entrapped in a phagocytic
Another group of noteworthy cytokines vacuole (phagosome), which
are endogenous pyrogens which elicit fever in fuses with lysosomes forming the
the host. Examples of endogenous pyrogens phagolysosome. The lysosomes
include interleukin – 1, I nterleukin – 6 and release their powerful lytic
tissue necrosis factor. All are produced by enzymes which digest the particle.
host macrophages in response to pathogens. (Figure 13.19). Phagocytosis is
• Complement system conducted by blood monocytes,
The complement system is a part of the neutrophils and tissue macrophages.
immune system, consists of a series of Phagocytosis may be enhanced
proteins that interact with one another by a variety of factors collectively
in a highly regulated manner, in order to referred to as opsonins which consist
eliminate pathogens. Complements are of antibodies and various serum
soluble proteins and glycoproteins mostly components of complement.
produced by hepatocytes. More than 20 ii. Phagocytic cells use two basic
types of complements are present in serum mechanisms for the recognition
found circulating normally in human body of microorganisms. Opsonin
in inactive forms (called as zymogens or dependent and opsonin independent
proenzymes). Complement activation is iii. Phagocytes use pathogen recognition
triggered by an antibody when it is bound to receptors to detect pathogen
the antigen. It can also be triggered by some associated molecular patterns on
components of innate immunity. Thus the microorganisms. Toll like receptors
complement system works in both innate are a distinct class of pathogen
and acquired immunity. recognition receptors.
232

1
Bacterium becomes
attached to membrane
evaginations called
pseudopodia.
2
Bacterium is ingested,
forming phagosome.
3
Phagosome fuses with
lysosome.
4
Bacterium is killed and
then digested by
lysosomal enzymes.
5
Digestion products are
released from cell.
(a) (b)
Figure 13.20: (a) Scanning electron micrograph of alveolar macrophage phagocytosis of

E. coli bacteria on the outer surface of a blood vessel in the lung pleural cavity. (b) Steps
in the phagocytosis of a bacterium.
D. Inflammation 2. An increase in capillary permeability

Tissue damage caused by a wound or by facilitates an influx of fluid and cells
an invading pathogenic microorganism from the engorged capillaries into
induces a complex sequence of events the tissue. The fluid that accumulates
collectively known as inflammatory (exudate) has much higher protein

response. Inflammation can either be acute content. Accumulation of exudate
or chronic. The gross features were described contributes to tissue swelling (edema)
over 2000 years ago and are still known as
Infobits
the cardinal signs of inflammation: redness
(rubor), warmth (calor), pain (dolor), Reactive Nitrogen Species: Highly
swelling (tumor), and loss of function cytotoxic antimicrobial compounds
(functiolaesa) formed by the combination of nitric
The cardinal signs of inflammation reflect oxide and superoxide anion within
the three major events of an inflammatory phagocytes such as neutrophils and
macrophages.
response.
Reactive Oxygen Species (ROS):
1. Vasodilation (an increase in the Highly reactive compounds such as
diameter of blood vessels) of nearby superoxide anion O2, hydroxyl radicals
capillaries occurs as the vessels that (OH)(OH–), hydrogen peroxide
carry blood away from the affected area (H2O2), and hypochlorous acid
constrict. This results in engorgement (HClO) that are formed from oxygen
of the capillary network. The engorged under many conditions in cells and
capillaries are responsible for tissue tissues, including microbe-activated
redness (erythema) and an increase in innate responses of phagocytic cells;
temperature. have anti-microbial activity.
233

3. Influx of phagocytes from the capillaries 2) Specificity
into the tissues is facilitated by increased The establishment of immunity by one
capillary permeability. As phagocytic organism does not provide protection
cells accumulate at the site and begin to against another unrelated organism. After
phagocytoses bacteria, they release lytic an attack of measles we are immune to
enzymes, which can damage nearby further infection but are susceptible to
healthy cells. The accumulation of dead polio or mumps viruses. Thus the body can
cells, digested material and fluid forms differentiate specifically between the two
substances called pus. organisms.
13.4.3 Acquired Immunity
3) Diversity
Lower animal forms possess so called innate The immune system is able to generate
or non-specific immune mechanisms such an enormous diversity of molecules such
as phagocytosis of bacteria by specialized as cellular receptors and soluble proteins,
cells. Higher animals have evolved an including antibodies that recognize trillions
adaptive or acquired immune response. of different foreign substances.
This acquired immune response provides a
flexible, specific and more effective reaction 4) Discrimination between self and
to different infections. nonself
The specific immune system almost
• Definition of Acquired (Adaptive)
responds selectively to non self and produces
Immunity
specific responses against the stimulus.
Acquired (adaptive)immunity refers to This is possible because host cells express
the type of specific immunity that a host a unique protein on their surface, making
develops after exposure to a suitable a ntigen. them as residents of that host or as self. Thus
• Important features of acquired the introduction of materials lacking that
immunity unique self marker results in their attack by
the host.
This is the immunity one develops
throughout life time. Adaptive or acquired 13.4.4 Humoral and Cellular Immunity
immunity has four important features Two branches or arms of specific immunity
namely (1) Memory (2) Specificity are recognized: humoral (antibody
(3) diversity and (4) discrimination between mediated) immunity and cellular (cell
self and non self. mediated) immunity (Figure 13.20).
1) Memory Humoral (antibody mediated) immunity
We rarely suffer twice from diseases such The antigen specific arm of the humoral
as measles, mumps, chicken pox, whooping immunity consists of the B cells. Each B cell
cough and so on. The first contact with an expresses a unique antigen binding receptor
infectious organism clearly imprints some on its membrane. The B cell receptor (BCR)
memory so that the body is effectively is membrane bound antibody molecule.
prepared to repel any later invasion by that When a naive B cell first encounters the
organism. antigen that matches its membrane bound
234

Antigens
Foreign Viruses Bacteria Parasites Fungi

Proteins
1
Intemalized antigen
digested by cell
2
Altered self-cell
presents antigen
Class II Class I
MHC MHC
TH cell TC cell
3
T cell receptors
recognize antigen bound
to MHC molecules
Activated 6
TH cell Activated CTLs
4 recognize and kill
Binding antigen-MHC altered self-cells
activates T cells
Cytotoxic T lymphocyte (CTL)
5
Activated TH cell secretes
Cell-mediated response cytokines that contribute to
activation of B cells, TC cells,
Humoral response and other cells
Antigen
7 8
B cell B cells interact with antigen Ab-secreting Antibody binds antigen
and differentiate into plasma cells and facilitates its clearance
antibody secreting plasma cells from the body
Figure 13.21: Overview of the humoral and cell-mediated branches of the immune
system. In the humoral response, B cells interact with antigen and then differentiate
into antibody-secreting plasma cells. The secreted antibody binds to the antigen
and facilitates its clearance from the body. In the cell-mediated response, various
subpopulations of T cells recognize antigen presented on self-cells. TH cells respond
to antigen by producing cytokines. TC cells respond to antigen by developing into
cytotoxic T lymphocytes (CTLs), which mediate killing of altered self-cells (Example:
virus-infected cells).
Passive Immuno- antibody, the binding of the antigen to the

therapy: Treatment of antibody causes the cell to divide rapidly.
an infectious disease Its progeny differentiate into memory
by administration B cells and antibody secreting plasma
of previously generated antibodies cells. A single plasma cell can secrete
specific for the infectious pathogen. more than 2000 molecules of antibody
per second. Circulating antibodies bind to
235

microorganisms, toxins and extracellular 13.5 Antigens
viruses, neutralizing them or tagging them
Substances capable of inducing a specific
for destruction by phagocytes and other
immune response are called antigens. The
mechanisms.
molecular properties of antigens and the
The cellular (cell mediated) immunity way in which these properties ultimately
consists of the T cells. Each T cell expresses contribute to immune activation are central
antigen receptors called T cell receptors to our understanding of the immune system.
(TCRS). Unlike membrane bound antibody
on B cells, which can recognize antigen alone, 13.5.1 Immunogenicity Versus
T cell receptors can recognize only antigen Antigenicity
that is bound to MHC molecules. There Two properties are exhibited by antigens;
are two major types of MHC molecules. they are immunogenicity and antigenicity.
Class I MHC molecules are expressed by all Immunogenicity is the ability of an antigen
nucleated cells. Class II MHC molecules are to induce a humoral and / or cell mediated
expressed only by antigen presenting cells immune response.
such as dendritic cells, macrophages and B B cells + antigen effector B cells (Plasma
cells. When a naive T cell encounters antigen cells) + memory B cells
combined with an MHC molecule on a cell,
T cells + antigen effector T cells (TC, TH
the T cell proliferates and differentiates
cells) + memory T cells
into memory T cells and various effector
T cells (helper T cells, cytotoxic T cells and Although a substance that induces a
regulatory T cells). Specific kinds of T cells specific immune response is usually called
directly attack target cells infected with an antigen, it is more appropriately called
viruses or parasites, transplanted cells or an immunogen. Antigenicity is the ability
organs and cancer cells. T cells can induce of an antigen to combine specifically with
target cell suicide (apoptosis), lyse targets the final products of the above responses.
cells, or release chemicals (cytokines) (antibodies and/or cell surface receptors).
that enhance specific immunity and non All immunogens are antigens but all
specific defences such as phagocytosis and antigens are not immunogens. Some small
inflammation. molecules called haptens are antigenic but
incapable, by themselves, of inducing a
13.4.5 Types of Specific Immunity specific immune response. In other words
Specific immunity can be acquired by haptens lack immunogenicity. Examples of
natural means actively through infection haptens are dinitrophenol, penicillin and
or passively through receipt of preformed m-amino benzene sulphonate.
antibodies as through colostrum. Specific 13.5.2 Factors that Influence
immunity can be acquired by artificial Immunogenicity
means actively through immunization or
Immunogenicity is not an intrinsic property
passively through receipt of preformed
of an antigen but rather depends on a number
antibodies as with antisera.
of properties of the particular biological
system that the antigen encounters. The
236

factors that influence immunogenicity can with proteins or polysaccharides (examples-
be divided under two categories. lipoprotein or nucleo - protein). For

1.
Contribution of the immunogen to example, attachment of tyrosine chains to
immunogenicity the weakly immunogenic protein gelatin
markedly enhances its immunogenicity.
2. Contribution of the biological system to
immunogenicity D. Susceptibility to antigen processing
and presentation
1. Contribution of the immunogen to
immunogenicity The development of both humoral and
Immunogenicity is determined in part, cell mediated immune responses requires
by the following four properties of the interaction of T cells with antigen that has
immunogen. been processed and presented together with
MHC (Major Histocompatibility Complex)
A. Foreignness molecules. To TH cells, the antigen must
The immune system normally discriminates be presented with class II MHC molecules
between self and non self, so that only on an antigen presenting cell; to TC Cells
molecules that are foreign to the host the antigen must be presented with class
are immunogenic. For example, albumin I MHC molecule on an altered self cell. 2.
isolated from the serum of a rabbit and Contribution of the biological system to
injected back into the same or another immunogenicity
rabbit will not induce an immune response
Even if a macromolecule has the properties
but the same protein when injected into
that contribute to immunogenicity, its ability
other vertebrate species (rat) will induce an
to induce an immune response will depend
immune response.
on the following properties of the biological
B. Molecular size system that the antigen encounters.
There is a correlation between the size of a
A. Genetic constitution of the host a nimal
macromolecule and its immunogenicity. The
best immunogens tend to have molecular The genetic constitution (genotype) of an
mass approaching 100,000 daltons (Da). immunized animal plays an important role
Generally, substances with a molecular in determining whether a given substance
mass less than 5000-10000 Da are poor will stimulate an immune response. Genetic
immunogens; however a few substances control of immune responsiveness is largely
with a molecular mass less than 1000 Da made by genes mapping within the MHC
have proven to be immunogenic.
B. Immunogen dosage and route of
C. Chemical composition and complexity administration
Proteins are the most potent immunogens Whether an immunogen will induce an
with polysaccharides ranking second. In immune response also depends on the dose
contrast, lipids and nucleic acids of an and mode of administration. A quantity of an
infectious agent generally do not serve as immunogen that has no effect when injected
immunogens unless they are complexed intravenously may evoke a good antibody
237

response when injected subcutaneously, coupling of a hapten to a large protein called
particularly if it is accompanied by an a carrier, yields an immunogenic hapten-
adjuvant. carrier conjugate.
C. Adjuvants 13.5.5 Cross-Reactivity

The response an immunogen is often When two antigens possess structurally
enhanced if it is administered as a mixture similar antigenic determinants, the
with adjuvants. Adjuvants are substances antibodies obtained to one of these antigens
that enhance the immunogenicity of an tend to react with the other antigen. These
antigen. Example: Freund’s incomplete reactions are called cross reactions.
antigen, Freund’s complete antigen,
Mycobacterium tuberculosis, Aluminum Infobits
potassium sulphate (alum) and Bacterial
Penicillin Allergy: New antigens are
lipopolysaccharide (LPS).
produced by altering epitopes. This can
be done by conjugating haptens to the
13.5.3 Epitopes
molecule. A classic example in human
Immune cells do not medicine is the allergic response of
interact with or recognize some persons to penicillin. A derivative
an entire immunogen of penicillin, penicilloic acid acting as
molecule instead; a hapten, can couple with body protein
lymphocytes recognize discrete sites on and elicit an immune response that
the macromolecule called epitopes or can be harmful, even life threatening,
antigenic determinants. Epitopes are the thus excluding this antibiotic from use
immunologically active regions of an in certain individuals.
immunogen that bind to antigen specific
membrane receptors on lymphocytes or to
secreted antibodies. Antigenic epitopes may 13.6 Antibodies
consist of a single epitope or have varying The first real chemical information
number of the same epitope on the same regarding the structure of antibodies was
molecule (Example: polysaccharides). provided by Tiselius and Kabat in the early
1940s. They demonstrated that the gamma
13.5.4 Haptens and the Study of globulin fraction of serum proteins that
Antigenicity migrated most slowly in electrophoresis
The pioneering work of Karl Landsteiner contained most of the serum antibodies.
in the 1920s and 1930s created a simple, This section deals with the structural
chemically defined system for studying and biological properties of antibodies
the binding of an individual antibody to (immunoglobulins).
a unique epitope on a complex protein
Definition of antibodies
antigen. Landsteiner employed various
haptens(small organic molecules that are Antibodies are glycoproteins present in
antigenic but not immunogenic). Chemical serum gamma globulins produced by
238

B-lymphocytes (B cells) or Plasma cells in approximately 25000 Da and are composed
response to exposure to antigen. Antibodies of about 220 amino acids. Light chains are
are also known as immunoglobulins. They common to all immunoglobulin classes
react specially with that antigen in vivo or and are of two types – kappa (κ) or lambda
in vitro and are hence a part of the adaptive (λ) - based on their structural differences.
immune response specifically, humoral A given immunoglobulin molecule may
immunity. contain either identical κ or λ chains but
13.6.1 Structure of an Immunoglobulin never both.
1. Basic unit b) Heavy chains
The basic structural unit (monomer) of an Heavy chains have a molecular weight
immunoglobulin molecule consists of four of approximately twice that of light
polypeptide chains linked covalently by chains (57000-70000 Da) and twice the
disulfide bonds (Figure 13.21). The four- number of amino acids (about 440). Five
chain structure is composed of two identical antigenically distinct isotypes of heavy
light (L) and two identical heavy (H) chains are recognized-gamma (γ), alpha
polypeptide chains. Every immunoglobulin (α), mu (μ), delta (δ) and epsilon (ε) – based
can be represented by the general formula on structural differences in the carboxy
(H2L2)n. terminal portion of heavy chains. The
heavy chains isotypes form the basis of five
a) Light chains classes of immunoglobulin molecules – IgG
Light Chains have a molecular weight of (contains γ chain), IgA (contains α chain),
Heavy chain Light chain

µ,γ,α,δ, or ∈ κ or γ
+ NH
NH
3
3 +
S
S
+ NH
3 +
NH
3
H
V
VH
S
S
S
L
VL
S
V
Hinge
S
CH
Hl
S
S
Antigen
C
l
S
S
–S binding
– -S
-S –S–S–
L
–
S
C
CL
–S
S
–S–S–
CO
S
–
S
4 –S–S– O–
21 C OO
CH2
CH2
Biological
–S–S– activity
CHO CHO
–S–S–
CH3
CH3
–S–S–
COO– COO–
Figure 13.25: Structure of Immunoglobulin
239

IgM (contains μ chain), IgD (contains δ 3. Regions
chain) and IgE (contains ε chain). Five Each heavy and light chain consists of
heavy chain classes of immunoglobulin can two segments, the variable region and the
be easily remembered as GAMDE. Heavy constant region. The variable (V) region
chain classes are again subdivided into shows a wide variation in amino acid
subclasses of molecules. sequence in the amino terminal portion of
i. Four known subclasses of the γ chain the molecule.
exist – γ1, γ2, γ3 and γ4 - which yield 4. Domains
IgG1, IgG2, IgG3 and IgG4.
Each immunoglobulin chain consists of
ii. Two subclasses of the α chain are a series of globular regions enclosed by
known – α1 and α2 - which yield disulphide bonds. Each heavy chain consists
IgA1 and IgA2. of four or five domains - one in the variable
iii. Two subclasses of the μ chain are region (VH ) and three or four in the constant
known – μ1 and μ2 - which yield region (CH1, CH2, CH3, and CH4). Each
IgM1 and IgM2. light chain consists of two domains – one
iv. No subclasses of the δ and ε (IgD and in the variable region (VL)and one in the
IgE) are known. constant region (CL).
2. Disulfide bonds 5. Fragments.
Disulfide bonds hold together the Proteolytic (peptide bond -splitting)

four polypeptide chains in normal enzymes such as papain and pepsin are used
immunoglobulin molecules and are of to degrade immunoglobulin molecules into
two types namely interchain bonds and definable fragments to facilitate study of
intrachain bonds. their structure Figure (13.22).
Disulfide L chain
bonds
-s-
s- -s-s-
-s- -s-s-
s-
F(ab)2
H chain Pepsin
digestion
- -s-
-s -s-s- s-
-s -s-s-
+
Fc fragments
Papain
digestion
Mercaptoethanol
reduction
Fab Fab
-s-
s- s- + + + +
-s-
-s-s-
-s-s- HS HS SH SH
SH SH L chains
Fc SH SH
H chains
Figure 13.22: Prototype structure of IgG, showing chain structure

and interchain disulfide bonds
240

6. Hinge region 2. It is present in blood plasma and
Hinge region is the portion of heavy chain tissue fluids. It has a monomeric
between the CH1 and CH2 domains. It is structure.
highly flexible and allows for movement 3. IgG class acts against bacteria and
of the Fab arms in relation to each other. viruses by opsonizing the invaders
The S values (sedimentation coefficient and neutralizing toxins and viruses.
that is expressed in Svedberg units(s)) of 4. IgG molecules are capable of fixing
immunoglobulins range from 7S- 19S. complement, except for IgG4.
13.6.2 Immunoglobulin Function 5. It is the major antibody in the
secondary immune response and it
There are three major effector functions
has half life of 23 days.
that enable antibodies to remove antigens
and kill pathogens. Opsonization promotes 6. IgG is the only immunoglobulin
antigen phagocytosis by macrophages and molecule able to cross the placenta
neutrophils. Complement activation by IgM and provides natural immunity in
and IgG can activate a pathway that leads to utero and to the neonate at birth.
the generation of a collection of proteins that ii) IgA
can perforate cell membranes. Antibody-
It is present in the serum and in various
dependent cell-mediated cytotoxicity
bodily secretions and thus takes two forms –
(ADCC) can cause NK cell mediated death
serum IgA and secretory IgA (sIgA)
of target cells when antibody bound to the
target cells associates with Fc receptors of A) Serum IgA
natural killer (NK) cells. 1. It accounts for about 12% of serum
immunoglobulin.
HOTS 2. In humans, over 80% of serum IgA
exists in a monomeric form and the
Which antibody protects the new born remaining existing as polymers in the
for few months against infections? form of dimers, trimers or tetramers.
In polymeric IgA, the monomeric
13.6.3 Properties and Activities of units are linked by disulphide bonds
Immunoglobulin Classes and joining (J) chain.
Each immunoglobulin class differs in its 3. Serum IgA fixes complement via the
general properties, distribution in the body alternative pathway. It has a half life
and interaction with other components of of 5 days.
the host defensive systems. B) Secretory IgA
i) IgG 1. Secretory IgA is the
primary immunoglobulin of
1. IgG is the major immunoglobulin in
mucosal associated lymphoid tissue
human serum, accounting for 80%
(MALT). It is also found in saliva,
of the immunoglobulin pool.
tears, and breast milk.
241

2. It consists of two monomeric units 2. It has a pentameric structure
plus J chain and secretory component consisting of five monomeric
(Figure 13.29). units linked by J chain and
3. The dominant subclass of sIgA is disulphide bonds at the Fc fragment
sIgA2 which is unique for its absence (Figure 13.23).
of a covalent bond between the light 3. It is the predominant antibody in
and heavy chains. In this subclass, the primary immune response to
light chains are linked by disulphide most antigens and the predominant
bonds. antibody produced by the fetus.
4. It has a half life of 5-6 days. It is 4. It is the first immunoglobulin
responsible for local immunity. made during B cell maturation
5. The sIgA molecules protect mucosal and individual IgM monomers are
surfaces by reacting with the expressed on B cells, serving as the
surface of potential pathogens and antibody component of the B cell
interfering with their adherence and receptor (BCR).
colonization. It also plays a role in 5. IgM tends to remain in the
the alternative complement pathway. bloodstream, where it agglutinates
(clumps) bacteria, activates
iii) IgM
complement by the classical pathway
1. IgM accounts for about 5-10% of the and enhances the ingestion of
serum immunoglobulin pool. pathogens by phagocytic cells.
Secretory
component
Heavy chain
Light chain
J Chain
lgM Secretory lgA
Figure 13.27: Structural models of IgM and secretory IgA. IgM has a pentameric structure
linked by J chain at the Fc fragment. Secretory IgA has a dimeric structure plus J chain
plus secretory component and is shown in the dominant IgA2 subclass, which is unique
for its absence of a convalent bond between the light and heavy chains. Light chains are
linked by disulfide bonds.
242

6. It has a half life of approximately monomeric structure. It is also called
5 days. reagin or reaginic antibody.
iv) IgD 2. The skin sensitizing and anaphylactic

antibodies belong to this class.
1. IgD accounts for about less than 1%
of the total immunoglobulin pool. 3. The Fc portion of IgE can bind to
Fc receptors specific for IgE that
2. One unique structural feature is the
are found on mast cells, eosinophils
presence of only a single H-H inter
and basophils. Thus these cells can
chain bond along with two H-L
become coated with IgE molecules.
interchain bonds.
When two cell- bound IgE molecules
3. It has a monomeric structure similar are cross linked by binding to the
to that of IgG. same antigen, the cells degranulate.
4. IgD antibodies are abundant in This degranulation releases
combination with IgM on the surface histamine and other mediators of
of B cells and thus are part of the inflammation.
B cell receptor complex. Therefore 4. IgE also stimulates production of an
their function is to signal the B cell excessive number of eosinophils in the
to start antibody production upon blood (eosinophilia) and increased
initial antigen binding. rate of movement of the intestinal
5. It has a half life of 2-3 days. contents (gut hypermotility) which
aid in the elimination of helminthic
v) IgE
parasites. IgE has a half life of 2-3
1. IgE accounts for only 0.004% of days.
serum immunoglobulin. It has a
Polyclonal Antibody: A mixture of antibodies produced by a variety

of B-cell clones that have recognized the same antigen. Although all
of the antibodies react with the immunizing antigen, they differ from
each other in amino acid sequence.
Breast Milk: Breast milk is uniquely suited to the human infant’s nutritional
needs and is a live substance with unparalleled immunological and anti-inflammatory
properties that protect against a host of illnesses and diseases for both mothers and
children. All five classes of immunoglobulins have been found in human milk, but by
far the most abundant type is IgA, specifically the form known as secretory IgA.
Antitetanus Serum: Antitetanus serum, also known as tetanus immune globulin
(TIG) is made up of antibodies against the tetanus toxin. It is used to prevent tetanus
in those who have a wound that is at high risk and have not been fully vaccinated
with tetanus toxoid. It is also used to treat tetanus along with antibiotics and muscle
relaxants. It is given by injection into a muscle.
243

Infobits
i) Isotype
Isotypic determinants are constant region determinants that collectively define each
heavy chain class and subclass and each light chain type and subtype within a species.
Each isotype is encoded by a separate constant region gene and all members of a species
carry the same constant region genes. Within a species, each normal individual will
express all isotypes in the serum. Different species inherit different constant region
genes and therefore express different isotypes. Therefore, when an antibody from one
species is injected into another species, the isotypic determinant will be recognized
as foreign, inducing an antibody response to the isotypic determinants on the foreign
antibody.
ii) Allotype
Although all members of a species inherit the same set of isotype genes, multiple
alleles exist for some of the genes. These alleles encode subtle aminoacid differences,
called allotypicdeterminnatsthat occur in some.
The unique amino acid sequence of the VH and VL domains of a given antibody can
function not only as an antigenic binding site but also as a set of antigenic determinants.
Therefore, the idiotypic determinants are generated by the conformation of the heavy
and light chain variable regions. Each individual determinant is called an idiotope
and the sum of the individual idiotopes is the idiotype. Anti-idiotype antibody is
produced by injecting antibodies that have minimal variation in their isotypes and
allotypes, so that the idiotypic difference can be recognized.
13.6.4 Antigenic Determinants on 13.7 Antigen – Antibody Reactions

Immunoglobulins
Antigen and antibody combine with each
Since antibodies are glycoproteins, they can other specifically and in an observable
themselves function as potent immunogens manner. The exquisite specificity of
to induce an antibody response. Such anti-Ig antigen-antibody interactions has led to the
antibodies are powerful tools for the study development of a variety of immunological
of B cell development and humoral immune assays. These assays can be used to detect
response. The antigenic determinants or the presence of either antibody or antigen.
epitopes, on immunoglobulin molecules These assays are also helpful in diagnosing
fall into three major categories: isotypic, diseases, monitoring epidemiological
allotypic and idiotypicdeteminants, which surveys and identifying molecules of
are located in characteristic portions of the biological or medical interest. Antigen-
molecule. antibody reactions in vitro are known as
serological reactions.
244

13.7.1 Three stages of Antigen – with its homologous antibody and
Antibody Reactions vice versa.
a) Primary stage 2. An entire molecule reacts and not
fragments.
The reactions between antigen and antibody
occur in three stages. The primary stage 3. There is no denaturation of the
is the initial interaction between the two antigen or the antibody during the
without any visible effects. This reaction reaction.
is rapid and obeys the general laws of
4. The combination occurs at the
physical chemistry and thermodynamics.
s urface.
The reaction is reversible. The combination
between antigen and antibody is effected 5. TBoth antigen and antibody
by the weaker intermolecular forces such participate in the formation of
as electrostatic forces, hydrogen bonds, Van agglutinates or precipitates.
der Waals forces and hydrophobic forces.
13.7.3 Measurement of Antigen and
b) Secondary stage Antibody
The primary stage is followed by the Many methods are available for the
secondary stage leading to demonstrable measurement of antigens and antibodies
events such as precipitation, agglutination, participating in the primary, secondary
lysis of cells, killing of live antigens, and tertiary reactions. Measurement
neutralization of motile organisms, may be in terms of mass (Example: mg
complement fixation and enhancement of Nitrogen) or more commonly as units or
phagocytosis. titre. The antibody titre of a serum is the
highest dilution of the serum which gives
c) Tertiary stage
an observable reaction with the antigen in
Some antigen-antibody reactions occurring the particular test. The titre of a serum is
in vivo initiate chain reactions that lead to influenced by the nature and quantity of the
neutralization or destruction of injurious antigen and the type and conditions of the
antigens or to tissue damage. These are test. Antigens may also be titrated against
the tertiary reactions and include humoral sera.
immunity against infectious diseases as well
The various tests used for detection of
as clinical allergy and other immunological
antigen and antibodies are given below:
diseases.
1. Precipitation tests
13.7.2 General Features of Antigen –
Antibody Reactions 2. Agglutintion tests
3. Complement Fixation test
Antigen-antibody reactions have the
following general characteristics: 4. Immunofluorescence
5. Radio immuno assay
1. The antigen-antibody reaction is
specific. An antigen combines only 6. Enzyme linked immuno sorbent assay
245

7. Western Blotting technique • Single Diffusion in One Dimension
8. Neutralization test (Oudin Procedure)
• Double Diffusion in One Dimensions
1. Precipitation reactions (Oakley-Fulthorpe Procedure)
When a soluble antigen combines with • Single Diffusion in Two Dimensions
its antibody in the presence of electrolytes (Mancini Procedure)
(NaCl) at a suitable temperature and pH,
• Double Diffusion in Two Dimensions
the antigen-antibody complex, forms an
(Ouchterlony Procedure)
insoluble (visible) precipitate and this
reaction is called precipitation. When Immunoelectrophoresis
instead of sedimenting, the precipitate Immunoelectrophoresis was devised by
remains suspended as floccules, the reaction Grabar and Williams (1953). This method
is known as flocculation. consists of two steps. The first step is agarose
• Applications of precipitation reactions electrophoresis of the antigen. Rectangular
trough is then cut into the agarose gel
The following types of precipitation tests are
parallel to the direction of the electric field
in common use:
and is filled with the antiserum. By diffusion,
a) Ring test lines of precipitation develop with each of
Examples of ring precipitation test are the separated compounds (Figure 13.24).
the C- r eactive protein test, Ascoli’s This method is used to detect normal and
thermoprecipitin and the grouping of abnormal serum proteins.
streptococci by the Lancefield technique.
b) Slide test
When a drop of antigen and a drop of 1
antiserum are placed on a slide and mixed by

shaking, floccules appear. The VDRL test for
syphilis is an example of slide flocculation. 2
c) Tube test
A quantitative tube flocculation test is used
for the standardization of toxins and toxoids. 3
Precipitation reaction in gels

There are several advantages in allowing 4
precipitation to occur in a gel rather than
in a liquid medium. The reaction is visible
as a distinct band of precipitation, which is 5
stable and can be stained for preservation,
if necessary. Imunodiffusion is usually
performed in 1% agarose gel. Different 6
modifications of the test are available.
Figure 13.28: Immunoelectrophoresis

246

1.
Semisolid agar layered on the glass b) Tube agglutination
slide. A well for antigen and a trough for This is a standard quantitative method for
antiserum cut out of agar.
measurement of antibodies. Widal test done
2. Antigen well filled with human serum.
3. Serum separated by electrophoresis. for typhoid and Weil Felix test done for
4. Antiserum trough filled with antiserum rickettsial infections are examples of Tube
to whole human serum. agglutination.
5. Serum and antiserum allowed to diffuse Latex agglutination test
into agar.
6. Precipitin lines form for individual Here latex particles are used as passive
serum proteins carriers for adsorbed soluble antigens.
• Counterimmunoelectrophoresis The most widespread application of latex
agglutination has been in the detection
• Rocket Electrophoresis
of rheumatoid factor. Latex agglutination
2. Agglutination reactions tests are also employed in the clinical
When a particulate antigen is mixed with its laboratory for detection of HBs Ag, ASO
antibody in the presence of electrolytes at a (Antistreptolysin O) and CRP (Carbohydrate
suitable temperature and pH, the particles Reactive Protein)
are clumped or agglutinated, and the Coombs test (antiglobulin test)
reaction is called agglutination. This test was devised by Coombs, Mourant
ADirect agglutination test and Race (1945) for the detection of
In the direct technique, a cell or insoluble anti-Rh antibodies that do not agglutinate
particulate antigen is agglutinated directly Rh-positive red blood cells in saline. The
by antibody. An example is the agglutination Coombs test may be of the direct or the
of group A erythrocytes by anti-A sera. indirect type.
Applications of coombs test
Indirect (Passive) agglutination test
1. Erythrocyte typing in blood banks.
Passive agglutination refers to agglutination
of antigen coated cells or inert p
articles (ben- 2. The evaluation of hemolytic disease
tonite or latex particles) which are passive of the newborn.
carriers of soluble antigens. An example is 3. The diagnosis of autoimmune
the latex agglutination for detection of rheu- hemolytic anemia.
matoid factor.
• Applications of agglutination
reactions
a) Slide agglutination
Slide agglutination is a routine test for
the identification of many bacterial isolates
from clinical specimens. It is also the
method used for blood grouping and cross
matching.
247

Summary neutrophils. They play a role in the defense
against parasitic organisms. Macrophages
Immunology began as a study of the response
and neutrophils are the accessory cells of
of the whole animal to infection. Over the
the immune system that phagocytose and
years, it has become progressively more
degrade antigens. Dendritic cells are antigen
basic, passing through phases of emphasis
presenting cells. They play an important
on serology, cellular immunology, molecular
role in TH cell activation by processing and
immunology and immunogenetics.
presenting antigen bound to class II MHC
The thymus and bone marrow are the molecules. Lymphocytes can be subdivided
primary lymphoid organs. The primary into B lymphocytes, T lymphocytes and
lymphoid organs provide sites for the null cells (NK cells). The two major
development and maturation of B and T subpopulations of T lymphocytes are T
lymphocytes. helper (TH) cells and T cytotoxic (TC) cells.
The secondary lymphoid organs Antibodies are a group of glycoproteins
function to capture antigen and provide present in the blood tissue fluids and
sites where lymphocytes interact with that mucous membranes of vertebrates. All
antigen and undergo clonal proliferation immunoglobulins have a basic structure
and differentiation into effector cells. composed of four polypeptide chains (two
The lymphatic system drains the tissue lights and two heavy) connected to each other
spaces and interconnects many organized by disulphide bonds. In any given antibody
lymphoid tissues. The spleen, lymphnodes molecule, the constant region contains one
and mucosal associated tissues (GALT and of five basic heavy chain sequences (γ, α,
SALT) are secondary lymphoid organs. μ, δ and ε) and one of two basic light chain
Lymph nodes are specialized to trap antigen sequences (k or γ). There are three major
from regional tissue spaces, whereas the effector functions of immunoglobulins
spleen traps blood- borne antigens. are Opsonization, Complement activation
The cells that participate in the and Antibody-dependent cell-mediated
immune response are white blood cells cytotoxicity (ADCC).
or leukocytes. All of the white blood cells
develop from a common pluripotent stem
cell in hematopoiesis. Lymphocytes are the
central cells of the immune system and are
responsible for acquired immunity. The
other types of white blood cells play ancillary
roles such as engulfing and destroying
microorganisms, presenting antigens and
secreting cytokines. Basophills and mast
cells are non phagocytic granulocytes that
play a role in allergic responses. Eosinophils
are motile phag ocytic cells. Their
phagocytic role is less important than that of
248

ICT CORNER
Immunology
How are we protected

from microbes?
STEPS:
• Use the link or Scan the QR code given below. “Cells Alive-Immunology” will
open. You can select any topic you wish. For example click “Making Antibodies”
• ‘Making Antibodies’ page will open. You can go through How ‘Lymphocytes
Produce Antibody’, ‘Antigen Processing’, etc….
• From the top of the page click on ‘Video’ and select ‘watch’ view video topics.
Select ‘Cytotoxic T Cells’.
• From the top select ‘Study’ and then ‘Quiz’ to answer the questions for the topic
you choose.
Step1 Step2
Step3 Step4
URL:
249

Evaluation B lymphocytes.
Multiple choice questions c. Erythrocytes, eosinophils, basophils,
monocytes, mast cells, platelets and
1. Who coined the term
T lymphocytes.
vaccine?
d. Erythrocytes, eosinophils,
a. Jenner
neutrophils, basophils, monocytes,
b. Pasteur
mast cells and NK cells.
c. Koch d. Roux
7. Which of the following is a correct
2. Who advanced the idea that immunity statement about NK cells?
was primarily due to white blood cells?
a. They kill target cells by phagocytosis
a. Metchinikoff b. Ehrlich and intracellular digestion.
c. Wright d. Kitasato b. They proliferate in response to
3. Which of the following does apply antigen.
uniquely to secondary lymphoid c. They kill target cells in an
organs? extracellular fashion.
a. Presence of precursor B and T cells. d. They are a subset of
b. Circulation of lymphocytes. polymorphonuclear cells.
c. Terminal differentiation. 8. Which of the following cells play an
d. Cellular proliferation. important role in the development of
allergies?
4. Which of the following is the major
a. Neutrophils b. Mast cells
function of the lymphoid system?
c. Monocytes d. Dendritic cells
a. Acquired immunity.
b. Innate immunity. 9. All of the following will act as opsonins
except
c. Inflammation.
a. Complement b. Antibody
d. Phagocytosis.
c. Acute – phase proteins
5. Lymph nodes taken from neonatally
d. Lactoferrin
thymectomized mice have unusually
few cells in the 10. Which of the following is not the
a. Paracortex b. Cortex important feature of acquired
immunity?
c. Medulla d. Thymus
a. Phgocytosis b. Memory.
6. The myeloid progenitor gives rise to
c. Specificity
a. Erythrocytes, neutrophils,
eosinophils, basophils, monocytes, d. Discrimination between self and
mast cells and platelets. non-self
b. Erythrocytes, eosinophils, basophils, 11. Cell mediated immunity is brought
monocytes, mast cells, platelets and about by
250

a. B cells b. T cells a. Immunogens b. Carriers
c. NK cells d. Null cells c. Epitopes d. haptens
12. Vaccines induce immunity that is 17. Basic structural unit of an
a. Naturally acquired active immunity. immunoglobulin molecule includes
b. Naturally acquired passive a. Identical λ light chains only.
immunity. b. One constant and three variable
c. Artificially acquired passive regions.
immunity. c. Two identical heavy and two
d. Artificially acquired active identical light chains.
immunity. d. A total of five domains.
13. Haptens 18. J chain is a glycopeptides chain
a. Require carrier molecules to be associated with which of the following
immunogenic. immunoglobulins?
b. Interact with specific antibody, even a. IgA b. IgG c. IgD d. IgE
if the haptens are monovalent. 19. Primary interactions between antigens
c. Cannot stimulate immune responses and antibodies involve all of the
without carriers. following except which?
d. All of the above. a. Van der Waals forces
14. The protection against small pox virus b. Hydrophobic forces
infection afforded by prior infection c. Electrostatic forces
with cowpox represents d. Covalent bonds
a. Antigenic specificity
20. When instead of the antigen, the
b. Antigenic cross reactivity.
antibody is adsorbed to carrier particles
c. Innate immunity.
in test for estimation of antigen, this
d. Passive protection. technique is known as
15. An adjuvant is a substance that a. Indirect agglutination
a. Enhances the immunogenicity of b. Direct agglutination
haptens. c. Reverse passive agglutination
b. Increases the chemical complexity d. Hemagglutination inhibition
of the immunogen.
c. Enhances the immune response to
the immunogen. 1. What is immunology?
d. Enhances the immunologic cross – 2. Define the term vaccination.
reactivity.
3. What are M cells?
16. Antigenic sites with which antibodies
react are called
251

4. What is the role of primary lymphoid 22. Briefly explain the various stages
organs and secondary lymphoid involved in phagocytosis.
organs?
23. Write short notes on interferons.
5. Define hematopoiesis.
24. Write short notes on primary immune
6. What are pluripotent stem cells? response/secondary immune response?
7. What is acquired immunity? 25. How do adjuvants function?
8. What is immunological memory? 26. Explain immunoglobulin structure and
function?
9. Define the term active/passive
immunity. 27. Describe briefly the structure and
function of thymus.
10. What is immunogenicity?
28. Describe the structure and function of
11. Define the term immunogen.
spleen/lymph node.
12. What are haptens?
29. Describe the characteristics of
13. What is antigenicity? macrophages
14. Define epitopes. 30. Write the characteristics of
B/T cells.
15. Define the term antibodies.
31. Briely explain the three major events in
16. What is opsonization?
the inflammatory response.
17. What is immunity/complement?
32. Briefly explain humoral immunity.
18. What is precipitation/agglutination?
33. Write an account of cell mediated
19. Write short notes on eosinophils/ immunity.
neutrophils.
34. Mention the properties of IgM.
20. Give a short account of natural killer
35. List out the general features of antigen-
cells.
antibody reactions.
21. How do intact mucous membranes
resist microbial invasion of the host?
252

Chapter 14
Microbial Genetics
Chapter Outline
14.1 Genetic Information is Stored in

DNA
14.2 Structure of DNA
14.3 DNA Replication
Microbial genetics provides powerful tools for

deciphering the regulation, as well as the functional and
pathway organization of cellular processes. The genetic
study of microbes has played a highly significant role in
the developments of Molecular Biology, Recombinant
DNA Technology and in the preparation of useful
products. Microbial Genetics makes microbes beautiful,
beneficial and bountiful.
Learning Objectives
After studying this chapter the student • To know Meselson and Stahl’s
will be able, experiment.
• To review the historical discoveries • To explain the steps of replication.
that led to establishing DNA as the • To know the enzymes and their roles
genetic material. involved in DNA replication.
• To identify the role of genetic
material. 14.1 Genetic Information is Stored
• To recognize the contributions in DNA
of Griffith, Avery, MacLeod, and Microorganisms are diverse in nature.
McCarty, and Hershey and Chase. A particular bacterium can be identified
• To explain the structure of DNA. based on certain characteristics. When a
• To recognize the contributions bacterial cell grows and divides, it gives
of Chargaff, Rosalind Franklin, rise to cells with similar characteristics.
Maurice Wilkins, Watson and Crick. Have you ever pondered as to why some
• To describe the Watson and Crick of the characteristics of progeny cells are
model of DNA. similar and a few dissimilar?
• To compare structure of DNA and In the middle of the 19th century it
RNA. was assumed that there was some particle
present somewhere in the cell which
253

was the controlling factor to carry the 1. Rough strain (R) – avirulent,
characteristics from one generation to non-capsulated strain, forming rough
another. colonies on culture media.
Genetics, a branch of science aims to 2. Smooth strain (S) – virulent,
understand the working of the controlling capsulated strain (resists phagocytosis),
factor. The factor governing the transfer forming smooth colonies on culture
of information is now very well known media.
as Gene. Gene can be defined as a unit of Griffith injected live smooth strain into
heredity which is transferred from parent mice which caused disease and killed
to progeny. it. When he injected live rough strain
Although there were experiments into mice, it did not cause disease and
to prove the inheritance pattern due to mice remained alive. He heat killed the
gene, there was no real understanding smooth strain and injected into mice, the
of the molecular nature of the gene. mice remained alive. But the experiment
Work of Frederick Griffith introduced gave surprising results when a mixture
the transforming principle which was of harmless live rough strain and heat-
further confirmed as Deoxyribonucleic killed smooth strain was injected
acid (DNA) by experiments of Avery, Mac into mice. Griffith observed that the
Leod and Mac Carty in 1944 followed by mice developed pneumonia and died.
Hershey and Chase in 1952. Further, when he analysed the blood
sample from dead mouse, he found that
14.1.1 Frederick Griffith’s Experiment
it contained live smooth strain. This
In 1928, British accidental discovery made Griffith to
bacteriologist conclude that the rough strain changed
Frederick Griffith (transformed) into smooth strain by
(Figure 14.1) was taking up a substance which he called a
trying to develop “transforming principle” from heat killed
a vaccine against smooth bacteria. This phenomenon
Figure 14.1:
pneumonia. In his is called “Bacterial Transformation”
Frederick Griffith
experiments Griffith Griffith’s experiment is summarized in
used two related strains of Streptococcus Figure 14.3.
pneumoniae (Figure 14.2).
Figure 14.2: Rough and Smooth colonies of Streptococcus pneumoniae

254

Live rough Live smooth Heat-killed Heat killed smooth strain and
strain strain smooth strain live rough strain
In blood sample,
living S cells are
found that can
reproduce,
yielding more S
cells
Mouse healthy Mouse dies Mouse healthy Mouse dies
Figure 14.3: Summary of Griffith’s experiment
(Figure 14.4) used the extracts of

HOTS heat-killed smooth bacteria and treated
it with enzyme protease, RNase, DNase
What did Griffith expect to happen to eliminate proteins, RNA and DNA
to mouse when he injected it with live respectively. Each of the treated extracts
rough strain and heat killed smooth were mixed with live rough bacteria and
strain injected into mice. The mice injected with
a mixture of DNase treated extract and
14.1.2 Oswald T. Avery, Colin live rough strain did not die. This partially
Mac Leod and Maclyn Mc Carty’s proved that DNA was responsible for
Experiment changing the rough strain of Streptococcus
pneumoniae bacteria into smooth bacteria.
Avery et al., experiment is summarized
in Figure 14.5. Later Hershey and
Chase’s experiment on T2 bacteriophage
confirmed that genetic information is
present in DNA.
These important early experiments and
many other lines of evidence have shown
that DNA bears the genetic information of
Figure 14.4: Avery et al., living cells and it is responsible for transfer
Griffith’s experimental results led to the of characteristics from one generation to
curiosity to explore the transforming another. This is true in all organisms, the
principle. Avery and his colleagues notable exceptions being RNA viruses
255

Extract (containing protein, RNA, DNA) of heat-killed smooth strain of
Streptococcus pneumoniae
Treated Protease RNase DNase

with
+ + +
Live rough strain Live rough strain Live rough strain
Mouse dies Mouse dies Mouse alive
S cells present: S cells present: S cells absent:

therefore, transformation therefore, transformation therefore, transformation
occurred. Transformation occurred. Transformation did not occur.
occurs in the absence of occurs in the absence Transformation does
proteins. of RNA. NOT occur
WITHOUT DNA.
Figure 14.5: Avery, Mc Cleod and Mac Carty’s experiment
Infobits
256

The entire genetic content of a cell is known as its genome and
the study of genomes is genomics. In eukaryotic cells, but not in
prokaryotes, DNA forms a complex with histone proteins to form
chromatin, the substance of eukaryotic chromosomes. A chromosome
may contain tens of thousands of genes. Many genes contain the information to make
protein product. DNA controls all of the cellular activities by turning the genes “on”
or “off.”
Types of Nucleic acids
DNA RNA
dsDNA Genetic RNA Non-genetic

ssDNA
Example: E.coli RNA:
Example:
Lamda (λ) mRNA, rRNA,
ØX174 phage dsRNA ssRNA
phage tRNA
Example: Example:
Rheovirus TMV
which store genetic information in RNA. • The sugar present in DNA is

The understanding of DNA’s role in deoxyribose sugar.
heredity has led to variety of practical • The nitrogenous bases present in DNA
applications including forensic analysis, are
paternity testing and genetic screening.
* Purines – Adenine (A), Guanine (G)
14.2 DNA Structure * Pyrimidines – Thymine(T),
• DNA is a polymer of simple monomeric Cytosine (C)
units, the nucleotides (Figure 14.6). • The nucleotide as a unit is formed by
• Each nucleotide is made up of three * Glycosidic bond between
components: nitrogenous base and sugar,
1. Nitrogenous base * Ester bond between phosphate
2. Sugar group and sugar
• Each of the nucleotides is bonded
3. Phosphate group
by a phosphodiester bond to form
• Nucleotide without phosphate group is a polynucleotide chain (strand)
known as nucleoside. (Figure 14.7a).
257

5′ end
Sugar
5′ 5′ 5′
Phosphate O O
HOCH2 OH HOCH2 OH
0
P 4′ C H H C 1’ 4′C H H C1’
1′ 3′ 2′ 3′ 2′
Base S B H H H H
4′ Sugar C C C C
OH H OH OH
Nucleotide
Deoxyribose Ribose
3′ 2′
3′ end O O
NH2
4
CH3
Nucleotide 3N 5 H N H N
N
5′ 2
N
6
O N O N O N
1
H H
0 S B
H
Pyrimidine Uracil (U) Thymine (T) Cytosine (C)
RNA only DNA only both DNA and RNA
1′
4′ Sugar Base Nucleoside NH2 O
6 7
1N 5 N
N N
P P P N H N
8
2 N9
3′ 2′ S B N 4
N N N
N
3 H2N
H
3′ end H H
Nucleoside triphosphate
Nucleoside Purine Adenine (A) Guanine (G)
Figure 14.6: Structure of nucleotide, nucleoside, deoxyribose, ribose and nitrogenous bases
• Two polynucleotide chains join variety of sequences that can be made from
together through hydrogen bonds the four nitrogenous bases is limitless, as
between nitrogenous bases, to form is the number of melodies possible with
double stranded DNA (Figure 14.7b). a few musical notes. RNA differs from
• Two hydrogen bonds exists between DNA by having a ribose sugar instead of
adenine and thymine and three deoxyribose and nitrogenous base Uracil
hydrogen bonds between guanine and instead of Thymine.
cytosine.
14.2.1 Watson And Crick Model of
• DNA is coiled in the form of a double DNA Double Helix
helix, in which both the strands of DNA
In the early 1950’s, Rosalind Franklin and
coil around an axis (Figure 14.7d & e).
Maurice Wilkins used the powerful method
• The further coiling of this axis upon
of X-ray diffraction to shed more light on
itself produces DNA supercoiling an
the structure of DNA. From the X-ray
important property of DNA structure.
Diffraction pattern it was deduced that
All DNA, whether large or small, possess DNA molecules are helical. In 1953 Watson
the same sugar phosphate backbone. What and Crick (Figure 14.8) postulated a three
distinguishes one DNA from another is dimensional model of DNA structure based
the length of the polymer and distribution on Franklin’s X-ray crystallographic studies.
of four bases along the backbone. The In recognition of their work leading to
258

5′ 3′
C
P
C G
Deoxyribose 5′
C G
3′ A T
A
P A T
C G
5′
Polymer
G C
Phosphodiester
3′ G C
G A T
P
bond
5′ C G
T A
T A
3′
T
P T A
5′ Nucleotide
3′ 5′
a) DNA polynucleotide chain b) Two DNA strands c) DNA ladder d) DNA e) DNA around
bonded by hydrogen double a central axis
bonds helix
Figure 14.7: Structure of a single polynucleotide chain of DNA, hydrogen bonding

between two DNA strands, Double helix around axis
the double helix model, Nobel prize was

awarded in 1962 to Watson, Crick and
Wilkins. According to Watson and Crick
model (Figure 14.9),
• The DNA consists of two helical
polynucleotide chains wound around the
same axis to form a right handed helix.
• The Purine and Pyrimidine bases of
Figure 14.8: Watson and Crick
both strands are stacked inside the
double helix. Figure with
14.8:their model
Watson and Crick
• Each nitrogenous base of one strand is and three hydrogen bonds are present
paired in the same plane with a base of between G and C (G C).
the other strand. • The hydrogen bonds provide chemical
• According to Watson and Crick stability essential to hold the two
rule Adenine base pairs with chains together.
Thymine and Guanine base pairs • The specific A equal to T and G
with Cytosine. equal to C base pairing is the basis
• Two hydrogen bonds are present for the complementarity concept.
between A and T (symbolised as A=T) This complementarity concept is very
259

5′ 3′
Diameter C G
20Å
T A
Minor
Groove G C
One complete
turn 34Å
A T
C G
Sugar-phosphate
backbone
A T
Major
groove 3′ 5′
Nitrogenous
base pair Antiparallel complementary
Strands
3.4 Å 5′ TACA 3′
Watson and Crick
DNA model
3′ ATGT 5′
Central axis
Figure 14.9: Watson and Crick DNA model, Antiparallel dsDNA
important in the process of DNA Chargaff measured the quantity of the

replication and gene expression. bases in DNA and noticed that the number
• The pairing of two strands creates a of Adenine is equal to the number of
major groove and minor groove on the Thymine and the number of Guanine is
surface of the duplex. equal to the number of Cytosine residues.
Hence the sum of Purine residues equal to
• The two strands are antiparallel, that is
the sum of the Pyrimidine residues.
their 5ʹ, 3ʹ phosphodiester bonds run
in opposite directions. Quantitatively A=T or A/T = 1
C=G or C/G = 1
• The vertically stacked bases are 3.4 Aº
A+G = T+C
apart.
• Each complete turn of double helix con-
HOTS
tain base pairs, which is 34 Aº units long.
14.2.2 Erwin Chargaff ’s Rule If percentage of adenine in one of the
In the late 1940s Erwin Chargaff and his DNA strand is 20. Can you determine
colleagues found that the four nucleotide the percentage of other bases. If yes
bases of DNA occur in different ratios in how?
the DNA of different organisms. Erwin
260

14.2.3 Alternative Forms of DNA Table 14.1: Properties of different forms
DNA is a remarkably flexible molecule. of DNA
Considerable rotation is possible around A form B form Z form
a number of bonds in sugar-phosphate
backbone and thermal fluctuations can Helical Right Right Left
produce bending, stretching and unpairing sense handed handed handed
of the strands. Watson and Crick model Diameter ∼ 26Aº ∼ 20 Aº ∼ 18 Aº
of DNA is called as B-DNA or B-form.
However DNA can exist in A or Z form. Base pairs 11 10 12
In 1979 Alexander Rich discovered Z form per helical
(Figure 14.10). Recently, several alternative turn
forms of DNA have been discovered C-form,
Distance 2.6 Aº 3.4 Aº 3.7 Aº
D-form and E-form. The B-form of DNA is
between
the most stable structure and is therefore
adjacent
the standard point of reference in any study
bases
of the properties of DNA (Table 14.1).
Bacterial genome size
• Bacterial genomes are typically expressed in Mb

• he length of Bacterial genomes are typically in the mm range
T
and therefore 1000X bigger than the typical bacterial size.
• The mass of Bacterial genomes are typically in 10−3 pg(picogram) range.
Conversions
1 Kb = 1 03 bp 1 bp ≈ 0.33nm 1 pg = 10−12 g
(base pairs) 1 kb ≈ 0.33μm 1pg = 978 Mb
1 Mb = 10 bp 6
1 Mb ≈ 0.33mm Number of base pairs =
1 Gb = 10 bp 9
1 Gb ≈ 0.33m mass in pg × (0.978 × 109)
1 kb ≈ 10−6 pg
1 Mb ≈ 10−3 pg
1 Gb ≈ 1 pg
Bacteria Virus, organelles Eukaryotes

Genomes Small(Mb) Tiny (Kb) Large (Gb)
Gene Density High High Low
Example E.coli 5000 genes Bacteriophages Homo sapiens
10-100 genes 25000 genes
261

14.3 DNA Replication
DNA is a marvelous device for the
stable storage of genetic information.
DNA replication is a process in which
copies of DNA molecules are faithfully
made. Here double stranded DNA
molecule is copied to produce two
identical dsDNA molecules. Replication
is an essential process because whenever
a cell divides, the two new daughter cells
must contain the same genetic information
Figure 14.10: Forms of DNA
in DNA as parent cell. DNA replication
occurs during the S (DNA synthesis)
phase and precedes cell division.
HOTS Watson and Crick proposed the
hypothesis of semiconservative replication.
Write the base sequence of According to them each DNA strand serves
complementary DNA and RNA strand as a template for the synthesis of a new
for the following. strand, producing two new DNA molecules
5ʹGCGCAATATTTCT3ʹ each with one old strand and one new
strand. (Figure 14.11).
Infobits
Max Delbruck suggested that there

Semiconservative +
could be three possible ways in which
DNA could replicate.
Semiconservative – DNA replication
that produce two copies of double Conservative +
stranded DNA (dsDNA) each containing

one old strand and one new strand.
Conservative – DNA replication that Random dispersive +
produces two daughter ds DNAs, one of
which consists of two original strands
Figure 14.11: Semiconservative,
whereas the other daughter DNA consists
conservative and dispersive replication
of two newly synthesised strands.
Dispersive – DNA replication in which the original dsDNA undergoes
fragmentation, the fragments synthesize complementary structure both of which
assemble to form two replicas.
262

14.3.1 Meselson and Stahl’s Experiment gradient centrifugation (Caesium
Mathew Meselson and chloride (CsCl) centrifugation).
Franklin Stahl in 1957 gave Expected results
experimental evidence for
• The heavy isotope 15N containing
semiconservative replica-
DNA, will reach equilibrium in a
tion process.
gradient point closer to the bottom of
Steps the tube.
1. E.coli cells were grown for many • 14N containing DNA will reach
generations in a medium containing equilibrium at a gradient point closer
radioactive isotope (heavy isotope) of to the surface of the tube.
nitrogen source 15N. Observed Results
2. After many generations all nitrogen
• After first generation the isolated DNA
containing molecules in E.coli cells,
occupied an intermediate density band.
including nitrogen bases of DNA
contained 15N. • After second generation two bands
3. The cells labeled with 15N were then were observed, one at intermediate
transferred to a medium containing density and the other at lighter density
only 14N (light isotope). Hence all corresponding to the 14N position in
subsequent DNA synthesised during the gradient.
replication contained 14N. These experimental results and other
4. Cell samples were removed at periodic experiments repeated by Meselson
time intervals from the growth medium. and Stahl with prokaryotes suggested
5. From each of the above samples, DNA that semiconservative mechanism of
was isolated and subjected to density replication is universal (Figure 14.12).
DNA replication is semiconservative

Observed results Predictions
Semiconservative Conservative Dispersive
Grow bacteria in 15N One band
Stock cuiture
Centrifuge sample
Parental
15
N DNA
in CsCI
Heavy DNA
15
N
Transfer cells to 14N
grow for one generation
One band
Grow 20 minutes
Centrifuge sample First

in CsCI Hybrid DNA replication
15
14
N
N–14N
Grow for second

generation in 14N Two bands
Light DNA Second

Grow 40 minutes 14
N replication
Centrifuge sample
in CsCI
Hybrid DNA
15
N–14N
14
14 NN
Figure 14.12: Meselson’s and Stahl’s experiment

263

14.3.2 Enzymes Involved in DNA Initiation
Replication • DNA replication is initiated at
DNA replication in E.coli requires many replication origin known as oriC (245
enzymes and proteins, each performing a base pairs in E.coli).
specific task. The entire complex is called • Dna A protein molecules bind to the
the DNA replicase system or replisome. The origin of replication.
major enzymes and proteins involved with • Helicase (DnaB ) denatures the DNA
their functions are tabulated in Table 14.2. helix by breaking the hydrogen bonds
between base pairs.
Table 14.2: Enzymes involved in DNA
replication • Many molecules of SSB (single stranded
binding) proteins bind cooperatively
Enzyme Function
to single stranded DNA, stabilizing
Helicase Unwinds DNA
the separated strands and preventing
DNA gyrase Relieves stress created by
renaturation.
unwinding
SSB protein Binds to single stranded • Gyrase (topoisomerase) releases the
DNA and stabilises it topological stress produced by helicase.
Primase Synthesis of RNA primer • RNA primers are synthesised by
DNA pol I Excision of primers primase.
and filling of gaps with The separated polynucleotide strands
nucleotides are used as templates for synthesis of
DNA pol III New strand elongation complementary strands. The area of the
DNA ligase Joins the nick DNA opened by helicase for DNA synthesis
is referred to as the replication fork. At the
Infobits replication fork there are four strands of
DNA, two are conserved and two are newly
• First in vitro synthesis of DNA synthesised. Replication may occur in either
with a template was carried out by a unidirectional or bidirectional manner
Kornberg in 1959. (Figure 14.13) from each origin. Bidirectional
• First in vitro synthesis of DNA replication can be explained as two replication
without template was carried out forks moving in opposite directions around
by H.G. Khorana in 1965. the circular chromosome. Both forks move
along the double helix away from the origin
of replication in opposite directions and
14.3.3 Events in DNA Replication in E.coli around the circular chromosome.
1. Initiation Elongation
2. Elongation • DNA synthesis proceeds in a 5ʹ3ʹ
direction( read as 5 prime to 3 prime).
3. Termination
• One strand is synthesised continuously
DNA replication is depicted in
and is known as leading strand.
Figure 14.14.
264

Unidirectional replication Bidirectional replication
Origin of Origin of
replication Movement of replication
replication fork Movement of
replication fork
Figure 14.13: Unidirectional and bidirectional replication

3′
5′ 3′ 5′
Newly
Synthesized
DNA strand 3′ 3′ 5′
3′
Direction of
5′ Replication fork 5′
Okazaki fragment 5′
Removal of RNA Primers
Lagging strand Leading strand
3′ 5′
5′ 3′ 5′ 3′
5′
3′ 5′ 3′ 5′ 3′ 5′ 3′
5′ 5′ Gaps between okazaki fragments
5′
Okazaki fragment filled by DNA polymerase
a) DNA Replication 5′
3′
5′
5′ 3′ 3′
O 5′ 3′ 5′ DNA fragments sealed by

3′ P O– OH 5′ DNA Ligase
O 5′
5′ 3′
3′
O
3′ P O 5′
b) Removal of RNA Primers
O
c) Action of DNA Ligase
Figure 14.14: a) DNA replication b) Removal of RNA primers

c) Action of Ligase and
• Other strand is synthesised Nobel Prize for discovering DNA
discontinuously and is known as polymerase in 1956.
lagging strand. • There are three known enzymes in
• The enzymes that are able to Escherichia.coli viz., DNA polymerase
synthesise new DNA strands on I, II and III.
a template strand are called DNA • All of the known DNA polymerases
polymerases. Kornberg was awarded can extend a deoxyribonucleotide
265

chain from a free 3ʹOH end, but none Okazaki fragments named after R. Okazaki.
can initiate synthesis. Okazaki fragments range in length from a
• Synthesis of DNA requires nucleoside few hundred to a few thousand nucleotides
triphosphates or nucleotides - depending on the cell types. Each okazaki
deoxyadenosinetriphosphate (dATP), fragment must be initiated by the action
deoxythymidinetriphosphate (dTTP), of primase. Once an Okazaki fragment has
deoxycytidinetriphosphate (dCTP), been completed its RNA primer is removed
deoxyguanosinetriphosphate (dGTP). and replaced with DNA by DNA polymerase
When a nucleoside triphosphate bonds I and the nick is sealed by DNA ligase.
to sugar in a growing DNA strand, it The fidelity of DNA replication is
loses two phosphates. maintained by (1) base selection by the
• RNA primer synthesised during polymerase, (2) a 3ʹ5ʹ proofreading
initiation is removed and replaced exonuclease activity that is part of most
with DNA by DNA polymerase I DNA polymerases, and (3) specific repair
systems for mismatches left behind after
• Sealing of the nick by DNA ligase which
replication.
catalyses the formation of phosphodiester
bond between a 3ʹ hydroxyl end of one
Infobits
DNA fragment and 5ʹ phosphate at the
end of another strand. RNA dependent DNA polymerases,
The elongation phase of replication also called reverse transcriptases, were
includes two distinct but related first discovered in retroviruses, which
operations convert their RNA genomes into
• Leading Strand Synthesis double-stranded DNA as part of their
life cycle. These enzymes transcribe
• Lagging Strand Synthesis
the viral RNA into DNA, a process
Leading strand synthesis begins with the that can be used experimentally to
synthesis of short (10 to 60 nucleotide form complementary DNA.
long) RNA primer at the replication origin.
Deoxyribonucleotides are added to this Termination
primer by DNA polymerase III. Leading Eventually, the two replication forks of
strand synthesis then proceeds continuosly, the circular E.coli chromosome meet at a
keeping pace with the unwinding of DNA at terminus region called Ter (for terminus).
the replication fork. The continuous strand The Ter sequence function as binding
or leading strand is one in which 5ʹ3ʹ sites for protein Tus (Termination
synthesis proceeds in the same direction as utilization substance). The Tus-Ter
replication fork movement. complex can arrest a replication fork
Lagging strand or discontinuous strand from only one direction. When either
is one in which 5ʹ3ʹ DNA synthesis replication fork encounters a functional
proceeds in the direction opposite to the Tus-Ter complex, it halts. The other fork
direction of fork movement. This strand is halts when it meets the first (arrested)
synthesised as short fragments, known as fork (Figure 14.15).
266

Parental
strand
Origin
Replication forks
Daughter
strand
Termination
of replication
Replication
proceeds in
both directions
Figure 14.15: Termination of replication in a circular DNA

14.3.4 Eukaryotic DNA Replication
Infobits
Replication in Eukaryotic cells is
more complex. The DNA molecules DNA molecules exists in circular form in
in Eukaryotes are considerably larger prokaryotic microorganisms, viruses and
than those in bacteria and are organized in organelles of eukaryotic organisms.
into complex nucleoprotein structures However not all DNA molecules are
(chromatin). The essential features of circular. The chromosomes of eukaryotic
DNA replication are same in eukaryotes organisms and of many viruses consists
and prokaryotes. However, some of linear DNA molecules. There are
interesting variations do occur. Initiation three general methods of replication of
of replication in all eukaryotes requires DNA molecule
a multisubunit protein. Multiple origins 1. Theta (θ) mode
of replication are probably a universal 2. Sigma (σ) mode
feature in eukaryotic cells. Like bacteria, 3. Linear mode
The two essential functions of genetic material are replication and

expression. Genetic material must replicate accurately so that progeny
inherit all of the specific genetic determinants (the genotype) of the
parental organism. A gene is a DNA sequence that encodes a protein,
rRNA, or tRNA molecule (gene product).
Gene expression usually involves transcription of DNA into messenger RNA and
translation of mRNA into protein. Genetic information encoded in DNA is expressed
by synthesis of specific RNAs and proteins, and information flows from DNA to RNA
to protein.
Expression of specific genetic material under a particular set of growth conditions
determines the observable characteristics (phenotype) of the organism. Bacteria have
few structural or developmental features that can be observed easily, but they have a
vast array of biochemical capabilities and patterns of susceptibility to antimicrobial
agents or bacteriophages. These latter characteristics are often selected as the inherited
traits to be analyzed in studies of bacterial genetics.
267

eukaryotes have several types of DNA Erwin Chargaff rules states that A = T
polymerases (Example: DNA polymerase and G = C. Watson and Crick postulated
α [alpha] and DNA polymerase δ [delta]). that DNA consists of two antiparallel
Some have been linked to particular chains in a right-handed double-helical
functions, such as the replication of arrangement. Complementary base pairs,
mitochondrial DNA. The termination A = T and G C, are formed by hydrogen
of replication on linear eukaryotic bonding within the helix. The basepairs
chromosomes involves the synthesis of are stacked perpendicular to the long
special structures called telomeres at the axis of the double helix. DNA can exist in
ends of chromosome. several structural forms. Two variations
of the Watson-Crick form, or B-DNA, are
HOTS A-DNA and Z-DNA.
Replication of DNA occurs with very
What will happen if single stranded high fidelity and at a designated time in the
binding proteins are not present cell cycle. Replication is semiconservative,
during replication of DNA? each strand acting as template for a new
daughter strand. It is carried out in
three phases: initiation, elongation, and
Summary
termination.
Many lines of evidence show that DNA The reaction starts at the origin and
bears genetic information. Frederick usually proceeds bidirectionally. DNA

Griffith showed transformation of is synthesized in the 5ʹ3ʹ direction by
bacteria. Avery, Mac Leod, Mc Carty DNA polymerases. At the replication fork,
experiment and further experiment by the leading strand is synthesized continu-
Hershey Chase confirmed that DNA is the ously in the same direction as replication
transforming principle. fork movement; the lagging strand is syn-
There are two types of nucleic thesised discontinuously as Okazaki frag-
acid: RNA and DNA. Nucleic acids are ments, which are subsequently ligated by
polymers of nucleotides, joined together DNA ligases.
by phosphodiester linkages between the Most cells have several DNA
5ʹhydroxyl group of one pentose and polymerases. In E. coli, DNA polymerase
the 3ʹhydroxyl group of the next. The III is the primary replication enzyme.
nucleotides in RNA contain ribose, and Replication of the E. coli chromosome
the common pyrimidine bases are uracil involves many enzymes and protein
and cytosine. In DNA, the nucleotides factors. Replication is similar in
contain deoxyribose sugar, and the eukaryotic cells, but eukaryotic
common pyrimidine bases are thymine chromosomes have many replication
and cytosine. The primary purines are origins.
adenine and guanine in both RNA and
DNA.
268

ICT CORNER
Bacterial DNA extraction
Lets separate DNA from

bacteria
STEPS:
• Scan the QR code
• Click DNA extraction on the left tab
• Select student lab notebook and click open Bacterial Extraction Protocol
• Press return and read students protocol on the left table
• Click producer and follow the steps
OBSERVATIONS :
• Select base pair interactions at the right side and join nitrogenous base
pairs as in DNA.
Step1 Step2 Step3
URL:
http://labcenter.dnalc.org/labs/dnaextraction/
dnaextractiond.html
269

Evaluation b. Double hydrogen
c. Vander Waal’s
d. Triple hydrogen
1. The genetic material of
8. Watson and Crick DNA model is
virus is
a. A form
a. DNA
b. RNA b. B form
c. a or b c. Z form
d. None d. D form
2. is used to denature RNA 9. In the first generation of Meselson and
a. DNase Stahl’s experiment, the results showed
b. Protease a hybrid band of DNA containing
c. Nuclease both 14N and 15N. Which of the
d. RNase following is the best interpretation of
3. In DNA molecule, the sugars these results?
a. Bond to nitrogenous bases by a. The results are consistent with
hydrogen bonds semi-conservative replication
b. Bond to nitrogenous bases by b. The results support conservative
glycosidic bonds replication
c. Bond to phosphate by hydrogen c. The results support both semi
bonds conservative and conservative
d. Bond to phosphate by glycosidic replication
bonds
d. Neither dispersive nor conservative
4. Which of the following is not present replication can take place.
in DNA
10. A form of DNA - A is
a. Adenine
b. Guanine a. Left Handed helix with
c. Uracil 20 nucleotide pairs per turn
d. None b. Right handed helix with
10 nucleotide pairs per turn
5. A nucleoside contains
c. Right handed helix with
a. Sugar
12 nucleotide pairs per turn
b. Nitrogenous base
c. Both a and b d. Left handed helix with
d. Only b 11 nucleotide pairs per turn
6. Glycosidic bond is present between 11. DNA polymerase is required for the
synthesis of
a. Phosphate and sugar
a. RNA from DNA
b. Sugar and nitrogenous base
b. DNA from RNA
c. Nitrogenous bases
c. DNA from DNA
d. All the above
d. RNA from RNA
7. The bond between adenine and
12. Okazaki segments are
thymine in a DNA double helix is
a. Segment of a chain of nucleotides
a. Hydrogen
270

removed during replication of Answer the following
DNA 1. Define gene
b. Segment of a chain of nucleotides 2. DNA is not always the genetic material,
formed during replication of DNA what are the exceptions?
c. Segments of gene which undergo 3. Define Nucleotide.
recombination
4. List any two difference between DNA
d. Segments of DNA capable of and RNA
replication 5. In what sense are the two strands of
13. DNA replication is aided by DNA antiparallel.
a. DNA Polymerase only 6. What is a nucleoside?
b. Both DNA polymerase and 7. Depict Erwin Chargaff rule by an
primase only equation.
c. DNA ligase only 8. Give examples of nitrogenous bases.
d. RNA Polymerase. 9. Draw the structure of Deoxyribose.
10. State Watson and Crick rule.
14. The semi-conservative mode of DNA
replication was proved by 11. List 2 characteristics of Z DNA
12. Define replication.
a. Beadle and Tatum
b. Meselson and Stahl 13. What is a template DNA?
c. Watson and Crick 14. Explain semiconservative replication.
d. H.G Khorana 15. List major events in replication
15. Enzymes involved in unwinding of 16. Write two main events during initiation
DNA at replication are of replication.
a. Ligases 17. What is the role of topoisomerase?
b. Helicases 18. What do you understand by leading
c. Endonucleases strand/lagging strand?
d. DNA Polymerases 19. What is RNA primer?
16. DNA replication is semiconservative 20. Name two enzymes that make primers
because the strand will for DNA synthesis
become half of the molecule 21. What is the origin of replication?
a. RNA, DNA 22. Label the following diagram of Griffith.
b. Template, finished
c. Sense, mRNA
d. Condon, anticodon
17. In DNA adenine is complementary 23. Point out the mistake in the following
base for and cytosine scheme:
is the complementary for Extracts of smooth strain + RNase
a. Guanine, thymine Mouse Alive
b. Uracil, guanine 24. Differentiate between right handed and
c. Thymine, guanine left handed DNA forms with any three
salient features.
d. Thymine, uracil
271

25. What are the types of DNA polymerases 40. Discuss the various bonds present in
present in E.coli? Write their functions. DNA double helix
26. Explain replication fork. 41. Discuss various forms of DNA double
27. Explain bidirectional replication. helix structure.
28. Explain continuous replication 42. Diagramatically explain the DNA
29. Define okazaki fragments. double helix structure
30. Why are RNA primers required 43. Draw a four base pair segment of a DNA
molecule, including each nucleotide
31. Describe what is meant by the and associated bonds involved in the
antiparallel arrangement of DNA maintenance of the double helix.
32. Why is one strand of DNA synthesised 44. Diagrammatically represent the results
discontinuously? of Meselson and Stahls experiment.
33. How is the faithfulness of DNA 45. Explain how Meselson’s and Stahl ruled
replication maintained? Write the name out dispersive model of replication.
of the enzymes with its function.
46. Tabulate the enzymes involved in DNA
34. Outline the experiment of Griffith. replication with their functions
35. Relate the experiments of Griffith and 47. Explain Elongation of DNA during
Avery. replication
36. Discuss Avery, Mc Cleod’s experiment.
37. What was the motive of Avery
and colleagues for conducting the
experiment.
38. Differentiate between R and S strains of
Streptococcus pneumoniae.
39. Describe the various characteristics
of the Watson and Crick double helix
model for DNA
Student Activity
1. Fun with beads – students will understand the concept of polymer – polynucleotide
and different sequences of DNA by preparing a chain of 20 beads of four different
colours.
2. Prepare a model of DNA
3. Supercoiling of DNA – students will hold the ends of the rubber band and twist it.
The two ends will be joined to feel the stress of coiling relieved due to supercoiling.
4. On paper replicate the following segment of DNA
5ʹATCGGCTACGTTCAC3ʹ
3ʹTAGCCGATGCAAGTG5ʹ
Show the direction of replication of the new strands and explain what the lagging
and leading strands are? Explain how this is semiconservative replication. Are the
new strands identical to the original segment DNA?
272

Glossary 13. Denature: To deprive something of its
1. Acute disease: A natural character and properties.
disease in which 14. Depyrogenation: Removal of
symptoms develop pyrogens from solutions mostly from
rapidly but lasts for injectable pharmaceuticals.
only a short time. 15. Dermatomycosis: A fungal infection
2. Aseptic techniques: Laboratory tech- of skin.
niques to minimize contamination. 16. Diatomaceous earth: A soft, crumbly,
3. Assimilation: The absorption and porous sedimentary deposit formed
digestion of nutrients by any biological from the fossil remains of diatoms.
system. 17. DNA amplification: The production of
4. Axenic: Pure cultures of micro multiple copies of a sequence of DNA.
organisms, which are not contaminated
18. Electromagnetic spectrum: The range
by any foreign organisms.
of wavelengths or frequencies over
5. Base Stacking: Stacking implies which electromagnetic radiation
vertical interactions between bases as extends.
they sit on top of one another.
19. Exudate: Low molecular weight
6. Bio-augmentation: The use of metabolites that enter the soil from
pollutant acclimated microbes or plant roots.
genetically engineered microbes for
20. Flake: A small flat thin piece of which
bioremediation.
has broken away or been peeled from
7. Coagulation: The action or process of a larger piece.
a liquid, especially blood, changing to
a solid or semi-solid state. 21. Fluoresence: The property of absorbing
light of short wave length and emitting
8. Coal-tar dyes: Liquid produced by
light of longer wave length.
distilling coal containing benzene
naphthalene, phenols, aniline and 22. Folliculitis: An infection of hair
many other organic chemical. follicles, often occurring as pimples.
9. Coliforms: Aerobic or facultatively 23. Fulminating: A condition that
anaerobic, Gram negative, non develops quickly and rapidly increases
endospore forming, rod shaped bacteria in severity.
that ferment lactose with acid and gas 24. Furuncle: A pus filled, painful
formation within 48 hours at 350°C. infection of a hair follicle.
10. Colony: A Colony is defined as a 25. Gene: A unit of heredity which is
visible mass of microorganism all transferred from parent to progeny.
originating from a single mother cell. 26. Genetic code: The mRNA codons and
11. Cover slip: A small, thin piece of glass the amino acids they encode.
used to cover and protect a specimen
27. Genetics: The study of heredity and
on a microscope slide.
variation of inherited characteristics.
12. Denaturation of DNA: Separation or
28. Genome: One complete copy of the
unwinding of dsDNA strands into
genetic information in cell.
single strands.
273
TN_GOVT_XI_Micro_Biology_Glossary.indd 273 16-02-2019 10.38.29 AM

29. Genomics: Study of genes and their 42. Lysis: Destruction of a cell by the
functions. rupture of the plasma membrane,
30. Genotype: The genetic makeup of an resulting in a loss of cytoplasm.
organisms. 43. Lysozyme: An enzyme capable of
31. Gestation: The development of hydrolyzing bacterial cell walls.
something over a period of time. 44. MHC: Major histocompatibility
32. Horizontal gene transfer: The transfer complex – The genes that code for
of genes between two organisms in histocompatibility antigens; also
the same generation. known as human leucocyte antigens.
33. Hypotonic environment: 45. Microaerophile: An organism that
Environment with higher water grows best in an environment with
concentration and less solutes. less molecular oxygen (O2) than is
34. Immunodiffusion test: A test normally found in air.
consisting of precipitation reactions
46. Molasses: It is a viscous product
carried out in an agar gel medium.
resulting from refining sugar cane or
35. Immunoelectrophoresis: The sugar beets into sugar.
identification of proteins by
electrophoretic separation followed 47. Monomer–A small molecule that
by serological testing. collectively combines to form
36. Inoculation loop: They are made of polymers.
platinum or nichrome wire. They are 48. Mucigel: Mucilage or complex
used to make smears. polysaccharide forming a layer
37. Inoculum: The material used to intro- around plant roots.
duce an organism into a certain medium 49. Neutralism: A lack of interaction
for growth or culture medium in which between two organisms in the same
microorganisms are implanted. ecosystem.
38. In vivo: Process taking place in a 50. Nick: It is discontinuity in a
living organisms. dsDNA molecule where there is
39. Ionizing radiation: Radiation no phosphodiester bond between
consisting of particles, X-rays, or adjacent nucleotides of one strand.
gamma rays with sufficient energy
51. Normal microbiota: The
to cause ionization in the medium
microrganisms that colonize a host
through which it passes.
without causing disease; also called
40. Latent infection: A condition in normal flora.
which a pathogen remains in the host
for long periods without producing 52. Occupational health: The branch of
disease. medicine dealing with the prevention
and treatment of job related injuries
41. Lymph: A colourless fluid containing
and illnesses.
white blood cells, which bathes
the tissues and drains through 53. Osmotic lysis: Rupture of the plasma
the lymphatic system into the membrane resulting from movement
bloodstream. of water into the cell.
274

54. Oxidation: The removal of electrons 67. Serological methods: Methods for
from a molecule. identifying microorganisms based on
55. Oxidation reduction: A coupled its reactions with antibodies.
reaction in which one substance is 68. Smear: A thin spread of bacterial
oxidized and one is reduced also suspension from a clinical spemicen
called redox reaction. or from a culture on a glass slide.
56. PCR: Polymerase chain reaction, a 69. Spectrophotometer: An apparatus for
technique using DNA polymerase measuring the intensity of light in a
to make multiple copies of a DNA part of the spectrum, especially as
template in vitro. transmitted or emitted by particular
57. Plasmolysis: Loss of water from a cell substances.
in a hypertonic environment. 70. Stab culture: A long straight wire
58. Polynucleotide: Chain of nucleotides. dipped in culture is punctured into
a solid medium usually to see the
59. Prevalence: The fraction of a
motility.
population having a specific disease
at a given time. 71. Topological stress: stress created
due to over winding or repeated
60. Progeny: Offspring, decendant of a
interwinding of DNA during
cell.
replication.
61. Protein sequencing: The practical
72. Topography: the arrangement of the
process of determining the amino acid
natural and artificial physical feature
sequence of all or part of a protein or
of an area.
peptide.
73. Toxigenic: (especially of a bacterium)
62. Protocooperation: An association
producing a toxin or toxic effect.
of mutual benefit to two or more
species but without the cooperation 74. Turbo blower: It is a fan that blows
or without being obligatory for their the air.
existence or the performance of some 75. Vaccine: A preparation of
function. killed, inactivated, or attenuated
63. Pustule: A small pus filled elevation microorganisms or toxoids to induce
of skin. artificial immunity.
64. Renaturation/Annealing: Process in 76. Vacuoles: A space or vesicle within
which ssDNA or ssRNA pair to form the cytoplasm of a cell enclosed by a
double stranded DNA. membrane and typically containing
fluid.
65. Salmon-GAL (6 chloro 3- indolyl –β – D
galactopyranoside): It is a chromogenic 77. Vegetative cells: A bacterial cell
substrate capable of detecting LacZ growing actively under favorable
gene encoded β galactosidase. conditions.
66. Semi-transparent: Partially admitting 78. Virulence: The degree of a
the passage of light through its pathogenicity of a pathogenic
substance. microorganism.
275

References Pearson Education.
1. Medical Microbiology: Mims.c, 15. Precotts Microbiology: Willey, Sherwood,
Dockrell.H.M, Goering.R.V., Ivan Roit., Woolverton (2014), Mc Graw-Hill, 9th
Waklin.D., Zukerman.M (2005). Third Edition.
edition, Elsevier Mosby, London. 16. Microbial Ecology: Atlas and Bartha (1997),
2. Medical Microbiology: Mackie and Pearson Publishers.
McCartney (1983) Churchill Livingston, 17. Plant Pathology: R.S Mehrotra (1998), Tata
ELBS Edition. McGraw-hill Publishing Company Limited,
3. Microbiology: Davis.B.D, D ulbecco.R., New Delhi.
Eisen.H., Ginsberg.H., (1980). Third edi- 18. Brock Biology of Microbiology: Michael
tion, Harper International Edition. T. Madigan and John M. Martinko (2006),
4. Jawetz, Melnick, & Adelberg’s Medical Eleventh Edition. Pearson Prentice Hall.
Microbiology: Brooks.G.F., Butel.J.S., 19. Essentials of Molecular Biology: Pamela
Morse.S.A. (1998), 21st Edition. Prentice Hanaratty and George M. Malacinski, 4th
Hall International Inc. Edition.
5. Ganong’s Review of Medical Physiology: 20. Microbial Genetics - David Freifelder
Barrett.K.E.,Barman.S.M., Boitano.S.C., (1935) Boston, MA : Jones and Bartlett,
and Brooks.H.L. 25 Edn. McGraw Hill
th c1987.
Education India Private Limited. New 21. Priciples of Microbiology: Ronald Atlas
Delhi. (2015), 2nd Edition. Mc Graw Hill India.
6. Microbiology: Michael J. Pelczar, E.C.S 22. Textbook of microbiology: R. Anantharayan
Chan, Noel E Krieg( 1993), 5th edition, Mc and C.K. Jayaram Paniker(2005). VII
Graw-Hill. Edition, Orient Longman Private Limited.
7. Environmental Microbiology: Raina 23. Microbiology: A Systems approach-
M.Maier,Ian L.Pepper,Charles P.Gerba Marjorie Kelly,Cowan, III Edition
(2009), 2nd Edition, Acadamic press. 24. Microbiology: Essentials and applications-
8. Soil Microbiology: Prof. N.S. Subha Larry Mackane/Judy Kancle,II Edition.
Rao (2009), Fourth Edition of Soil 25. Microbiology: Daniel Lim II edition
Microorganisms and Plant Growth, Oxford
26. Foster
& IBH Publishing Co. Pvt. Ltd, New Delhi.
27. Microbial Genetics - David Freifelder
9. Environmental Microbiology: K. Vijaya
(1935) Boston, MA :
Ramesh (2004), MJP Publishers.
28. Jones and Bartlett, c1987. 2nd edition
10. Soil Microbiology: R.R.Mishra (2014), CBS
Narosa Publications.
Publishers and distributors Pvt. Ltd.
29. Microbiology : A systems approach –
11. A Textbook of Microbiology. Dr. R.C.
Marjorie Kelly, Cowan (2011), III Edition,
Dubey and Dr. D.K. Maheshwari (1999).
McGraw - Hill Publications.
Revised edition. Chand and company Pvt.
Ltd. New Delhi. 30. Microbiology : Essentials and applications
– Larry Mackane/Judy Kandel (1996), II
12. Practical Microbiology: Dr. R.C. Dubey
Edition, McGraw – Hill Publications.
and Dr. D.K. Maheshwari(2002). Revised
edition. Chand and company Pvt. Ltd. New 31. Microbiology : Daniel V.Lim (1998), II
Delhi. edition, McGraw – Hill Publications.
13. Industrial Microbiology: A. H. Patel (2012), 32. Microbiology : An evolving Science Joan
2nd Edition, Mac Millan publishers India L. sonczewskl,John W. Foster (2113). 3rd
Ltd. Edition, W.W. Norton Publications.
14. Microbiology: Gerard J. Tortora, Berdell R. 33. Kuby Immunology. Janis Kuby (1997). 3rd
Funke, Christine L. Case (2004). 8th Edition. Edition. W. H Freeman and Company.
276
TN_GOVT_XI_Micro_Biology_Reference.indd 276 16-02-2019 10.38.50 AM

Microbiology Weblinks
Chapter - 1 Chapter – 9
Web link: http://www.britannica.com / Can microbes clean up oil
biography/Alexender-Fleming https://youtu.be/a_HWLFzgQiM
Chapter - 2 Composting
Working of compound microscope https://youtu.be/VNgFXvL9ZH8
https://youtu.be/cmzWDkOYTjM Chapter – 10
Chapter – 3 soil horizons types
Gram Staining https://youtu.be/OEvLuucpYw8
https://youtu.be/L9bats-vGDY Chapter – 11
Endospore Staining Nitrogen fixation
https://youtu.be/o1uYmUW4qe8 https://www.youtube.com/
Chapter – 4 watch?v=qzh7ZzJQJ84
Quick review of sterilization Late blight of potato
https://youtu.be/ZDmP14twN8g https://youtu.be/2Y77KEYuw_g
Chapter – 5 Chapter – 12
Streak Plate Bacterial meninigitis
http://youtu.be/NDMNGnxCZ1Q https://youtu.be/HhWjA1xq3Ig
Bacterial colony description Chapter – 13
https://youtu.be/gH--8YWdyyk Agglutination Reaction
Chapter – 6 https://www.youtube.com/
Photosynthesis watch?v=3W67OH3v2lU
https://youtu.be/1Dn_zdAZN0I Coomb’s Test

https://www.youtube.com/
Chapter – 7
watch?v=sUHsX3xrlFM
Bacterial flagellum
Chapter – 14
https://youtu.be/PIOfMifowP4
Structure of DNA
Chapter – 8
https://youtu.be/F5JazhVvlm4
Classification of microbes
Difference between prokaryotic and
https://youtu.be/W2nNIRUs6Wo eukaryotic DNA
Taxonomy and Classification https://youtu.be/0CoZT6hYemk
https://youtu.be/yCMDHd44ekQ
277
TN_GOVT_XI_Micro_Biology_WebLinks.indd 277 16-02-2019 10.39.14 AM

Microbiology – Class XI
List of Authors and Reviewers
Domain Experts Manimegali K
PGT, Govt Model HSS,
Dr. Sundararajan T Saidapet, Chennai
HOD (Retd) Dr.ALM PG IBMS,
University of Madras, Chennai Pameela Fathima H
PGT, JGGHSS, Thiruvottiyur,
Reviewers Thiruvallur District
Dr. Arunagirinathan N Parvin Zeenath Anwar
Assistant professor (Retd), Department PGT, St Mary’s Girls, HSS,
Microbiology, Presidency College Perambur, Chennai-11
Dr. Carol D
Assistant professor Academic Coordinator
Loyola College, Nungambakkam, Chennai Angeline Ruby G
Authors Assistant Professor
SCERT, Chennai-6
Dr. Niren Andrew S
Professor Department of Microbiology, ICT Coordinators
Madras Christian College, Chennai
Rajesh Kumar N
Dr. Linnett Naveena M B.T. Asst., CCMA GGHSS School, Coimbatore.
Assistant professor
Ethiraj College, Chennai Janakiraman M
B.T. Asst., PUMS, Mattayampatti, Salem.
Mary Sheela J
Assistant professor
Ethiraj College for Women, Chennai
Gajalakshmi S
PGT, KBC GGHSS, Redhills,
Thiruvallur District
Ramachandran N
PGT, GHSS, Kovur,
Kancheepuram District
Art and Design Team

Illustration
Jaishree A
Pugazharasi N
Layout
Winmac Solutions, Chennai
In-House QC
Manohar
Wrapper Design
Kathir Arumugam
Co-ordination
Ramesh Munisamy
Typist
Suresh M
This book has been printed on 80 G.S.M.
Elegant Maplitho paper.
Printed by offset at:
278
TN_GOVT_XI_Micro_Biology_Acknowledgement.indd 308 16-02-2019 10.40.16 AM

11th Micro Biology English-Medium Text

Загружено:

Сведения о документе

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

11th Micro Biology English-Medium Text

Загружено:

Авторское право:

Доступные форматы

GOVERNMENT OF TAMIL NADU

HIGHER SECONDARY FIRST YEAR

A publication under Free Textbook Programme of Government of Tamil Nadu

Department of School Education

Untouchability is Inhuman and a Crime

TN_GOVT_XI_Micro_Biology_FM.indd 1 16-02-2019 10.43.09 AM

NOT FOR SALE

State Council of Educational

Printing & Publishing

Tamil NaduTextbook and Educational

TN_GOVT_XI_Micro_Biology_FM.indd 2 16-02-2019 10.43.09 AM

Chapter 1 Introduction to Microbiology����������������������������������������������� 01

TN_GOVT_XI_Micro_Biology_FM.indd 3 16-02-2019 10.43.09 AM

Chapter Outline Presents a complete overview of the chapter

Learning Goals to transform the classroom processes into

Amazing facts, Rhetorical questions to lead students

Directions are provided to students to conduct activities

Infographics Visual representation of the lesson to enrich learning �

To motivate the students to further explore the content

ICT To enhance digital Science skills among students

Glossary Explanation of scientific terms

Evaluation Assess students to pause, think and check their understanding

Career corner List of professions particular to that chapter

References List of related books for further details of the topic

Web links List of digital resources

TN_GOVT_XI_Micro_Biology_FM.indd 4 16-02-2019 10.43.10 AM

Microbiologists are biological scientists microbiologists study the relationship

TN_GOVT_XI_Micro_Biology_FM.indd 5 16-02-2019 10.43.10 AM

TN_GOVT_XI_Micro_Biology_FM.indd 6 16-02-2019 10.43.10 AM

TN_GOVT_XI_Micro_Biology_FM.indd 7 16-02-2019 10.43.10 AM

Para Medical Courses and certificate courses in Tamilnadu Government Medical

1 Year Certificate Courses

2 Years Diploma Courses

TN_GOVT_XI_Micro_Biology_FM.indd 8 16-02-2019 10.43.10 AM

Job Prospects similarities between microbiology

TN_GOVT_XI_Micro_Biology_FM.indd 9 16-02-2019 10.43.10 AM

TN_GOVT_XI_Micro_Biology_FM.indd 10 16-02-2019 10.43.10 AM

1.1 Groups of Microorganisms Protozoa

1.2 Contributors to Microbiology

Microorganisms - Bacteria, Fungi, Algae,

TN_GOVT_XI_Micro_Biology_CH01.indd 1 16-02-2019 9.54.21 AM

TN_GOVT_XI_Micro_Biology_CH01.indd 2 16-02-2019 9.54.21 AM

1.2.1 Antony Van Leeuwenhoek

TN_GOVT_XI_Micro_Biology_CH01.indd 3 16-02-2019 9.54.21 AM

Figure 1.2: Leeuwenhoek’s Microscope

theory of spontaneous generation. He strongly

TN_GOVT_XI_Micro_Biology_CH01.indd 4 16-02-2019 9.54.22 AM

TN_GOVT_XI_Micro_Biology_CH01.indd 5 16-02-2019 9.54.22 AM

Koch’s Thread Method

TN_GOVT_XI_Micro_Biology_CH01.indd 6 16-02-2019 9.54.23 AM

Figure 1.7: Koch’s postulates for infectious diseases

TN_GOVT_XI_Micro_Biology_CH01.indd 7 16-02-2019 9.54.23 AM

Usefulness of Koch’s Postulates

TN_GOVT_XI_Micro_Biology_CH01.indd 8 16-02-2019 9.54.23 AM

Figure 1.11: Selman Abraham Waksman

Table 1.1: Branches of Microbiology

Based on Taxonomical characteristics

TN_GOVT_XI_Micro_Biology_CH01.indd 9 16-02-2019 9.54.23 AM

Microbial Cytology The study of microscopic and sub microscopic details

Evolutionary Microbiology The study of the evolution of microbes

Microbial Taxonomy The study of naming and classification of microorganisms

Microbial Systematics The study of the diversity and genetic relationship of

TN_GOVT_XI_Micro_Biology_CH01.indd 10 16-02-2019 9.54.24 AM

TN_GOVT_XI_Micro_Biology_CH01.indd 11 16-02-2019 9.54.24 AM

Chapter 1 Introduction to Microbiology�� 01