Std12 Microbiology EM WWW - Tntextbooks.in

GOVERNMENT OF TAMIL NADU
HIGHER SECONDARY SECOND YEAR
MICROBIOLOGY
THEORY & PRACTICAL
A publication under Free Textbook Programme of Government of Tamil Nadu
Department of School Education

Untouchability is Inhuman and a Crime
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Government of Tamil Nadu
First Edition - 2019
Published under New Education Syllabus
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II

Contents
Chapter 1 Developments in Microbiology ............................................. 01

Chapter 2 Microscopy ............................................................................. 12
Chapter 3 Control of Microorganisms by Chemical Methods ............. 28
Chapter 4 Microbial Metabolism ............................................................ 45
Chapter 5 Food Microbiology ................................................................. 68
Chapter 6 Industrial Microbiology ......................................................... 85
Chapter 7 Medical Bacteriology ........................................................... 114
Chapter 8 Medical Parasitology ............................................................ 151
Chapter 9 Medical Mycology ................................................................ 188
Chapter 10 Medical Virology ................................................................ 206
Chapter 11 Immunology ....................................................................... 230
Chapter 12 Microbial Genetics ............................................................. 249
Practical ................................................................................................... 291
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III

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.
To motivate the students to further explore the content

digitally and take them to virtual world
Evaluation Assess students to pause, think and check their understanding
Career corner List of professions particular to that chapter
IV

Chapter
1 Developments in Microbiology
microorganisms are
Learning Objectives necessary to live on
After studying this chapter the the planet and their
students will be able to, extraordinary diversity
of structure, function,
• Know the microbes in space habitat and applications
• Understand the emerging microbes are of paramount importance.
• Learn about genetically modified Microorganisms (or microbes)
microbes inhabiting every corner of the globe,
• Appreciate nanoparticles production are indispensable to life on Earth and
using microbes are responsible for some of the deadliest
• Know the important of microbiome human diseases and form the basis of
many industrial processes. This field
• Understand the applications of
of study could be considered as one of
automated machines in microbial
the most important areas of knowledge,
identification
considering that the bacteria in and on
our bodies outnumber our own cells.
Chapter Outline Microbiology, an organismal
discipline concerned with the properties
1.1 Microbes in Space of small forms of life or microorganisms.
1.2 Emerging Microbes Bacteria neatly fit this definition, but
what about fungi and algae? These
1.3 Immunology
two groups each contain members
1.4 Molecular Biology and Genetic that are far from microscopic. On the
Engineering other hand, certain animals, such as
1.5 Nanoparticles Production Using nematode worms, can be microscopic,
Microbes yet are not considered to be the domain
of the microbiologist. Viruses represent
1.6 Equipments
another special case; they are most
certainly microscopic (indeed, most are
The field of microbiology, critical submicroscopic), but by most accepted
to human beings, not only due to definitions they are not living. The
the infectious diseases caused by concept of microbiology is remarkably
these microbes but because “good” broad in covering bacteria, protozoa and
1
TN_GOVT_XII_Micro_Biology_CH01.indd 1 27-02-2019 15:14:45

the viruses, which differ profoundly their equipment device) facility and in the
structural and biological properties. Biostack experiments. Arriving in space
Microbiology is without aquestion, a without any protection, microorganisms
branch of biology that possesses both are confronted with an extremely hostile
unity and coherence. environment, characterized by an intense
The following list of specializations in radiation field of galactic and solar origin,
microbiology will provide an insight on high vacuum, extreme temperatures, and
the significance of microbiology in the microgravity.
world today:
HOTS
•• Medicine
•• Environmental science
1. W
hat are the sources of
•• Food production microorganisms in space?
•• Fundamental Research 2. How do bacteria survey in space?
•• Agriculture
•• Pharmaceutical Industry Some bacteria were found in
•• Genetic Engineering International Space Station and on the
The popular perception among the Mars Rover. Some bacteria and tiny
general public, however, remains one of microbes called tardigrades are able
infections and plagues. In reality, only a to survive for longer periods in space.
couple of hundred out of the half million This ability of surviving in extreme
or so known bacterial species, termed environmental condition leads to forward
as pathogens with the potential to cause contamination. Sea planktons and other
disease give rise to infections in humans microorganisms have been identified in
and dominate the microbial world. cosmonauts’ spacewalk samples.
In July 2016, Kate Rubins was the first to
1.1 Microbes in Space sequence DNA in space. NASA astronaut
The majority of experiments on Peggy Whitson amplified and sequenced
microorganisms in space were performed the DNA of bacteria that grew as colonies
using Earth-orbiting robotic spacecraft, in the petri plate on the surface on the space
Example: the Russian Foton satellites station. In June 2018, Professor George
and the European Retrievable Carrier Fox and his team have isolated genus
(EURECA) (121), or human-tended Bacillus from spacecraft assembly rooms
spacecraft, such as space shuttles (106, at the Jet Propulsion Laboratory. They
107) and space stations, Example: MIR have sequenced the complete genomes
and the International Space Station (ISS). of two strains, B. safensis FO-36bT and
Only twice, during translunar trips B. pumilus SAFR-032 and found that they
of Apollo 16 and 17 in the early 1970s, are resistant to radiation.
were microorganisms exposed to space Los Angeles in great news for India,
conditions beyond Earth’s magnetic scientists at NASA have named a new
shield, in the MEED (microbial ecology organism discovered by them after the
2

much loved APJ Abdul Kalam. Till date, Ultimately, it causes levels of blood-
the new organism – a form of bacteria – has clotting cells to drop. This leads to severe,
been found only on the International Space uncontrollable bleeding. The disease was
Station (ISS) and has been found on earth. known as Ebola hemorrhagic fever but is
Researchers at the Jet Propulsion Laboratory now referred to as Ebola virus. It kills up
(JPL) the foremost lab of NASA for work on to 90% of people who are infected.
inter-planetary travel discovered the new Ebola is a contagious as more common
bacteria on the filters of the international viruses like colds, influenza, or measles. It
space station (ISS) and named it Solibacillus spreads to people by contact with the skin
kalam to honour the late president, who or bodily fluids of an infected animal, like
was a renowned aerospace scientist. a monkey, chimp, or fruit bat. Then it
moves from person to person the same way
HOTS early on, Ebola can feel like the flu or other
illnesses. Symptoms show up 2 to 21 days
What could be source of microorgan- after infection and usually include: High
isms in space? fever, Headache, Joint and muscle aches,
Sore throat, Weakness, Stomach pain,
Lack of appetite. It causes bleeding inside
1.2 Emerging Microbes
the body, as well as from the eyes, ears,
Microbes and the diseases they cause and nose. Some people will vomit
have emerged as a major threat to human or cough up blood, have bloody diarrhea,
populations. Some of them like malaria and get a rash (Figure 1.1).
have been major pathogens since they
emerged in the depths of antiquity.
Emerging infection is defined as those
infections whose incidence in humans
has increased in the past two decades or
will increase in the future. It can be new,
reemerging, drug resistant infections.
Ebola
Ebola virus disease (EVD), also known
as Ebola hemorrhagic fever (EHF) or Figure 1.1: Ebola virus
simply Ebola, is a viral hemorrhagic
fever of humans and other primates caused Zika
by Ebola virus. Zika virus (ZIKV) is a member of
Ebola is a rare but deadly virus that the virus family Flaviviridae. It is spread
causes fever, body aches, and diarrhea, and by daytime-active Aedes mosquitoes,
sometimes bleeding inside and outside the such as A. aegypti and A. albopictus. Its
body. The virus spreads through the body; name comes from the Zika Forest of
it damages the immune system and organs. Uganda, where the virus was first

isolated in 1947. Zika virus is related
to the dengue, yellow fever, Japanese
encephalitis, and West Nile viruses.
Zika virus do not develop symptoms.
Symptoms are generally mild including
fever, rash, conjunctivitis, muscle and
joint pain, malaise, and headache, and
usually last for 2–7 days.
Zika virus is primarily transmitted
by the bite of an infected mosquito Figure 1.2: Zika virus
from the Aedes genus, mainly Aedes
aegypti, in tropical and subtropical mucus that is able to trap potential
regions. Aedes mosquitoes usually bite infectants. In the airways, mobile
during the day, peaking during early ciliate hairs work together to transport
morning and late afternoon/evening. contaminants away from vulnerable areas.
This is the same mosquito that transmits Tissues such as the skin, mucosal surfaces
dengue, chikungunya and yellow fever. and airways also contain populations of
immune cells that can respond to infectants
Zika virus is also transmitted from
that breach these physical defences.
mother to fetus during pregnancy, through
sexual contact, transfusion of blood and In its most complex forms, the
blood products, and organ transplantation. immune system consists of two branches:
An increased risk of neurologic the innate immune system that utilises
complications is associated with Zika virus certain ‘hard-wired’ strategies to provide
infection in adults and children, including a rapid, general, response when alerted
Guillain-Barré syndrome, neuropathy and by certain typical signals of infection
myelitis (Figure 1.2). (essentially forming a first-line of
defence); and the adaptive immune
1.3 Immunology system that is able to develop highly
specific responses (and a persistent
Immunology is the study of the immune
‘immune memory’) to target infection
system and is a very important branch of
with extraordinary accuracy. Both
the medical and biological sciences. The
systems work in close cooperation and,
immune system protects us from infection
to an important extent, the adaptive
through various lines of defence.
immune system relies upon the innate
Important initial barriers to infection immune system to alert it to potential
are physical (Example: the skin), targets, and shape its response to them.
enhanced by substances secreted by the
body, such as saliva and tears, that contain Vaccines currently in development
molecules that can neutralise bacteria. The include
internal mucosal tissues (Example: lungs •• A genetically-modified vaccine for the
& airways, and the gut) are coated with treatment of pancreatic cancer.

•• A therapeutic vaccine that increases (i.e. hybridomas) by fusing myeloma
the immune response against the HIV cells with desired antibody-producing
virus. splenocytes (Example: B cells). These B
•• A vaccine that protects infants against cells are typically sourced from animals,
meningococcal disease, a leading cause usually mice. After cell fusion, large
of meningitis. numbers of clones are screened and
•• An immunotherapeutic vaccine for the selected on the basis of antigen specificity
treatment of Alzheimer’s disease. and immunoglobulin class (Figure 1.3).
•• A recombinant vaccine to prevent

malaria.
Evolving science has increasingly
enabled researchers to explore both
promising therapeutic vaccines and
new preventative agents for infectious
diseases. Although the development
process is extremely complex, advances in
other scientific fields, such as genomics,
are being leveraged in the development of
new vaccines.
“Vaccines have been a major
contributor in saving countless lives
around the world,” said Castellani.
“Vaccinations contribute to the public
health at large, and they make good Figure 1.3: Monoclonal antibody
economic sense. The many exciting
candidates in the pipeline offer great hope 1.3.2 Stem Cell & Therapy
for a healthier, more productive future.”
Stem cells are biological cells that can
1.3.1 Monoclonal Antibodies differentiate into other types of cells &
they are found in multicellular organism.
mAb or moAb are identical Stem cells are a class of undifferentiated
immunoglobulins, generated from a single cells that are able to differentiate into
B-cell clone. These antibodies recognize specialized cell types. Commonly, stem
unique epitopes, or binding sites, on a single
cells come from two main sources:
antigen. Derivation from a single B-cell
clones and subsequent targeting of a single •• Embryos formed during the blastocyst
epitope is what differentiates monoclonal phase of embryological development
antibodies from polyclonal antibodies (embryonic stem cells) and
The traditional monoclonal antibody •• Adult tissue (adult stem cells).

(mAb) production process usually starts Both types are generally characterized
with generation of mAb-producing cells by their potency, or potential to

differentiate into different cell types such a donor (either from cord blood or bone
as skin, muscle, bone, etc., (Figure 1.4). marrow) are known to reconstitute the
Stem-cell therapy is the use of stem defective bone marrow and permanently
cells to treat or prevent a disease or overcome the disorder.
condition. Stem Cell Therapy (SCT) is the
treatment of various disorders, non-serious 1.4 Molecular Biology and Genetic
to life threatening, by using stem cells. Engineering
These stem cells can be procured from a lot Molecular biology- is the study of the
of different sources and used to potentially structure, function & makeup of the
treat more than 80 disorders, including molecular building blocks of life. It
neuromuscular and degenerative disorders. focuses on the interactions between the
Hematopoietic disorders various system of a cell, including the
(Example: leukaemia, thallassemia, interrelationship of DNA, RNA & Protein
aplastic anemia, MDS, sickle cell anemia, synthesis &how these interaction are
storage disorders etc.) affect the bone regulated. Bioscience, Molecular biology
marrow and manifest with various closely interrelate with the fields of
systemic complications. Stem cells from Biochemistry, Genetics & Cell biology.
Figure 1.4: Stem cells

6

Molecular biology is a specialised
branch of biochemistry, the study of
the chemistry of molecules which are
specifically connected to living processes.
Importance to molecular biology are
the nucleicacids (DNA and RNA) and
the proteins which are constructed using
the genetic instructions encoded in those
molecules. Other biomolecules, such
as carbohydrates and lipids may also be
studied for the interactions they have with
nucleic acids and proteins. Molecular
biology is often separated from the field
of cell biology, which concentrates on Figure 1.5: Genetic Engineering
cellular structures (organelles and the like),
molecular pathways within cells and cell Genetically Modified Organism (GMO)
life cycles.
Organism genome has been
engineered in the laboratory in order
Genetic Engineering
to favour the expression of desired
Genetic Engineering is the act of physiological traits or the production
modifying the genetic makeup of an of desired biological products. In
organism. Modification can be generated conventional livestock production,
by methods such as gene therapy, nuclear crop farming, and even pet breeding,
transplantation, transfection of synthetic it has long been the practice to breed
chromosome or viral insertion. select individuals of a species in order
The manipulation of genetic make up to produce offspring that have desirable
of living cells by inserting desired genes traits. In genetic modification, however,
through a DNA vector, is the genetic recombinant genetic technologies are
engineering. The gene is a small piece of employed to produce organisms whose
DNA that encodes for a specific protein. genomes have been precisely altered
The gene is inserted into a `vector DNA at the molecular level, usually by the
so that a new combination of vector DNA inclusion of genes from unrelated species
is formed. The DNA formed by joining of organisms that code for traits that
DNA segments of two different organisms would not be obtained easily through
is called recombinant DNA. The organism conventional selective breeding.
whose genetic make up is manipulated GMOs are produced through
using recombinant DNA technique, is called using scientific methods that
geneticall manipulated organism (GMO). include recombinant DNA technology and
Genetic engineering has many reproductive cloning. In reproductive
application in agriculture, animal science, cloning, a nucleus is extracted from a
industry and medicines (Figure 1.5). cell of the individual to be cloned and is

into the DNA (deoxyribonucleic acid)
Microbiome of another. Whole-genome replacement,
The human microbi- involving the transplantation of
ome is composed of one bacterial genome into the “cell body,”
communities of bacte- or cytoplasm, of another microorganism,
ria, viruses and fungi that have a greater has been reported, although this
complexity than the human genome it- technology is still limited to basic
self. Genome sequencing technologies scientific applications.
and metagenomic analysis has helped
in our understanding of human micro- 1.5 Nanoparticles Production Using
biome. This is useful in manipulation of Microbes
gut microbiome to be used in the treat-
ment of childhood diseases. Particles with one or more dimensions
of the order of 100 nm or less. There are
a large number of physical, chemical,
inserted into the enucleated cytoplasm of
biological, and hybrid methods
a host egg. The process results in the
available to synthesize different types of
generation of an offspring that is genetically
nanoparticles. Although physical and
identical to the donor individual. The
chemical methods are more popular in the
first animal produced by means of this
synthesis of nanoparticles, the use of toxic
cloning technique with a nucleus from an
chemicals greatly limits their biomedical
adult donor cell (as opposed to a donor
applications, in particular in clinical
embryo) was a sheep named Dolly, born
fields. Therefore, development of reliable,
in 1996. Since then a number of other
nontoxic, and eco-friendly methods for
animals, including pigs, horses, and dogs,
synthesis of nanoparticles is of utmost
have been generated by reproductive
importance to expand their biomedical
cloning technology. Recombinant DNA
applications. One of the options to achieve
technology, on the other hand, involves
this goal is to use microorganisms to
the insertion of one or more individual
synthesize nanoparticles.
genes from an organism of one species
Nanoparticles are biosynthesized
when the microorganisms grab target ions
Infobits
from their environment and then turn the
Gut Microbes metal ions into the element metal through
enzymes generated by the cell activities.
Human gut houses 1000 different species
It can be classified into intra-cellular and
bacteria comprising of two major phyla
extracellular synthesis according to the
namely Bacteroidetes and Firmicutes.
location where nanoparticles are formed.
The loss of balance in gut microbiota
The intracellular method consists of
may lead to dybiosis. This may cause
transporting ions into the microbial cell
health problems like bowel disorders,
to form nanoparticles in the presence
bowel diseases like inflammation,
of enzymes. The extracellular synthesis
allergies, obesity and diabetes.
of nanoparticles involves trapping the
8

metal ions on the surface of the cells and of confocal microscopy involves imaging
reducing ions in the presence of enzymes. either fixed or living cells and tissues that
The biosynthesized nanoparticles have have usually been labeled with one or
been used in a variety of applications more fluorescent probes.
including drug carriers for targeted delivery, Confocal microscopy, most
cancer treatment, gene therapy and DNA frequently confocal laser scanning
analysis, antibacterial agents, biosensors, microscopy (CLSM) or laser confocal
enhancing reaction rates, separation scanning microscopy (LCSM), is an optical
science, and magnetic resonance imaging imaging technique for increasing optical
(MRI). Many microorganisms can produce resolution and contrast of a micrograph by
inorganic nanoparticles through either means of using a spatial pinhole to block out-
intracellular or extracellular routes. This of-focus light in image formation. Capturing
section describes the production of various multiple two-dimensional images at different
nanoparticles via biological methods depths in a sample enables the reconstruction
following the categories of metallic of three-dimensional structures (a
nanoparticles including gold, silver, alloy process known as optical sectioning)
and other metal nanoparticles, oxide within an object. This technique is used
nanoparticles consisting of magnetic and extensively in the scientific and industrial
nonmagnetic oxide nanoparticles, sulfide communities and typical applications are
nanoparticles, and other miscellaneous in life sciences, semiconductor inspection
nanoparticles (Figure 1.6). and materials science.
Light travels through the sample under
a conventional microscope as far into
the specimen as it can penetrate, while a
confocal microscope only focuses a smaller
beam of light at one narrow depth level at
a time. The CLSM achieves a controlled
and highly limited depth of focus.
1.6.2 DNA Sequencing System

Figure 1.6: Advanced Nano particles
Sequencing means finding the order of
nucleotides on a piece of DNA. Nucleotide
1.6 Equipments
order determines amino acid order, and by
1.6.1 Confocal Microscopy extension, protein structure and function
Confocal microscopy offers several (proteomics). An alteration in a DNA
advantages over conventional optical sequence can lead to an altered or non
microscopy, including shallow depth of functional protein, and hence to a genetic
field, elimination of out-of-focus glare, disorder. DNA sequence is important to
and the ability to collect serial optical detect the type of mutations in genetic
sections from thick specimens. In the diseases and offer hope for the eventual
biomedical sciences, a major application development of treatment DNA.

Methods of sequencing This condition associated with many
1. Sanger dideoxy (primer extension/ chronic diseases, including diabetes,
chain-termination) method Lupus multiple sclerosis symptoms of
leaky gut are bloating, gas, cramps,
Most popular protocol for sequencing, very
inflammatory bowel disease, fatigue,
adaptable, scalable to large sequencing
food sensitivities, join pain, moodiness,
projects.
irritability, sleepless, skin problem and
2. Maxam-Gilbert chemical cleavage eczema, psoriasis.
method
(https://www.sciencedaily.com/releases/
DNA is labelled and then chemically 2018/06/180627160249.htm)
cleaved in a sequence dependent manner.
(http://tass.com/science/977591)
This method is not easily scaled and is
rather tedious.
Evaluation
It provides an important tool for
determining the thousands of nucleotide Multiple choice questions
variations associated with specific genetic 1. Size of the Nono
diseases, like Huntington’s, which may particles varies from
help to better understand these diseases a. Less than 10nm
and advance treatment. b. 100nm or less
Summary c. 100nm or more
d. none of these
Microorganisms (or microbes)
inhabiting every corner of the globe, 2. is an example for
are indispensable to life on Earth and optical imaging technique
are responsible for some of the deadliest a. CLSM b. LCSM
human diseases and form the basis of c. both a and b d. TEM
many industrial processes. Microbiology, 3. First genetically produced animal by
an organismal discipline concerned with cloning technique is _______
the properties of small forms of life or
a. Shally b. Dolly c. bally d. Vally
microorganisms. Microbes could help
solve crimes. Arriving in space without 4. EHF stands for
any protection, microorganisms are a. Ebola hemorrhagic fever
confronted with an extremely hostile b. Ebola heart fever
environment, characterized by an c. Ebola human fever
intense radiation field of galactic and
d. none of these
solar origin, high vacuum, extreme
temperatures, and microgravity. 5. ISS stands for
Emerging infection is defined as those a. International space station
infections whose incidence in humans b. Indian space station
has increased in the past two decades or
c. Indian standard system
will increase in the future. It can be new,
reemerging, drug resistant infections. d. None of these
10

Answer the following 6. Give the importance of stem cells?
1. Short note on Microbes in space. 7. List out the various instruments used
2. Brief account on Monoclonal in Diagnostic microbiology.
antibody. 8. Short note on genetically modified
3. What is r.DNA technology? foods.
4. Discuss on Emerging microbes. 9. Brief note on sequencing methods.
5. Describe about Nano particles and its 10. Write about Vaccines and its
importance and its important in the importance.
field of medicine?
11

Chapter
2 Microscopy
sophisticated compound
Learning Objectives light microscopes
After studying this chapter the are routinely used
students will be able to, in microbiology
laboratories. In the
• Identify the principle components previous year, we have
of Phase Contrast, Fluorescence learnt about light microscopes that includes
and Electron Microscope. bright field and Dark-field microscopes.
• Understand the optics in different This year we are going to learn about other
light microscope and image types of light microscopes such as phase
formation mechanism. contrast and fluorescence microscopes.
• Know the principle, working Yet another well advanced microscope
mechanism of Phase Contrast, which uses electron as source rather than
Fluorescent Microscope and light – the electron microscope is also
Electron Microscope. discussed in detail in this chapter.
• Differentiate Light and Electron
Microscope. 2.1 Phase Contrast Microscope
• Appreciate the applications of Frits Zernike a Dutch Physicist invented
Phase Contrast, Fluorescence and the Phase Contrast Microscope and was
Electron Microscopes. awarded Nobel Prize in 1953. It is the
microscope which allows the observation
of living cell. This microscopy uses
Chapter Outline special optical components to exploit
2.1 Phase Contrast Microscope fine differences in the refractive indices
of water and cytoplasmic components of
2.2 Fluorescence Microscope living cells to produce contrast.
2.3 Electron Microscope
2.1.1 Principle
Microscopes are specialized optical The phase contrast microscopy is based on
instruments designed to produce the principle that small phase changes in
magnified visual or photographic images the light rays, induced by differences in the
of objects or specimens that are too small thickness and refractive index of the different
to be seen with naked eye. Today, more parts of an object, can be transformed into
12

differences in brightness or light intensity. his plate is located at the back
T
The phase changes are not detectable to focal plane of the objective lenses.
human eye whereas the brightness or light The phase plate has two portions,
intensity can be easily detected. in which one is coated with light
retarding material (Magnesium
2.1.2 Optical Components of PCM fluoride) and the other portion
The phase contrast microscope is similar devoid of light retarding material but
to an ordinary compound microscope in can absorb light. This plate helps to
its optical components. It possesses a light reduce the phase of the incident light
source, condenser system, objective lens (Figure 2.2).
system and ocular lens system (Figure 2.1).
2.1.3 Working Mechanism of Phase
A phase contrast microscope differs
Contrast Microscopy
from bright field microscope in having,
i. Sub-stage annular diaphragm (phase The unstained cells
condenser) cannot create contrast
under the normal
n annular aperture in the diaphragm
A
microscope. However,
is placed in the focal plane of
when the light passes
the sub-stage which controls the
through an unstained
illumination of the object. This is
cell, it encounters regions in the cell
located below the condenser of the
with different refractive indexes and
microscope. This annular diaphragm
thickness. When light rays pass through
helps to create a narrow, hollow cone
an area of high refractive index, it deviates
of light to illuminate the object.
from its normal path and such light
ii. Phase – plate (diffraction plate or rays experience phase change or phase
phase retardation plate) retardation (deviation). Light rays pass
through the area of less refractive index
Figure 2.1: Components of phase contrast microscope

13

Phase
plate
Bacterlum Ray devlated by Deviated ray is Deviated and

specimen is 1/4 1/2 wavelength undeviated rays
wavelength out out of phase. cancel each other
of phase. out.
Figure 2.2: Production of contrast in phase contrast microscopy by phase plate
Phase-contrast
Specimen now image
Phase plate
appears dark.
Light refracted by
1/2 in total
Phase plate
1/4 wavelength
Specimen
Approximate
Slide effect of
specimen.
Light refracted by
1/2 (specimen)
Specimen
Unobstructed light
Light source (phase unaltered
by specimen)
Annular ring
Light source
Figure 2.3: Optical path of Phase contrast microscopy
remain non-deviated (no phase change).

Figure 2.3 shows the light path in phase HOTS
contrast microscope.
How does phase contrast microscope
differ from Bright Field microscope?
14

The difference in the phases between in the phase of light. The phase contrast
the retarded (deviated) and un-retarded microscope has special devices such as
(non-deviated) light rays is about ¼ of annular diaphragm and phase plate, which
original wave length (i.e., λ/4). Human convert these minute phase changes into
eyes cannot detect these minute changes brightness (amplitude) changes, so that a
contrast difference can be created in the
final image. This contrast difference can be
Infobits
easily detected by human eyes.
Whenever light (or any wave in In phase contrast microscope, to get
general) goes from one medium to contrast, the diffracted waves have to be
another, some of the energy of the separated from the direct waves. This
wave is “reflected” back through the separation is achieved by the sub-stage
first medium cut the same angle as the annular diaphragm.
incident wave and some of the energy The annular diaphragm illuminates
is refracted (bent). Through the the specimen with a hollow cone of light.
second medium when light goes from Some rays (direct rays) pass through the
a low refractive index medium to a thinner region of the specimen and do not
high refractive index medium such as undergo any deviation and they directly
air to water the reflection undergoes enter into the objective lens. The light
a 180° phase change. Light waves that rays passing through the denser region
are in phase (that is, their peaks and of the specimen get regarded and they
valleys exactly coincide) reinforce run with a delayed phase than the non-
one another and their total intensity deviated rays. Both the deviated and non
increases. deviated light has to pass through the
Light waves that are out of phase phase plate kept on the back focal plane
by exactly one-half wavelength cancel of the objective to form the final image.
each other and result in no intensity. The difference in phase (Wavelength)
That is darkness wavelengths that gives the contrast for clear visibility
are out of phase by any amount will of the object. Figure 2.4 Microscopic
produce some degree of cancellation image comparing phase and bright field
and result in brightness less than microscopy.
maximum, but more than darkness.
Thus, contrast is provided by 2.1.4 Applications
differences is light intensity that result •• Phase contrast microscope enables
from differences in refractive indices the visualization of unstained living
in parts of the specimen that put light cells.
waves indices in parts of the specimen
•• It makes highly transparent objects
that put light waves more or less out
more visible.
of phase. As a result, the specimen
appears as various levels of darks
against a bright background.
15

Figure 2.4: (a) Saccharomyces under bright field microscope (b) Saccharomyces under
phase contrast microscope
•• It is used to examine various properties of the light microscope with

intracellular components of living cells fluorescence technology.
at relatively high resolution.
•• It helps in studying cellular events such
as cell division.
•• It is used to visualize all types of cellular
movements such as chromosomal and
flagellar movements.
2.2 Fluorescence Microscope

Fluorescence microscope is a very
powerful analytical tool that combines the
magnifying properties of light microscope Figure 2.5: Principle of fluorescence
with visualization of fluorescence.
Fluorescence microscope is a type of
Infobits
light microscope which instead of utilizing
visible light to illuminate specimens, uses British scientist Sir George G. Stokes
a higher intensity (lower wavelength) first described fluorescence in 1852. He
light source that excites a fluorescence observed that the fluorophore emitted
molecule called a fluorophore (also red light, when it was illuminated by
known as fluorochrome). Fluorescence is ultraviolet excitation. Stokes noted
a phenomenon that takes place when the that fluorescence emission always
substances (fluorophore) absorbs light occurred at a longer wavelength than
at a given wavelength and emits light at of the excitation light. This shift
a higher wavelength. Thus, fluorescence towards longer wavelength is known
microscopy combines the magnifying as stokes shift.
16

The fluorophore absorbs photons is focused onto the fluorescent specimen.
leading to electrons moving to a higher The emissions from the specimen are in
energy state (excited state). When the turn, passed back up through the objective
electrons return to the ground state by where magnification of the image occurs
losing energy, the fluorophore emits light and through the dichroic mirror.
of a longer wavelength (Figure 2.5). Three This light is filtered by the barrier filter,
of the most common fluorophores used which selects for the emission wavelength
are Diamidino – phenylindole (DAPI) and filters out contaminating light from the
(emits blue), FITC (emits green), and arc lamp or other sources that are reflected
Texas Red (emits red). off from the microscope components.
Finally, the filtered fluorescent emission
2.2.1 Principle is sent to a detector where the image can
be digitized.
Light source such as Xenon or Mercury
Arch Lamp which provides light in a wide
2.2.2 Components of Fluorescence
range of wavelength, from ultraviolet to
Microscope
the infrared is directed through on exciter
filter (selects the excitation wavelength). The main components of the fluorescent
This light is reflected toward the sample by microscope resemble the traditional
light microscope. However, the two main
a special mirror called a dichroic mirror,
difference are the type of light source
which is designed to reflect light only at
used and the use of the specialized filter
the excitation wavelength. The reflected
elements (Figure 2.6).
light passes through the objective where it
Infobits
Some substances have the property

of luminescence. They emit light
of one colour when exposed to
light of a different colour. If light
emission occurs within one millionth
of a second of light exposure, the
luminescence is phosphorescence.
The colour of the emitted light has
a longer wavelength than the colour
of the exciting light. For example,
fluorescein isothiocyanate (FITC) is
excited by blue light and emits green
light; rhodamine isothiocyanate is
excited by green light and emits red
light. Figure 2.6: Components of fluorescence
microscope
17

Light source
Fluorescence is called
Fluorescence microscopy requires a very
“cold light” because it
powerful light source such as a Xenon or
does not come from
Mercury Arch Lamp. The light emitted
a hot source like an
from the Mercury Arc Lamp 10–100
incandescent light bulb.
times brighter than most incandescent
lamps and provides light in a wide range
of wavelengths from ultra-violet to the 2.2.3 Working Mechanism
infrared. Lasers or high-power LEDs were The specimen to be observed are stained
mostly used for complex fluorescence or labeled with a fluorescent dye and then
microscopy techniques. illuminated with high intensity ultra violet
Filter elements light from mercury arc lamp. The light
passes through the exciter filter that allows
A typical fluorescence microscope consists
only blue light to pass through. Then the
of three filters: excitation, emission and
blue light reaches dichroic mirror and
the dichroic beam splitter.
reflected downward to the specimen. The
Excitation filters: It is placed within specimen labeled with fluorescent dye
the illumination path of a fluorescence absorbs blue light (shorter wavelength) and
microscope. Its purpose is to filter out all emits green light. The emitted green light
wavelength of the light source, except for goes upward and passes through dichroic
the excitation range of the fluorophore in mirror, reflects back blue light and allows
the sample or specimen of interest. only green light to pass the objective lens,
Emission filters: The emission filter then it reaches barrier filter which allows
is placed within the imaging path of a only green light. The filtered fluorescent
fluorescence microscope. Its purpose is emission is sent to a detector where the
to filter out the entire excitation range image can be digitized Figure 2.7.
and to transmit the emission range of the
fluorophore in the specimen.
Dichroic filter or beam splitter: The
dichroic filter or beam splitter is placed in
between the excitation filter and emission
filter, at 45° angle. Its purpose is to reflect
the excitation wavelength towards the
fluorophore in the specimen, and to
transmit the emission wavelength towards
the detector.
18

Figure 2.7: (a) Fluorescence microscope (b) Fluorescent stained tubercule bacilli
2.2.4 Application
Infobits
•• Fluorescence microscope has become
The Two Types of Fluorescence one of the most powerful techniques
Microscopes includes diascopic in biomedical research and clinical
fluorescene and episcopic fluorescene. pathology.
Diascopic Fluorescence: K. Reichert •• Fluorescence microscope allows the
and O. Heimstadt demonstrated a use of multicolour staining, labeling
fluorescence microscope using auto of structures within cells, and the
fluorescent specimens in 1911. measurement of the physiological state
This first type of fluorescence of a cell.
microscopy used transmitted light. •• Fluorescence microscope helps in
Light from the illumination source observing texture and structure of coal.
first passes through an excitation filter •• To study of porosity in ceramics, using
and subsequently to the specimen a fluorescent dye.
through a dark field condenser. This
•• To identify the Mycobacterium
eliminates most of the excitation light
tuberculosis.
from the imaging side of the system.
Episcopic Fluorescence: In episcopic 2.3 Electron Microscope
fluorescence microscopy, the excitation
Examining the ultra structure of cellular
light comes from above the specimen
components such as nucleus, plasma
through the objective lens. This is the
membrane, mitochondria and others
most common form of fluorescence
requires 10,000X plus magnification
microscopy today. In this microscope,
which was just not possible using Light
objective lens acts as both condenser
Microscopes. This is achieved by Electron
and objective. Quartz objective lenses
microscopes which have greater resolving
are required for deep ultraviolet
power than light microscopes and can
excitation.
. obtain higher magnifications.
In an electron microscope, a focused
electron beam is used instead of light to
19

Table 2.1: Difference between light and electron microscope
S.No Light microscope Electron microscope
1. Light is the illuminating source The beam of electrons is the

illuminating source
2. Specimen preparation takes usually Specimen preparation takes usually
few minutes to hours. Live or dead takes a few days. Only dead or dried
specimen may be seen specimen are seen
3. Condenser, objective and eye pieces All lenses are electromagnetic
lenses are made up of glasses
4. Specimen is stained by coloured dyes Specimen is coated with heavy metals
in order to reflect electrons
5. It has low resolving power (0.25µm to It has high resolving power (0.001µm),
0.3 µm). It has a magnification of 500X about 250 times higher than light
to 1500X microscope. It has a magnification
more than 100,000X
6. Vacuum is not required Vacuum is essential for its operation
7. Image is seen by eyes by through Image is produced on fluorescent
ocular lens screen or photographic plate
examine objects. Electrons are considered

In 1924, a French sci-
as radiation with wavelength in the range entist, Dr. De Broglie,
0.001–0.01 nm compared to 400–700 nm showed that an elec-
tron beam behaved
wavelength of visible light used in an opti- like waves and had a wavelength
much shorter than the sizes of mole-
cal microscope. Optical microscopes have
cules and atom when accelerated.
a maximum magnification power of 1000,
and resolution of 0.2 μm compared to Types of Electron Microscopes
resolving power of the electron microscope •• Transmission electron microscopes

(TEM)
that can reach 1,000,000 times and resolu-
•• Scanning electron microscopes (SEM)
tion of 0.2 nm. Hence, electron microscopes •• Scanning transmission electron
deliver a more detailed and clear image microscopes (STEM)
The electron microscope was invented
compared to optical m
icroscopes. Table 2.1
in 1931 by two German scientists, Ernst
differentiate
electron m
icroscope from Ruska and Max Knoll. Ernst Ruska later
received Nobel Prize for his work in 1986.
light microscope.
20

The Transmission Electron Microscope diffracted beam. The image is magnified
(TEM) was the first type of Electron and focused onto an imaging device,
Microscope to be developed. such as a fluorescent screen, on a layer of
photographic film, or to be detected by a
2.3.1 Principle sensor.
The fundamental principle of electron Sample Preparation
microscope is similar to light microscope.
Preparation of specimens is the most
In electron microscope, a high velocity
complicated and skillful step in EM. The
beam of electrons is used instead of
material to be studied under electron
photons. In the electron gun, electrons are
microscopy must be well preserved,
emitted from the surface of the cathode
fixed, completely dehydrated, ultrathin
and accelerated towards the anode by and impregnated with heavy metals that
high voltage to form a high energy sharpen the difference among various
electron beam. All lenses in the electron organelles.
microscope are electromagnetic. Charged
The material is preserved by fixation
electrons interact with the magnetic fields
with glutaraldehyde and then with
and magnetic force focuses an electron
osmium tetroxide. The fixed material
beam. The condenser lens system controls
is dehydrated and then embedded in
the beam diameter and convergence
plastic (epoxy resin) and sectioned with
angles of the beam incident on a specimen.
the help of diamond or glass razor of
The image is formed either by using
ultra-microtome.
the transmitted beam or by using the
Figure 2.8: Interaction between electron beams with specimen
21

In TEM, sample sections are 2.3.2 Working Principle and
ultrathin about 50–100 nm thick. These Instrumentation of TEM
sections are placed on a copper grid
The optics of the TEM is similar
and exposed to electron dense materials
to conventional transmission light
like lead acetate, uranylacetate,
microscope. A transmission electron
phosphotungstate. In SEM, samples can
microscope has the following components,
be directly imaged by mounting them on
an aluminum stub. 1. Electron gun
2. Condenser lens
Electron–Sample Interactions
3. Specimen stage
Interaction of electron beam with the
sample results in different types of 4. Objective lens and projector lens
electrons: Elastic scattered electrons, 5. Screen/photographic film/Charged
Inelastic scattered electrons, secondary Coupled Device (CCD) camera
electrons and backscattered electrons. Electron Gun consists of a tungsten
Almost all types of electron interactions filament or cathode that emits electrons
can be used to retrieve information about on receiving high voltage electric
the specimen. Depending on the kind of current (50,000–100,000 volts). A
radiation or emitted electrons which are
high voltage between the electron
used for detection, different properties
source (cathode) and an anode plate
of the specimen such as topography,
is applied leading to an electrostatic
elemental composition can be concluded.
field through which the electrons are
The following Figure 2.8 shows the
accelerated.
interaction of the electron beam with the
specimen. The emitted electrons travel through
vacuum in the microscope column. Vacuum
In Transmission electron microscope
is essential to prevent strong scattering
(TEM), a beam of electrons is transmitted
of electrons by gases. Electromagnetic
through an ultrathin specimen, interacting
with the specimen as it passes through. condenser lenses focus the electrons into
An image is formed from the interaction a very thin beam. Electron beam then
of the transmitted unscattered electrons travels through the specimen and then
through the specimen. through the electromagnetic objective
lenses. In a TEM microscope, the sample
Secondary electrons are mainly used
is located in the middle of the column. At
in scanning electron microscope (SEM)
the bottom of the microscope, unscattered
for imaging the surface topography of
electrons hit the fluorescent screen giving
biological specimens. The interaction
image of specimen with its different parts
of electron beam with samples results in
secondary electrons and backscattered displayed in varied darkness, according
electrons that are detected by standard to their density. The image can be studied
SEM equipment. directly, photographed or digitally
recorded. Figure 2.9 show the arrangement
22

Electron gun
Anode
Condenser lens
Specimen
Objective
aperture lens
Intermediate lens
Projetor lens
Fluorescent
screen
Figure 2.9: (a) Transmission microscope (b) Components of TEM (c) image under TEM
of components for transmission electron •• Crystallographic arrangement of

microscope. atoms
Information that can be obtained using •• Composition: elements and the
TEM include, their relative amounts
•• Topography: surface features,
texture 2.3.3 Working Principle and
Instrumentation of SEM
•• Morphology: shape and size of the
particles It is first built by Knoll in 1935. It is used
to study the three dimensional images of
23

Electron gun
Electron beam Anode
Magnetic lens
Backscattered
electron detector Secondary
electron detector
Stage
Specimen
Figure 2.10: (a) Scanning electron microscope (b) Components of SEM (c) image under SEM
the surfaces of cells, tissues or particles.

Foldscope – origami The SEM allows viewing the surfaces
based paper microscope of specimens without sectioning. The
A foldscope is an optical specimen is first fixed in liquid propane
microscope that can be at-180°C and then dehydrated in alcohol
assembled from simple components, at-70°C. The dried specimen is then
including a sheet of paper and a lens. coated with a thin film of heavy metal,
It was developed by an Indian Manu such as platinum or gold, by evaporation
Prakash. It consists of the following in a vacuum provides a reflecting surface
parts which are as follows: Lens stage, of electrons. In SEMs, samples are
sample stage, panning guide, ramp, positioned at the bottom of the electron
lens and magnetic cuppler. It has the column and the scattered electrons (back-
magnification of 140X and maximum scattered or secondary) are captured by
of 2400X electron detectors.
24

Table 2.2: Difference between SEM and TEM
S.no Properties SEM TEM

1. Types of electrons It is based on scattered It is based on transmitted
electrons that are emitted electrons
from the surface of a
specimen
2. Sample preparation Sample can be of any Laborious sample prepa-
thickness and is coated ration is required. The
with a thin layer of a heavy sample has to be cut into
metal such as gold or thin section s so as to
palladium and mounted on allow electrons to pass
an aluminum slab through it and are sup-
ported on TEM grids
3. Resolution The resolution is up to 20nm TEM has much higher
resolution than SEM. It
can resolve objects as
close as 1nm
4. Magnification The magnifying power of The magnifying power of
SEM is up to 100,000X TEM is up to 5,000,000X
5. Image formation SEM provides a 3 dimen- TEM provides a 2 di-
sional image. Secondary or mensional image. Trans-
back scattered electrons are mitted electrons hit a
captured, detected and dis- fluorescent screen giving
played on computer screen rise to a shadow image.
The image can be studied
directly by the operator
or photographed with a
camera
6. Application SEM is used to study the TEM is used to
topography and atomic study the interior of
composition of specimens cells, the structure
of protein molecule,
the organization of
molecules in viruses and
cytoskeletal filaments
and the arrangement of
protein molecules in cell
membranes
25

In SEM, there are several magnified visual or photographic images
electromagnetic lenses, including of objects or specimens that are too
condenser lenses and one objective lens. small to be seen with naked eye. Frits
Electromagnetic lenses are for electron Zernike a Dutch Physicist invented the
probe formation, not for image formation Phase Contrast Microscope and was
directly, as in TEM. Two condenser awarded Nobel Prize in 1953. It is the
lenses reduce the crossover diameter of first microscopic method which allows
the electron beam. The objective lens the observation of living cell. The image
further reduces the cross-section of the of the aperture is formed at the rear focal
electron beam and focuses the electron plane of the objective. In this plane there
beam as probe on the specimen surface is a phase shifting element or phase plate.
(Figure 2.10). Objective lens thus functions Deviated rays from object form structures
like a condenser. This is in contrast due to different refractive index. Light
to TEM where objective lens does the waves that are in phase (that is, their peaks
magnification. Major difference between and valleys exactly coincide) reinforce one
SEM and TEM are given in Table 2.2. another and their total intensity increases.
SEMs are equipped with an energy Light waves that are out of phase by exactly
dispersive spectrometer (EDS) detection one-half wavelength cancel each other
system which is able to detect and display and result in no intensity. Fluorescence
most of the X-ray spectrum. The detector microscopy is a very powerful analytical
normally consists of semiconducting tool that combines the magnifying
silicon or germanium. properties of light microscopy with
visualization of fluorescence. Examining
Scanning transmission electron
the ultra structure of cellular components
microscopy (STEM) combines the
such as nucleus, plasma membrane,
principles of transmission electron
mitochondria and others requires 10,000x
microscopy and scanning electron
plus magnification which was just not
microscopy and can be performed on
possible using Light Microscopes. It is first
either type of instrument. Like TEM,
built by Knoll in 1935. It is used to study the
STEM requires very thin samples and looks
three dimensional images of the surfaces
primary at beam electrons transmitted by
of cells, tissues or particles. The SEM
the sample. One of its principal advantages
allows viewing the surfaces of specimens
over TEM is in enabling the use of other of
without sectioning. The specimen is first
signals that cannot be spatially correlated
fixed in liquid propane at-180°C and then
in TEM, including secondary electrons,
dehydrated in alcohol at-70°C. Scanning
scattered beam electrons, characteristic
transmission electron microscopy
X-rays, and electron energy loss.
(STEM) combines the principles of
transmission electron microscopy and
Summary scanning electron microscopy and can be
Microscopes are specialized optical performed on either type of instrument.
instruments designed to produce One of its principal advantages over TEM
26

is in enabling the use of other of signals a. Objective lens
that cannot be spatially correlated in TEM, b. Electromagnetic lens
including secondary electrons, scattered c. Glass lenses
beam electrons, characteristic X-rays, and d. Condensor lenses
electron energy loss.
8. is used to illuminate
specimen in fluorescent microscope
Evaluation
a. Mercury arc lamp b. Sunlight
Multiple choice questions c. Tungsten lamp d. LED
1. Who invented PCM/ 9. Which among the following help us
a. Robert Koch in getting a 3-D picture of specimen?
b. Frits Zernike a. TEM
c. George Strokes b. SEM
d. Alexander Fleming c. Compound microscope
2. The component d. Simple microscope
that makes the difference between 10. Dye used to stain specimen in
phase contrast microscope and Bright fluroscent microscopic view
Field microscope is a. Acridine dye b. Rezazurin
a. Objective b. Phase plate c. Methylene Blue d. Flurochrome
c. Condenser d. Occular
3. Tumor cells can be diagnosed by Answer the following
a. PCM 1. Principle of PCM
b. BFM 2. Write about the special features of
c. Light Microscope TEM
d. Electron Microscope 3. List out the dyes used for fluorescent
4. In electron microscope light source is microscopy
a. Electric light b. Electron Beam 4. What are the different types of filters
used in fluorescent microscopy
c. Sun light d. Fluorescent
light 5. Define flurochromes.
5. What is the medium used in electron 6. Write the functions of Barrier filter
microscope? 7. Write about the components of PCM
a. Air b. Water 8. What are the Application of EM
c. Vacuum d. Light 9. Write about TEM – Introduction and
6. are mainly used Principle
in scanning electron microscope 10. Write a brief account on the
a. Transmitted electrons application of fluorescent microscopy
b. Primary electrons 11. Explain in detail about the principle
c. Secondary electrons construction and working of PCM.
d. Elastically scattered electrons 12. Explain about the Principle,
7. Lenses used in TEM Components and Mechanism of EM.
27

Chapter
3 Control of Microorganisms by
Chemical Methods
3.6 Antibiotics
Learning Objectives
3.7 Antimicrobial Susceptibility Testing
After studying this chapter the
3.8 Drugs Resistance Mechanisms
students will be able to,
• Defines the terms disinfectants, Control of microorganisms
antiseptics and antibiotics is essential in order to
• Describe major groups of prevent the transmission
antimicrobial chemical agents and of diseases, infection,
spoilage and to or remove
uses of disinfectants.
unwanted microbial
• Describe the factors related to contamination. Microorganisms are
effective disinfectants. controlled by means of physical agents and
• Discuss the classification of chemical agent. In 11th standard, we learnt
antibiotics and their mode of action. different physical methods of sterilization.
• Know the procedure used in Control by chemical agents refers to the use
antimicrobial susceptibility testing of disinfectants, antiseptics, antibiotics and
chemotherapeutic antimicrobial chemicals.
in clinical laboratory.
This chapter describes various chemical
• Knows the resistance mechanisms agents, their mode of action, and their
developed by pathogens against evaluation.
antibiotic or chemotherapy drugs.
Use of chemicals to sterilize objects
and to control microbial pathogen from
Chapter Outline causing diseases has been in practice since
centuries. A large number of chemicals
3.1 Disinfectants, Antiseptics and are now available for this purpose.
Antibiotics Commercial products which incorporate
these chemicals are used in a variety of
3.2 Factors Influencing the Antimicrobial conditions and they usually differ in their
Activity of Chemical Agents mode of action. No single chemical agent is
3.3 Mode of Action of Chemical Agents best for any and all purposes. Hence several
classes of chemicals have been identified
3.4 Major Groups of Antimicrobial and new compounds are developed that
Chemical Agents possess destructive properties in terms of
3.5 Evaluation of Antimicrobial their suitability for practical application.
Chemical Agents
28

3.1 Disinfectants, Antiseptics and Following Table gives few examples of
Antibiotics antimicrobial chemical agents that destroy
Disinfection is the elimination of unwanted microorganisms.
microorganisms from inanimate objects or
Disinfection Antiseptics Antibiotics
surfaces. The term disinfectant is used for an
Chlorine, Phenol, Penicillin,
agent used to disinfect inanimate objects or Copper Tincture of Streptomycin
surfaces but is generally toxic to use on human Iodine
tissues. Antiseptic refers to an agent that kills
Basic terms used in chemical control
or inhibits growth of microorganisms but is
of microorganism are mentioned in
safe to use on human tissues.
Table 3.1 and Table 3.2 Describes the
Antibiotics are produced by
microorganisms kill or inhibit the growth difference between chemical disinfectant
of other microbes. against bacteria.
Table 3.1: Basic terms used in Chemical sterilization

Term Meaning
Disinfection The selective elimination of certain undesirable microorganisms to prevent
their transmission directed against their metabolism or structure; applies to
the use directly on inanimate objects.
Antisepsis Prevention of the growth or activity of microorganisms by inhibition
or killing; applies to the use of chemicals on living tissue
-cide Suffix used to denote agents, usually chemical, that kill.
Commonly used terms are bactericide, fungicide, virucide, and algicide. The
term germicide is used if the agents kill pathogens but not necessarily spores.
An agent that kills bacterial spores is a sporicide.
-static Suffix used to denote agents, usually chemical, that prevents growth but
do not necessarily kill the organism or bacterial spores. Commonly used
terms include bacteriostatic and fungistatic.
Term Action Examples

Algicide Agent that kills algae Copper sulfate
Bactericide Agent that kills bacteria Chlorohexidine, ethanol
Biocide Agent that kills living organisms Hypochlorite (bleach)
Fungicide Agent that kills fungi Ethanol
Germicide Chemical agent that Formaldehyde, silver,
specifically kills pathogenic mercury
microorganisms
Sporicide Agent that kills bacterial Glutaraldehyde
endospores
Virucide Inactivates viruses so that they Cationic detergents (qua-
lose the ability to replicate ternary ammonium salts
of acetates, chlorides)
29

Table 3.2: Difference between Bactercidal and Bacteriostatic
Bactercidal Bacteriostatic
Bactercidal refers to antibiotics that Bacteriostatic refers to antibiotics that prevent
kill bacteria the growth of bacteria
Action is irreversible Action is reversible
Inhibit the cell wall formation of Inhibit DNA replication and protein synthesis
bacteria of bacteria
Do not work with the immune system Work with the immune system of the host to
of the host prevent the growth and reproduction of bacteria
MBC refers to the concentration of MIC is the m inimum drug concentration which
the drug required to kill 99.99% of the inhibits the bacterial growth
bacterial population
Examples include betalactam antibiot- Examples include tetracyclines, spectinomycin,
ics, cephalosporins, and vancomycin chloramphenicol, sulfonamides, etc.
3.2 Factors Influencing the e. Number of organisms present

Antimicrobial Activity of Chemical The larger the number of organisms,
Agents the greater will be the time required for
The following factors will affects the disinfection.
activity of a disinfectant or antiseptic and f. The kinds of microorganisms present
these should be borne in mind during use. - Presence of spores
a. The Concentration and kind of a Spores are exceptionally resistant to the
chemical agent used great majority of disinfection.
The higher the concentration of the
g. Mode of action of antimicrobial agent
germicide the greater will be the rate of
killing. This is particularly important with The nature of the material bearing the
the phenolic group of compounds, whose microorganism.
activity falls off very rapidly with dilution.
3.3 Mode of Action of Chemical
b. Time of exposure to the agent
Agents
In general germicidal activity is increased
with time and a sufficient exposure is Chemical agents act on microorganisms
imperative for efficient disinfection. by:
c. Temperature at which the agent is used •• They may damage the lipids and
An increase of temperature will also raise proteins of the cytoplasmic membrane
the rate of killing. of microorganisms.
•• They may denature microbial enzymes
d. Presence of Organic matter
and other proteins usually by disrupting
Most germicides are reduced in activity
by the presence of organic matter and the hydrogen and disulfide bonds that
particularly by the presence of proteins give the protein its 3-D shape. This
such as those in body fluids. blocks metabolism function.
30

Chemical disinfectants are grouped presence of organic material, and remain
by the types of microbes and infectious active on surfaces long after application.
agents they are effective against. However, they have a disagreeable odour
High-level germicides kill vegetative and can cause skin irritation.
cells, fungi, viruses and endospores and Hexachlorophene is one of the most
can ultimately lead to sterilization. popular antiseptics because it persists
Intermediate-level germicides cannot on the skin once applied and reduces
kill all viruses and are less effective against skin bacteria for a long period. However,
endospores. it can cause brain damage and is now
used in hospital nurseries to control and
Low-level germicides kill vegetative
outbreak of gram positive (Staphylococcal)
cells and some enveloped viruses, but are
infections. It is mainly used in soaps and
ineffective against endospores.
creams. It is an ingredient of various
dermatological preparation used for skin
3.4 Major Groups of Antimicrobial
disorders.
Chemical Agents
A large number of chemical agents are in 2. Alcohols
common use. Some of the more common Alcohols are among the most widely
groups are listed below. used disinfectant and antiseptic. They
are bactericidal and fungicidal but not
1. Phenol and Phenolics
sporicidal. Alcohols can destroy the lipid
Phenol was the first widely used chemical component of enveloped viruses. The
antiseptic and disinfectant. In 1867, two most popular alcoholic germicides
Joseph Lister employed carbolic spray to are ethanol and isopropanol. They act
reduce the risk of infection in surgical by denaturing proteins and dissolving
theatres. Phenol derivatives called membrane lipids. The recommended
phenolics contain altered molecules optimum concentration of ethanol is
of phenol useful as antiseptics and 70%, but concentration between 60% and
disinfectants. The phenolics damage cell 95% are employed to kill germs as well.
membranes and inactivate enzymes of Thermometers and small instruments are
microorganisms, while denaturing the disinfected by soaking in alcohol for 10 to
proteins. Phenolics includes cresols, such 20 minutes.
as Lysol, as well as several bisphenols,
such as hexachlorophene. Today phenol 3. Halogens
and phenolics such as cresol, xylenol, Halogen compounds are broad spectrum
and orthophenyl phenol are used compounds that are considered low
as disinfectants in laboratories and toxicity, low cost and easy to use. Among
hospitals. the halogens, iodine and chlorine are
The commercial disinfectant Lysol is important antimicrobial agents. Small
made of mixture of phenolics. Phenolics quantities of drinking water can be
are tuberculocidal, effective in the disinfected with halazone tablets.
31

a. Iodine 4. Heavy Metals
Iodine compound are broad spectrum For many years the ions of heavy metals
and considered effective for a variety of such as mercury, silver, arsenic, zinc, and
bacteria, mycobacteria, fungi and viruses. copper were used as germicides. More
The alcoholic tincture of iodine is highly recently these have been superseded
active against gram positive organisms by other less toxic and more effective
and so is used as a skin antiseptic. It stains germicides. Many heavy metals are more
the skin. Iodine combines with microbial bacteriostatic than bactericidal. There are
protein and inhibits their function. a few exceptions. 1% solution of Silver
nitrate is often applied to the eyes of
b. Chloride
infants to prevent ophthalmic gonorrhea.
Chloride is also used as a gas to maintain
Silver sulfadiazine is used on burns.
a low microbial count in drinking water.
Copper sulfate is an effective algicide used
Chlorine together with ammonia called
in lake and swimming pools to retard the
chloramines are used to sanifige glasswall
growth of algae.
and eating ulenits. Sodium hypochlorite
(NaOCl) is one of the most widely used
In many hospitals,
chlorine containing disinfectants. Low
Erythromycin is used
concentrations (2-500ppm) are active
instead of Silver nitrate
against vegetative bacteria, fungi and most
because it is effective
viruses. Rapid sporicidal action can be
against Chlamydia as well as Neisseria.
obtained around 2500ppm, however this
concentration is very corrosive so should be
limited in its use. High concentrations are Heavy metals combine with sulfhydryl
also irritating to the mucous membranes, (SH) groups of proteins and inactivate
eyes and skin. Chlorine compounds are them. High concentration of metallic
rapidly inactivated by light and some salts, particularly those of mercury, silver
metals so fresh solutions should always and copper coagulate cellular proteins that
be used. Hypochlorites should never be results in damage or death of the microbial
mixed with acids or ammonia as this will cell. The most toxic heavy metals are the
result in the release of toxic chlorine gas. mercury, silver, and copper.
c. Iodophores 5. Quaternary Ammonium Compounds

They combinations of iodine and (Quats)
organic molecules are called Iodophores. The most widely used surface active agents
They include wescodine, betadine and are the cationic detergents, especially
previdone. These iodophore contains the quaternary ammonium compounds
surface active agents. They cause less (quats).
irritation to the skin than free Iodine Quaternary Ammonium compounds
and do not stain. They are used for are strongly bactericidal against Gram
cleaning wounds and as a general purpose positive bacteria and less active against
laboratory disinfectant for discarded jars. gram negative bacteria. These include
32

agents such as cetrimide, bromide and It is less irritating than formaldehyde and
benzalkonium chloride. Their antibacterial is used to disinfect hospital and laboratory
activity is antagonized by soaps and certain equipments. Glutaraldehyde usually
organisms like Pseudomonas. They are disinfects objects within about 10 minutes
useful for washing cutlery in catering but may require as long as 12 hours to
industry and for cleaning wounds in destroy all spores.
hospitals. Savlon, a popular antiseptic, is a
mixture of cetrimide and chlorohexidine Infobits
and is active against Gram negative bacteria.
They are used as skin disinfectants and as a Disinfection of Rooms
preservative of ophthalmic solution. Fumigation with gaseous disinfectants
The combined properties of was at one time commonly performed
germicidal activity and low toxicity, high after a room had been occupied by a
solubility in water, stability in solution patient with an infections disease.
and non-corrosiveness have resulted in Sulphur di oxide, generated by burning
many applications of quaterneries as sulphur was the popular agent for this
disinfectants and sanitizing agents. purpose but it is effective only if the
Quats are also fungicidal, amoebicidal, relative humidity is 60 percent or more.
and virucidal against enveloped
viruses. They do not kill endospores or These are highly reactive molecules that
mycobacteria. combine with nucleic acids and proteins
and inactivate them. They disrupt the
Infobits function of cell organelles and kill the cells
probably by cross linking and alkylating
If you mouthwash bottle fills with foam
the molecules. These are sporicidal and
when shaken, the mouthwash probably
can be used as chemical sterilants.
contains a quat.
7. Gaseous Sterilization
6. Aldehydes Gaseous disinfectants (alkylating
Aldehydes are highly effective, broad agents) are used for the sterilization or
spectrum disinfectant. The most which disinfection of hospital equipment that is
typically achieve its anitimicrobial action bulky or heat labile. The most widely used
by denaturing proteins and disrupting gases are ethylene oxide, formaldehyde
nucleic acids. Commonly used aldehydes and β Propiolactone.
are formaldehyde and glutaraldehyde.
Formaldehyde is usually dissolved in water Ethylene oxide (EtO)
or alcohol before use for moldehyde is used Ethylene oxide has a boiling point of
as a surface disinfectat and a fumigant 10.8°C. It is highly inflammable and
and has been used to decontaminate in explosive in pure form, but is safe to
animate objects. A concentration 2% of handle when mixed with Carbon dioxide.
glutaraldehyde is an effective disinfectant. It is powerful in the killing of all bacteria,
33

including tubercule bacilli and spores. It not difficult to eliminate. It destroys
is an effective sterilizing agent because it microorganisms more readily than
rapidly penetrates packing materials, even ethylene oxide but does not penetrate
plastic wrappers. To be potent, however, materials well and may be carcinogenic.
the humidity and temperature must be For these reasons, BPL has not been used
carefully controlled within narrow limits. as extensively as EtO.
It is highly toxic on contact with the
skin or mucous membrane. Materials 3.5 Evaluation of Antimicrobial
that have been sterilized with ethylene Chemical Agents
oxide must be set aside in detoxification Testing of antimicrobial agents is a complex
chambers for a few days to allow the process regulated by two different federal
gases to dissipate. It is frequently used to agencies.
sterilize heart lung machines and plastic
The U.S. Environmental Protection
items like catheters.
Agency regulates disinfectants, where as
agents used on humans and animals are
Recently vapour
under the control of the Food and Drug
– phase hydrogen
Administration.
peroxide has been
used to decontaminate Testing of antimicrobial agents often
biological safety cabinets. begins with an initial screening test to see
if they are effective and at what required
concentrations.
Formaldehyde
Laboratory techniques for the
It is highly bactericidal. Formaldehyde evaluation of antimicrobial chemical
is used as 40% formalin with humidity agents are conducted by one of the
at around 50%. It causes irritation. It is following three general procedures. In
used occasionally to fumigate rooms and each procedure, the chemical agent is
disinfect respirators. tested against a specified microorganisms
Betapropiolactone (BPL) referred to as the test organism.
This is occasionally employed as a Chemical Methods of Microbial Control
sterilizing gas in the liquid form. It has
been used to sterilize vaccines, tissue i. Chemical agents are used on living
grafts, surgical instrument and enzyme as tissue (as antiseptics) and on inanimate
a sterilants of blood plasma, water, milk objects (as disinfectants).
and as a vapour – phase disinfectant in ii. Few chemical agent achieve sterility.
enclosed spaces, short-term inhalation Evaluating a disinfectant
exposure to betapropiolactone causes
1. The phenol coefficient is the comparison
server irritation of the eyes, nose, throat
of one chemical’s disinfecting action
and respiratory tract in humans.
with that of phenol, applied for the same
BPL decomposes to an inactive form length of time on the same organisms
after several hours and is therefore under identical conditions.
34

2. In the use-dilution test, a series of tubes compound in relation to phenol. Phenol
contain increasing concentrations of coefficient is calculated by dividing the
disinfectant; the more the chemical concentration of test disinfectant at which
can be diluted and still be effective, the it kills the organism by the concentration
higher its rating. of phenol at which it kills the organism in
3. In the filter paper method, a disk of 10 minutes and not in 5 minutes under the
filter paper is soaked with a chemical same conditions. This method is used for
and placed on an inoculated agar plate; evaluating the efficiency of water-miscible
a clear zone of inhibition indicates disinfectants.
effectiveness.
Series of 10 test tubes with 2ml of
Agar Plate Method distilled water is taken (Figure 3.1).
Phenol is added to first test tube and
A plate of agar medium is inoculated with dilution is made by transferring 1ml to
the test organism and the chemical agent
next tube up to 5 dilutions. Similarly
is placed on the surface of the medium.
commercial disinfectant is also diluted.
The chemical solution is first impregnated
Pure culture of test organisms, such as
in absorbent papers or confined by
a hollow cylinder placed on the agar Staphylococcus aureus or Salmonella
surface. Following incubation, the plate is typhi, is added to test tubes. Subcultures
observed for a zone of inhibition around from these tubes incubated at 37°C for
the chemical agent. This is particularly 48 hours are examined for the presence or
suiTable for semisolid preparations. absence of growth at intervals of 5, 10 and
15 minutes. The highest dilution that kills
Tube Dilution Methods the bacteria after 10 minutes, but not after
Appropriately diluted water soluble liquid 5 minutes is used to calculate the phenol
substances are dispensed into sterile test coefficient (Table 3.3),
tubes and are inoculated with a measured Table 3.3: Illustration of phenol coefficient
amount of the test organism. At specified determination
intervals, a transfer is made from this
tube into tubes of sterile media that are Chemical Presence of Growth in
then incubated and observed for the Agent and Subcultures (minutes)
appearance of growth. It is necessary in Dilution 5 10 15
this type of procedure to ascertain whether Phenol
the inhibitory action is bactericidal and
1:80 - - -
not bacteriostatic. This approach can
also be used to determine the number 1:90* + - -
of organisms killed per unit time by 1:100 + + -
performing a plate count on samples taken
Test Chemical
at appropriate intervals.
1:400 - - -
Phenol Coefficient Test 1:450 +
+ - -
Phenol coefficient is a measure of 1:500 + + -
the bactericidal activity of a chemical
35

Figure 3.1: Phenol coefficient test
Phenol dilution of 1:90 showed at discovery and development of Penicillin

5 minutes but no growth at 10 minutes that a truly wide ranging search for
Test Chemical dilution of 1:450 should antibiotics was initiated.
growth at 5 minutes but not growth at
10 minutes phenol coefficient of test Antibiotics are not
chemical = 450/90=5. effective against viral
infections such as the
3.6 Antibiotics common cold.
The term ‘antibiotic’ was derived from

‘antibiosis’ which refers to the suppression Historical Development
of microorganisms due to secretion of The first chemotherapeutic agent,
toxic or inhibitory compounds by other discovered by Paul Ehrlich, was Salvarsan,
microorganisms. Although antibiosis has used to treat syphilis.
been observed by many scientific workers Alexander Fleming discovered the
fairly frequently towards the end of the first antibiotic, penicillin, in 1929; its first
nineteenth century, it was not until the clinical trails were done in 1940.
36

Antibiotics are produced by species of Broad – spectrum antibiotics
Streptomyces, Bacillus, Penicillium and These attack different kinds of microbial
Cephalosporium. pathogens and therefore find wider
medical use. Antibacterial antibiotics of
Infobits broad – spectrum are effective against
both Gram positive and Gram negative
1904 Ehrlich found that the dye try pan
bacteria. They also attack pathogens
red was active against the trypanosome
belonging to Mycobacteria, Rickettsia, and
that causes African sleeping sickness
Chlamydia. Similarly, broad – spectrum
and could be used therapeutically.
antifungal antibiotics attack different type
of fungal pathogens.
Drugs such as the sulfonamides are
sometimes called antibiotics although they Narrow – spectrum antibiotics
are synthetic chemotherapeutic agents Narrow – spectrum antibiotics are
which are not microbially synthesized. categorized as those that are effective
only against a limited variety of microbial
Classification of Antibiotics
pathogens. These antibiotics are quite
The antibiotics are usually classified on valuable for the control of microbial
the basis of: pathogens that fail to respond to other
•• Target group of microorganisms antibiotics. For example, vancomycin
is a narrow spectrum glycopeptide. It
•• Antimicrobial spectrum and
is an effective bactericidal agent for
•• Mode of action gram – positive penicillin resistant
Classification based on target group bacterial pathogens belonging to genera
of microorganisms Staphylococcus, Bacillus, and Clostridium.
Based on the target group, the antibiotics
3.6.1 Mode of Action of Antibiotics
can be classified as antibacterial, antifungal
and antiviral. The mode of action of antibiotics varies
as they damage pathogens in several ways
Classification based on Antimicrobial
(Flowchart 3.1). Some of the important
spectrum
actions of therapeutic drugs in microbial
Antimicrobial spectrum or antibiotic pathogens are as follows.
spectrum refers to the range effectiveness
Cell wall synthesis Protein synthesis
of antibiotics on different kind of
Nucleic acid synthesis Cell membrane
microorganisms, i.e. the range of different
disruption Metabolic pathways blockage
kind of microorganisms that can be
inhibited, killed, or lysed by a particular
1. Inhibition of Cell Wall Synthesis
type of antibiotic.
The most selective therapeutic antibiotics
The susceptibility of microorganisms
are those that interfere with the synthesis
to individual antibiotic varies significantly
of bacterial cell walls. These drugs posses
and on account of this, the antibiotics can
a high therapeutic index because bacterial
be classified in two groups as,
37

Mode of Action
Nucleic Cell Metabolic

Cell wall Protein
acid membrane pathways
synthesis synthesis
synthesis disruption blockage
Flowchart 3.1: Mode of action of antibiotics
cell walls have a unique structure which selectively toxic as other drugs. This is due
is not found in eukaryotic cells. The to the fact that prokaryotic and eukaryotic
important cell wall attacking drugs are nucleic acid synthesis mechanisms do
Penicillin, Cephalosporin, Ampicillin, not vary greatly. Example Quinolones,
Methicillin and Vancomycin. Novobiocin, Actinomycin and Rifampin
2. Inhibition of Protein Synthesis 4. Disruption of Cell Membrane

Many therapeutic antibiotics discriminate There are some antimicrobial drugs or
between prokaryotic and eukaryotic antibiotics that act as cell membrane
ribosomes and inhibit protein synthesis. disorganizing agents. Polymixins are such
The therapeutic index of these drugs is drugs of clinical importance.
fairly high, but not as favourable as that E.g. Polymixin B and Polymixin
of cell wall synthesis inhibitors. Several E (colistin)
of these drugs are medically useful and
effective research tools because they block 5. Blocking Metabolic Pathways
individual steps in protein synthesis.
Some therapeutic drugs act as
Some therapeutic drugs bind to 30S while
antimetabolites and block the functioning
others attach to 50S ribosomal subunits.
of metabolic pathways. They competitively
Example Streptomycin, Chloramphenicol,
inhibit the key enzymes in the metabolic
Tetracyclin and Erythromycin
pathway. Example Sulfonamides,
Trimethoprim, Dapsone and Isoniazid
3. Inhibition of Nucleic Acid Synthesis
(Figure 3.2).
Some antimicrobial drugs or antibiotics
inhibit nucleic acid synthesis. These are not 3.7 Antimicrobial Susceptibility
Testing
A chemotherapeutic
Antimicrobial susceptibility tests
agent destroys
are used to determine the type and
or inhibit the
quantity of antimicrobial agents used in
intracellular parasite
chemotherapy. One of the most important
by penetrating the cells and tissues of
functions of a clinical laboratory is to
the host in effective concentrations.
determine the antimicrobial susceptibility
38

Figure 3.2: Mechanism of Antibiotic action
of pathogens refers to the limitation Filter paper discs containing known

of pathogens to grow in the presence concentrations of antimicrobial agents are
of effective antibiotics. There are two placed onto the surface of an agar plate
methods that can be used to determine (Muller – Hinton agar medium) inoculated
the susceptibility of a potential pathogen with the test bacterium (Figure 3.3). The
to antimicrobial agents. They are: plate is incubated for 16 to 18 hours, and
•• Disk diffusion method the zones of inhibition are read around
each paper disc. During the incubation
•• Tube dilution method
periods, the antimicrobial agent diffuses
through the agar, and a concentration
Disc Diffusion Method (Kirby – Bauer
gradient of agent is established. (Figure)
Test)
At some point in this gradient, growth
William Kirby and Alfred Bauer, in of the susceptible bacteria is suppressed,
1966 first introduced the principle and no growth is observed within a
of measuring zones of inhibition circular zone around disc. The size of a
around antibiotic discs to determine zone of inhibition must be compared to
antimicrobial agent susceptibilities. It is a standard Table for that particular drug
a rapid, convenient method to determine before accurate comparisons can be made.
the susceptibilities of microorganisms to Thus, enabling to classify pathogens as
antimicrobial agents and a most common susceptible (S), intermediate or resistant
procedure used in susceptibility testing (R) to a drug. The procedure is highly
in clinical laboratory. regulated and controlled by the clinical
39

Figure 3.3: Microorganisms in Petri plate showing sensitivity and resistance towards
antibiotics
and laboratory standards institute (CLSI) growth is determined by the turbidity in

and must be accompanied by a rigorous each tube. Tubes containing moderate to
quality assurance program including high concentrations of the antimicrobial
performance by certified and/or licensed agent would normally expected to have no
personnel when the results are to be growth.
reported in clinical settings.
Minimal Inhibitory Concentration
Tube Dilution Method (Mic) Test
In this method, the test bacterium is The potency of an effective antimicrobial
inoculated into broth tubes containing agent is expressed in terms of minimal
serial dilution of antimicrobial agent. inhibitory concentration (MIC). It is
The inoculated cultures are incubated the smallest amount of drug that will
for a suiTable period of time (usually inhibit the growth of pathogen. The
24 hours), and the presence or absence of MIC is determined by serial dilutions of
40

antimicrobial agents in tubes with standard The lowest concentration of drug for
amount of bacteria. Turbidity (cloudiness) which no growth occurs is the minimum
after incubation indicates bacterial growth bactericidal concentration.
and lack of turbidity indicates that the The tube dilution method is considered
growth of bacteria is inhibited. accurate for determining susceptibility of a
pathogen to precise quantities antimicrobial
Etest agent. However, the method is time
This is another test to determine the consuming, expensive, and not practical
minimum inhibitory concentration where for use in most clinical laboratories for
a plastic strip containing a gradient of the routine susceptibility testing.
antimicrobial agent is used (Figure 3.4). An
elliptical zone of inhibitory concentration Infobits
can be noted with the help of a scale
What is CRE?
printed on the strip.
CRE, which stands for carbapenem
resistant Enterobacteriaceae, is the most
fearsome family of germs because it is
resistant even to last-resort antiboitcs.
3.8 Drugs Resistance Mechanisms

Some microbes respond predictably
to certain drugs making selection of
treatment easy. Other microbes may vary
in their responses, and laboratory tests are
usually required to ensure that the selected
therapy is appropriate. Chemotherapeutic
effectiveness depends upon the sensitivity
of the pathogen to the agent. Antibiotic
Figure 3.4: E – test resistance, however, may develop in
microbes within the population. In fact, the
The Minimal Bactericidal history of chemotherapy has been closely
Concentration (MBC) Test paralleled by the history of drug resistance.
None of the therapeutic drugs
MBC test is similar to MIC, the minimal
(antibiotic) inhibits all microbial pathogens
bactericidal concentration test is used to
and some microbial pathogens possess
determine the amount of antimicrobial
natural ability to resist to certain antibiotics.
agent required to rather kill the pathogen Bacteria become drugs resistant using
inhibit its growth. In MBC test, samples several different resistance mechanisms. A
taken from MIC tubes are transferred to particular type of resistance mechanism is
drug free plates. Bacterial growth in these not confined to a single class of drugs. Two
subcultures indicates that some bacterial bacteria may employ different resistance
cells have survived antimicrobial drug. mechanisms to counter the same antibiotic.
41

Figure 3.5: Drugs resistance mechanisms
Summary
Chemical control refers to the use of
disinfectants, antiseptics, antibiotics
and chemotherapeutic antimicrobial
chemicals. Disinfection is the elimination
of microorganism, but not necessarily
endospores, from inanimate objects or
surface. A disinfectant is an agents used to
disinfect inanimate objects but generally
However, bacteria acquire drugs resistance to toxic to use on human tissues. An
using resistance mechanisms such as antibiotic is a metabolic product produced
reduced permeability to antibiotic, efflux
by one microorganisms that inhibits or
(pumping) antibiotic out of the cell, drugs
kills other microorganism. Synthetic
inactivation through chemical modification,
chemicals that can be used therapeutically.
target modification and development of a
resistant biochemical pathway (Figure 3.5). An agent that is static in action inhibits
the growth of microorganism. An agent
Infobits that is cidal in action kills microorganism.
Selective toxicity means that the chemical
Methicillin-resistant staphylococcus being used should inhibit or kill the
aureus (MRSA) is a bacteria that intended pathogen without seriously
is resistant to many antiobiotics. harming the host. A broad spectrum agent
Staph and MRSA can cause a variety is one generally effective against a variety
of problems ranging from are skin of gram positive & gram negative bacteria.
infections and sepsis to pneumonia to A narrow spectrum agent generally works
blood stream infections. against just Gram positive, gram negative
or only a few bacteria.
42

Evaluation 6. In the disk-diffusion assay, a large
zone of inhibition around a disk to
Multiple choice questions
which a chemical disinfectant has
1. Identify the term been applied indicates of
that describes a the test microbe to the chemical
disinfectant that can disinfectant.
kill bacteria:
a. Susceptibility or Sensitivity
a. Bactericidal
b. Resistant
b. Bacteriostatic
c. Intermediate
c. Pathogenic
d. None of these
d. Bacteriosis
7. Which of the following agents are
2. Which of the following is not a used as a preservative in ophthalmic
disinfectant containing a heavy solution?
metal?
a. Alcohol
a. Silver b. Mercury
b. Quaternary ammonium salts
c. Zinc d. Chlorine
c. Phenol
3. Which of the following is most
d. Aldehydes
effective for sterilizing mattresses and
plastic petriplates? 8. Which of the following chemical lack
penetrating power?
a. Chlorine
a. Phenol
b. Ethylene oxide
b. Iodine
c. Glutaraldehyde
c. Ethylene oxide
d. Ultraviolet radiation
d. Beta-propiolactone
4. is used
to prevent infection by killing or 9. Polymyxins inhibits the growth of the
inhibiting pathogen growth on animal microbes by carrying get which of the
tissue. following actions?
a. Bacteriostatic agent b. Sanitizer a. Inhibition of cell-wall synthesis
c. Disinfectant d. Antiseptic b. Damage to cell membrane
5. Which of the following refers to a c.

Inhibition of nucleic acid and
germicide that can kill vegetative cells protein synthesis
and certain enveloped viruses but not d.
Inhibition of specific enzyme
endospores? systems
a. High-level germicide 10. All of the following are sporicidal
b. Intermediate-level germicide except
c. Low-level germicide a. Glutaraldehyde b. Ethylene oxide
d. Sterilant c. Formaldehyde d. Alcohol
43

Answer the following 7. Give an account on Gaseous
1. Define Disinfectants/antiseptics/ sterilization ?
antibiotics. 8. Describe the test is used to evaluate
2. Difference between Bacteriostatic antimicrobial agent ?
and Bactericidal ? 9. How antibiotics the therapeutic drugs
3. What is Iodophores ? acts on target microorganisms?
4. Explain the mode of action of chemical 10. Through disc diffusion method
agents against microorganisms? how an antibiotic sensitivity of
microorganism is evaluated and
5.
Listout the major groups of
explain the test ?
antimicrobial chemical agents with
an example. 11. What is E – test?
6. Give examples of antimicrobial

chemical agent which act as both
disinfectant and antiseptics ?
44

Chapter
4 Microbial Metabolism
4.3 Generation of ATP

Learning Objectives
4.4 Carbohydrate Catabolism
4.5 Tricarboxylic Acid Cycle
4.6 Electron Transport Chain
• Identify the role of ATP in cellular
activities. 4.7 Lipid Catabolism
• Define metabolism and describe the 4.8 Protein Metabolism
fundamental differences between 4.9 Fermentation
catabolism and anabolism.
4.10 Enzymes
• Explain oxidation – reduction
reaction.
All living organisms are
• List and provide examples of three
constantly in need of
types of phosphorylation reactions
energy to function. The life
that generates ATP.
support activity of even the
• Describe the Carbohydrate, most structurally simple
Lipid, Protein and its pathways organism involves a large
(Glycolysis, Krebs cycle, electron number of complex biochemical reactions.
transport chain) Living cells carry out three major types
• Electron transport chain and of processes namely Chemical Process,
chemiosmotic model for ATP Transport Process and Mechanical
generation. Process. In chemical processes, energy is
• Understand about the types of required to synthesize complex biological
fermentation and its products. molecules from much simpler molecules.
• Describe the mechanism of Transport processes require energy to take
enzymatic activity and significance up nutrients, eliminate waste, and maintain
of microbial enzymes. ion balance. Mechanical processes require
energy to change the physical location of
structures within cells. Even during resting
Chapter Outline state, a substantial amount of energy is
needed for fundamental functions of
4.1 Metabolism cells. All living system obeys the laws of
4.2 Energy of Chemical Reaction thermodynamics. This law analyzes energy
45

changes in a collection of matter called 4.1 Metabolism
system (a cell or a plant). The term Metabolism refers to the sum
The energy exchanges between the of all bio chemical reactions that occur
system and the surrounding balance each within a living cell. Chemical reaction
other. All chemical reactions in cells involve either release energy or require energy.
energy transformation. (For example: Metabolism can be viewed as an energy
Photosynthetic bacteria transform balancing act. It can be divided into two
radiant energy into chemical energy). In classes of chemical reactions namely
living cells thermodynamic changes are Catabolism and Anabolism.
essential for biological function such as
growth, reproduction, photosynthesis and Catabolism: It is called catabolic or
respiration. Microorganisms obtain degradative reactions because complex
energy and nutrients for their survival and organic compounds are broken down
reproduction through metabolism. The into simples ones. Catabolic reactions
microbial species and ecological niche can are generally hydrolytic reactions. It is
often be differentiated from each other enzyme regulated chemical reaction that
based on metabolic characteristics. The release energy and they are exergonic
metabolic reaction often allows the use of Example: Break down of sugar into Carbon
micro organisms in fermentation process dioxide and water in cells.
and biogeochemical cycle. Anabolism: It is called anabolic or
biosynthetic reactions because complex
Three fourth of the organic molecules are formed from
energy is derived simples ones. Anabolic process often
from carbohydrate involves dehydration, are bio synthesis
that we consume and reactions (Figure 4.1). It is enzyme
Glucose is the major fuel for all living regulated energy requiring reaction and
organisms. they are endergonic. Examples: Formation
of proteins from amino acids.
Figure 4.1: Catabolic and anabolic reactions

46

Catabolic reactions furnish the energy energy. ATP consists of an adenosine unit
needed to drive anabolic reactions. composed of adenine, ribose with three
This coupling of energy requiring and phosphate groups. In ATP and some other
energy releasing reactions is made phosphorylated compounds, the outer
possible through the molecule Adenosine two phosphate groups are joined by an
tri-phosphate (ATP). anhydride bond.
Some of the other high energy
4.2 Energy of Chemical Reaction nucleotides involved in biochemical
Light energy is trapped by phototrophs processes are given in Table 4.1.
during photosynthesis, in which it is Table 4.1: High energy nucleotides involved
absorbed by bacteriochlorophyll and in biosynthesis
other pigments and converted to chemical
energy for cellular work. The energy is Name of the
Biosynthesis
required by the bacterium for synthesis Nucleotide
of cell wall or membrane, synthesis of Uridine triphosphate Polysaccharide
enzymes, cellular components repair (UTP)
mechanism growth and reproduction. Cytidine triphosphate Lipid
(CTP)
Some change of energy occurs whenever
Guanidine triphosphate Protein
bonds between atoms are formed or
(GTP)
broken during chemical reactions. When
a chemical bond is formed, energy is Nutrients are broken from highly
required. Such a chemical reaction which reduced compounds to highly oxidized
requires energy is called an endergonic compounds within the cells. Much of
reaction, meaning that energy is directed the energy released during oxidation -
inward. When a bond is broken, energy is reduction reactions is trapped within the
released. A chemical reaction that release cell by the formation of ATP. A phosphate
energy is an exergonic reaction, meaning group is added to ADP with the input of
that energy is directed outward. energy to form ATP.
During chemical reaction energy ATP + H2O
is either released or absorbed and the ADP + pi (ΔG° = −7.3 K Cal/mol)
quantum of energy liberated or taken up ATP + H2O
is that is useful energy and is referred to AMP + ppi (ΔG° = −10.9 K Cal/mol)
Free Energy Change (ΔG) of the reactions. ATP is ideally suited for its role as an
energy currency. It is formed in energy
4.2.1 High Energy Phosphate trapping and energy generating processes
Adenosine Tri-Phosphate (ATP) is the such as photosynthesis, fermentation,
principal energy carrying molecule of all and aerobic respiration. In bacterial and
cells and is indispensable to the life of the archeal cells, most of the ATP is formed
cell. It stores the chemical energy released on the cell membrane, while in eukaryotes
by some chemical reactions, and it provides the reactions occur primarily in the
the energy for reactions that require mitochondria (Figure 4.2).
47

Figure 4.2: Structure of ATP
4.2.2 Oxidation – Reduction Reactions 4.3.1 Substrate Level Phosphorylation

Oxidation is the removal of electrons (e−) It is a metabolic reaction that results in
from an atom or molecule and is often an the formation of ATP or GTP by the direct
energy producing reaction. Reduction of transfer of a phosphoryl group to ADP
a substrate refers to its gain or addition or GDP from another phosphorylated
of one or more electrons to an atom or compound.
molecule. Oxidations and reduction are
always coupled. In other words, each
Alkaline phosphatase
time one substance is oxidized, another is
is a heat sensitive
simultaneously reduced.
enzyme in milk
F2 + 2e− 2F− which is used as an
H2 + 2e− 2H+ + 2e− indicator in Pasteurization.
NAD+ + 2H+ + 2e− NADH + H+
4.3.2 Oxidative Phosphorylation
4.3 Generation of ATP
In this reaction, electrons are transferred
Much of energy released during oxidation
reduction reaction is trapped within the from organic compounds to molecules of
cell by the formation of ATP. A phosphate Oxygen (O2) or other inorganic molecules
group is added ADP with the input of through a series of different electron
energy to form ATP. The addition of a carriers (Example: NAD+ and FAD. Then
phosphate to a chemical compound is the electrons are passed through a series
called phosphorylation. of different electron carriers to molecules
of oxygen. The process of oxidative
Organism uses three different
phosphorylation occurs during electron
mechanisms of phosphorylation to
generate ATP from ADP. They are transport chain (Figure 4.3).
48

Figure 4.3: Phosphorylation
4.3.3 Photo Phosphorylation is the breakdown of carbohydrate molecule

It occurs only in photosynthetic to produce energy and is therefore of great
cells which contain light trapping importance in cell metabolism. Glucose is
pigments. Example: In photosynthesis, the most common carbohydrate energy
photosynthetic pigment, Chlorophyll source used by cells.
is involved in the synthesis of organic To produce energy from glucose,
molecules especially sugars, with the microorganism use two general processes
energy of light from the energy poor namely Respiration and Fermentation.
building blocks like Carbon dioxide
and water. In phototropic bacteria 4.4.1 Cellular Respiration
(purple, green sulphur bacteria, Respiration is defined as an ATP generating
Cyanobacteria), photosynthetic pigments process in which organic molecules are
bateriochlorophylls are involved in ATP oxidized and the final electron acceptor
production. is an inorganic compound. In aerobic
respiration, the final electron acceptor is
4.4 Carbohydrate Catabolism Oxygen and in anaerobic respiration the
Most microorganisms oxidize final electron acceptor is an inorganic
carbohydrates as their primary source of molecule like NO3, SO42− other than
cellular energy. Carbohydrate catabolism Oxygen.
49

The aerobic respiration of glucose Glycolysis. Net energy production from
typically occurs in three principal stages. each glucose molecule is two ATP molecules
They are The Glycolysis pathway consists of two
phases. They are
Glycolysis
1. The preparatory/Investment phase,
Krebs cycle
where ATP is consumed
Electron transport chain
2. The pay off phase where ATP is
Glycolysis produced (Figure 4.4).
Glycolysis is the process of splitting of sugar 1. In the preparatory stage, two molecules
molecule, where the glucose is enzymatically of ATP are utilized and then glucose
degraded to produce ATP. Glycolysis is the is phosphorylated, restructured, and
oxidation of glucose to pyruvic acid with split into two 3 carbon compounds
simultaneous production of some ATP namely Glyceraldehyde-3-phosphate
and energy containing NADH. It takes and Dihydroxyacetone phosphate.
place in the cytoplasm of both prokaryotic 2. In pay off phase or energy conserving
and eukaryotic cells. Glycolysis occurs stage, the two 3 carbon molecules are
in the extra mitochondrial part of the cell oxidized in several steps to 2 molecules
cytoplasm. Glycolysis was discovered by of pyruvic acid are produced and two
Emden, Meyerhof and Parnas. So, this molecules of NAD+ are reduced to NADH,
cycle is shortly termed as EMP pathway, in thus four molecules of ATP are formed by
honour of these pioneer workers. This cycle substrate level phosphorylation.
occurs in animals, plants and large number Two molecules of ATP are needed
of microorganisms. Glycolysis does not to initiate Glycolysis and four
require oxygen, it can occur under aerobic molecules of ATP are generated at the
or anaerobic condition. Glycolysis is a end of the process. Therefore, the net
sequence of ten enzyme catalyzed reactions. gain of Glycolysis is two ATP for each
molecule of glucose oxidized.
Aerobic condition
Glycolysis Respiration Alternatives to Glycolysis
Glucose Pyruvate CO2+ H2O
O2
Many bacteria have another pathway in
Anaerobic condition
Glycolysis Fermentation addition to Glycolysis for the oxidation of
Glucose Pyruvate Fer-
mented products
glucose. Some of the common pathways
that occur in most of the bacteria are
C6H12O6 +2 NAD + 2 ADP + 2 P
• Pentose phosphate pathway (PPP)
2 CH3 COCOOH + 2 ATP +
or Hexose Mono Phosphate shunt
2 NADH + 2 H+
• Entner –Doudoroff Pathway
(Pyruvic acid)
Since glucose is a six carbon molecule HOTS
and pyruvate is a three carbon molecule,
two molecules of pyruvate are produced
Does Glycolysis require Oxygen?
for each molecule of glucose that enters
50

Figure 4.4: Glycolysis Pathway
51

compound, called acetyl group, attaches
Strips used in Glu- to coenzyme A through a high-energy
cometer a chemical bond, the resulting is a complex known
called glucose oxidase as acetyl coenzyme (acetyl CoA). During
which reacts with the this reaction, pyruvic acid is also oxidized
glucose in the blood sample and is con- and NAD+ is reduced to NADH by
verts it into an acid called gluconic acid. pyruvate dehydrogenase complex. This
multi enzyme complex is responsible for
4.5 Tricarboxylic Acid Cycle the conversion of pyruvate to acetyl-coA.
Therefore (PDHC) contribute to linking
TCA cycle was first elucidated by
the Glycolysis metabolic pathway to the
in 1937 “Sir Hans Adolf Krebs, a
citric acid pathway.
Germen biochemist. It is also known
as Tricarboxylic acid cycle, Citric acid
cycle or Amphibolic cycle. In prokaryotic Pyruvate dehydroge-
cells, the citric acid cycle occurs in the nase deficiency is a
cytoplasm; in eukaryotic cells it takes common cause of lactic
place in the matrix of the mitochondria. acidosis in new born
and often present with poor feeding.
The process oxidizes glucose
derivatives, fatty acids, and amino acids
to carbon dioxide (CO2) through a series Pyruvate dehydrogenase complex is
of enzyme controlled steps. The purpose a complex of three enzymes that convert
of the Krebs cycle is to collect high energy pyruvate into acetyl-coA by a process called
electrons from these fuels by oxidizing pyruvate decarboxylation (Figure 4.5).
them, which are transported by activated Acetyl-CoA may then be used in the citric
electron carriers such as NADH and FADH2 acid cycle to carryout cellular respiration,
to electron transport chain. The Krebs and this complex links the Glycolysis
cycle is also the source for the precursor metabolic pathway to the TCA cycle.
of many other molecules and is therefore The Krebs cycle generates a pool
an amphibolic pathway (both anabolic and of chemical energy (ATP, NADH, and
catabolic reactions take place in this cycle) FADH2) from the oxidation of Pyruvic acid
and therefore, it can be used for both the and it loses one carbon atom as CO2 and
synthesis and degradation of bio molecules. reduces NAD+ to NADH. The resulting
Pyruvate + CoA-SH + NAD+ two carbon acetyl molecule is joined to Co
Pyruvate dehydrogenase complex enzyme A. Acetyl CoA transfers its acetyl
group to a 4C compound (oxaloactate)
Acetyl CoA + Co2 + NADH to make a 6C compound (Citrate) and
Pyruvate cannot enter the Krebs cycle the Coenzyme A is released which goes
directly. In a preparatory step, it must back to the link reaction to form another
loses one molecule of CO2 and becomes molecule of acetyl CoA. Oxaloacetate is
a two-carbon compound. This process is both the first reactant and the product of
called decarboxylation. The two-carbon the metabolic pathway (creating a loop).
52

Figure 4.5: Krebs cycle
After citrate has been formed, the 4.6 Electron Transport Chain
cycle machinery continues through seven
An electron transport chain consists of
distinct enzyme catalyzed reactions
a sequence of carrier molecules that are
that produce in order isocitrate, α –
ketoglutarate, succinyl CoA, succinate, capable of oxidation and reduction. In
fumarate, malate and oxaloacetate. Eukaryotic cell, the ETC is contained in
the inner membrane of mitochondria
At the end of Krebs cycle, each pyruvic
acid produces 2 CO2, 1 ATP (substrate or chloroplast membrane, whereas in
level phosphorylation), 3 NADH and 1 prokaryotic cells, it is found in plasma
FADH2. Then NADH and FADH2 can be membrane or cytoplasmic membrane.
oxidized by electron transport chain to The ETC is carried out through a series
provide more ATPs. of electron transporters embedded in the
53

inner mitochondrial membrane that transfer The mitochondrial system is arranged
electrons from electron donors NADH into three complexes of electron carriers.
and FADH2 to acceptor such as molecular They are
Oxygen. In the process, protons are pumped 1. Flavoproteins: These proteins contain
from the mitochondrial matrix to the inner flavin, a coenzyme derived from
membrane space, and eventually combine riboflavin (Vit B12). One important
with O2 and H+ to form water (Figure 4.6). flavoprotein is flavin mono nucleotide.
2. Ubiquinones (coenzyme Q): These
HOTS
are small non protein carriers.
3. Cytochromes: These are proteins
Why each NADH makes 3 ATPs and
each FADH2 makes 2 ATPs? with iron containing group, capable of
existing alternately as reduced (Fe2+)
As the electrons flow through the chain, and oxidized form (Fe3+). Cytochromes
much of their free energy is conserved involved in ETC include cyt (b),cyt c1,
in the form of ATP. The process by cyt c, cyt a, cyt a3.
which energy from electron transport is The first step in electron transport
used to make ATP is called as oxidative chain is the transfer of high energy
phosphorylation. Respiratory chain is an electrons from NADH to FMN. This
electron transport chain where a pair of transfer actually involves the passage of
electrons or hydrogen atoms containing hydrogen atom with 2 e − to FMN, which
electron from the substrate oxidized is then picks up an additional H + from
coupled to reduction of oxygen to water. the surrounding aqueous medium. As
Figure 4.6: Electron Transport Chain and Chemiosmotic mechanism of ATP

54

a result of the first transfer, NADH is gradient has potential energy called
oxidized to NAD+, and FMN is reduced proton motive force.
to FMNH 2.
The proton diffuses across the
In the second step, FMNH 2 passes 2 membrane through protein channels that
H+ to the other side of the mitochondrial contain an enzyme called ATP synthase.
membrane and passes 2 e − to coenzyme When this flow occurs, energy is released
Q. As a result, FMNH 2 is oxidized and is used by the enzyme to synthesize
to FMN. Coenzyme Q also picks up ATP from ADP and phosphate.
additional 2H+ from the surrounding
At the end of the chain, electrons
aqueous and releases to other side of the
join with protons and O2 in the matrix
membrane.
fluid to form H2O. Thus O2 is the final
In the next step, electrons are passed electron acceptor. ETC also operates in
successively from coenzyme Q to cyt b1, photophosphorylation and is located in
cyt c1, cyt c, cyt a, cyt a3. Each cytochrome thylakoid membrane of Cyanobacteria
in the chain is reduced, as it picks up (BGA), and of eukaryotic chloroplasts.
electrons and is oxidized as it gives up Overview of Aerobic respiration
electrons. The last cytochrome cyt a3 (Figure 4.7):
passes its electrons to molecular O2 which
Electron transport chain regenerates
picks up protons from the surrounding
NAD and FAD which can be used
medium to form H2O.
again in Glycolysis and Krebs cycle.
FADH2 derived from the Krebs cycle
is another source of electrons. Thus at the Various electrons transfer in the
electron transport chain generates
end of ETC, NADH pumps three protons
about 34 ATP, (10 NADH = 10 × 3 =
(synthesizes 3ATPs) whereas FADH2
30 + 2 FADH2 = 2 × 2 = 4).
pumps only two protons (synthesizes
2ATPs). A total of 38 ATP molecules is
generated from one molecule of
4.6.1 Chemiosmotic Mechanism of ATP glucose oxidized in prokaryotes,
whereas in eukaryotes,
Chemiosmotic mechanism of ATP
36 molecules of ATP is generated
synthesis was first proposed by the because in eukaryotes, some
Biochemist, Peter Mitchell in 1961. In energy is lost when electrons are
ETC, when energetic electrons from shuttled across the mitochondrial
NADH pass down the carriers, some of membranes that separate
the carriers (proton pumps) in the chain Glycolysis (in the cytoplasm)
pump [actively transport] protons across from the electron transport chain
the membrane to inner membrane space. (Table 4.2). There is no such
Thus in addition to a concentration separation exists in prokaryotes.
gradient, an electrical charge gradient is C6H12O6 + 6CO2 + 38ADP + 38Pi
created. The resulting electro chemical 6CO2 + 6H2O + 38 ATP
55

Figure 4.7: Overview of aerobic respiration
Table 4.2: Net gain of ATP produced during aerobic respiration of glucose in prokaryotes
Glycolysis Preparatory step
1. Oxidation of glucose to Pyruvic acid. 2 ATP (substrate level phosphorylation)
2. Production of 2 NADH 6 ATP (Oxidative phosphorylation in ETC)
Preparatory step 6 ATP (Oxidative phosphorylation in ETC)
1. Formation of acetyl CoA produces
2NADH
Krebs cycle
1. Oxidation of succinyl CoA to 2 ATP (Substrate level phosphorylation)
succinic acid
2. Production of 6 NADH 18 ATP (Oxidative phosphorylation in ETC)
3. Production of 2 FADH 4 ATP (Oxidative phosphorylation in ETC)
Total 38 ATP
1 NADH = 3 ATPs and 1FADH2 = 2 ATP

56

4.7 Lipid Catabolism 4.8 Protein Catabolism
Microorganisms frequently use lipids such Many microbes use protein as their
as triglyceride or triacylglycerol (esters of source of carbon and energy. Pathogenic
glycerol and fatty acids) as common reserve microorganisms secrete protease enzyme
energy sources. These can be hydrolyzed to that hydrolyze proteins and polypeptides
glycerol and fatty acid by microbial lipases. to amino acids which are then transported
The glycerol is then phosphorylated and into the cell and catabolized. Protease
oxidized to Dihydroxyacetone phosphate (Peptidase or proteinase) helps in
and then catabolized in the Glycolysis proteolysis (Figure 4.8). These proteolytic
pathway. Fatty acids from triacylglycerols enzymes break the long chains of proteins
and other lipids are often oxidized in the into peptides and eventually into amino
β-oxidation pathway. In this pathway fatty acids. The enzymes are classified based
acids are degraded to acetyl CoA (2C on the sites at which they catalase the
segment), then it enters into the TCA cycle. cleavage of proteins as exopeptidase and
endopeptidase.
Bubble Test: Bubbles The protein catabolism involves two
are a positive result reactions namely,
for the presence
• Deamination and
of catalase. If an
organism can produce catalase, it • Transamination
will produce bubbles of oxygen when Deamination is the removal of the
hydrogen peroxide is added to it. amino group from an amino acid.
Transamination is the transferring of
amino group from an amino acid to an
Infobits amino acid acceptor.
The organic acid resulting from
One of the major environmental
problems today is hydrocarbon deamination can be converted to pyruvate,
contamination resulting from the acetyl CoA or TCA cycle intermediates
activities related to the petrochemical to release energy. Excess nitrogen
industry. Several bacteria can use from deamination may be excreted as
hydrocarbon as a feed and reduce ammonium ion.
pollution.
Pseudomonas putida (Super Various metabolic
Bug) Alcanivorax borkumens,
processes such as
Mycobacterium, Brevibacterium,
Aspergillus, Penicillium, Candida blood coagulation,
lipolytica are the most active agents in fibrinolysis, comple-
petroleum degradation and they work ment activation, phagocytosis and
as primary degraders in oil spilled blood pressure control are regulated
environment. These organisms are by proteases.
mainly in involved in bioremediation
which reduce environmental pollution
57

Figure 4.8: Overall Metabolism of Protein , Carbohydrates and Lipids
4.9 Fermentation Anaerobes do not use an electron

transport chain to oxidize NADH to
In 1856 fermentation, reaction was first
demonstrated by Louis Pasteur in yeast. NAD+ and therefore use fermentation as
The study of fermentation and its practical alternative method to maintain a supply
uses is named as Zymology. Any energy of NAD+ for the proper function of
releasing metabolic process that takes normal metabolic pathways. Facultative
place only under anaerobic condition is anaerobes can use fermentation under
called fermentation. It can also be defined anaerobic condition and carryout aerobic
is a metabolic process that release energy respiration when oxygen is present.
from a sugar or other organic molecule. Fermentation reoxidizes NADH to NAD+
It does not require oxygen or an electron by converting pyruvic acid into various
transport system, and uses an organic organic acids.
molecule as the final electron acceptor. During fermentation, NADH is
Fermentation reaction yields only a small converted back into the coenzyme NAD+
amount of energy (2 ATP). so that it can be used again for Glycolysis
58

1
2ADP+2P 2ATP
Glucose
2 Pyruvate
NAD+ NADH 2CO2

NAD+
2
NADH
2 Acetaldehyde
2 Ethanol
3-
Figure 4.9: Biochemical exchange of NADH and NAD+
(Figure 4.9). Organic electron acceptors Milk is converted into fermented

such as pyruvate or acetaldehyde react products such as curd, yogurt and cheese.
with NADH to form NAD+, producing The fermentation of lactose in milk by
CO2 and organic solvent like ethanol. these bacteria produces lactic acid which
Fermentation can be classified as acts on milk protein to give yogurt its
Lactic acid fermentation and alcohol texture and characteristic tart flavour.
fermentation. Here lactase enzyme is produce by the
bacteria which convert the lactose into
lactic acid.
Aquifex (water
maker) of Aquificae Builds up of Lactic
is a diverse collection acid in muscle cells
of bacteria that live causes muscle cramp.
in harsh environmental settings.
These can produce water by oxidizing
hydrogen.
Homolactic acid fermentation
In this type of fermentation, organism
4.9.1 Lactic acid fermentation produces lactic acid alone. So it is referred
During Glycolysis, in the first step of lactic to as homolactic fermentation.
acid fermentation, a molecule of glucose Glucose + 2ADP + 2P actic acid
L
is oxidized to 2 molecules of pyruvic acid + 2 ATP
and it generates the energy. In the next
step pyruvic acid is reduced by NADH Heterolactic acid fermentation
to form lactic acid. Lactobacillus and In this type of fermentation, organism
Streptococcus are some of the lactic acid produces Lactic acid as well as other
producing genera (Figure 4.10). acids or alcohol. So it is known as hetero
59

Figure 4.10: Varoius products of microbial fermentation
fermentation or heterolactic and often The acetaldehydes are then reduced by

uses the pentose phosphate pathway. NADH to form ethanol. The ethanol and
G + ADP + p Lactic acid + ethanol CO2 produced by the yeast Saccharomyces
is used in alcoholic beverages and to raise
+ CO2 + ATP
bread dough respectively.
HOTS
4.10 Enzymes
Why do cells need to ferment when Life is an intricate meshwork involving
they get 2ATPs from Glycolysis? a perfect coordination of a vast majority
of chemical reactions. This is due to the
presence of some catalysts synthesized
4.9.2 Alcohol Fermentation inside the body of the organism. The
Alcohol fermentation begins with the term ‘enzyme’ was coined by Friedrich
Glycolysis which yields two molecules of Wilhen Kuhne (1878) to designate these
pyruvic acid and two molecules of ATPs. In biological catalysts. The name ‘enzyme’
the next step, the two molecules of pyruvic (en – in, zyme – yeast) literally means
acid are converted to two molecules of ‘in yeast’. The name of enzyme usually
acetaldehyde and two molecules of CO2. ends in – ase. Example: Cytochrome
60

dehydrogenase. The study of enzyme is 4.10.1 Characteristics of Enzymes
called Enzymology. Enzymes
Enzymes are proteins or large • are highly substrate specific
biomolecules that can catalyze certain • are reused at several times
biochemical reactions for metabolic • synthesized within the cells are
process within the cell. The substances that determined by genes
can speed up a chemical reaction without • speed up the chemical reaction
being permanently altered itself are called • decrease the activation energy
catalysts. Enzymes accelerate the rate of needed to start
chemical reactions. The molecule upon • act as a biocatalyst
which enzyme may act are called substrate
and the enzyme convert the substrate into Infobits
different molecules known as products.
The enzyme serves as biological catalyst Proteins have four levels of structure
(i) primary (sequence of amino
(Table 4.3).
acids), (ii) secondary (regular coils
or pleats linked by peptide bonds),
Tyrosinases are syn- (iii) tertiary overall three dimensional
thesized by Agaricus structure of a polypeptide linked by
bisporus, which is disulphide bonds) and (iv) quarterly
involved in melano-
structure (two or more polypeptides
genesis (pigmentation of skin and hair). chains). Like all proteins, enzymes are
composed of one or more long chain
of inter connected amino acids.
Table 4.3: Enzyme Classification Based on Type of Chemical Reaction

Class Type of Chemical Reaction Reactions Examples
Oxido- Oxidation-reduction in which Xred + Yox Xox + Yred Cytochrome oxidase,
reductase oxygen and hydrogen are lactate dehydrogenase
gained or lost
Transferase Transfer of functional groups, X − P + Y X+Y−P Acetate kinase,
such as an amino group, acetyl alanine deaminase,
group, or phosphate group transaminase,
phosphotransferase
Hydrolase Hydrolysis (addition of water) X − Y + H2O Lipase, sucrose
X − H + Y − OH
Lyase Removal of groups of atoms X−Y X+Y Oxalate
without hydrolysis decarboxylase,
isocitrate, lyase
Isomerase Rearrangement of atoms X−Y−Z X − Z − Y Glucose-phosphate
within a molecule isomerase, alanine
racemase
Ligase Joining of two molecules (using X + Y + ATP Acetyl-CoA
energy usually derived from X − Y + ADP + pi synthetase, DNA
breakdown of ATP) ligase
61

The region of an enzyme where
Low level of catalase substrate molecules bind and undergo
plays a major role in a chemical reaction is its active site.
greying process of Each active site is specially designed in
human hair. response to their substrate; as a result
most enzymes have specificity and can
only react with particular substances.
4.10.2 Structure of Enzymes
After the formation of enzyme substrate
Enzymes are generally globular proteins complex (Figure 4.12), forces exerted
that range in molecular weight from about on the substrate by the enzyme cause it
10,000 to several million. Each enzyme to react and become the product of the
possesses a unique sequence of amino acid intended reaction.
that causes it to fold into a characteristic Example: Sucrase catalyses the hydrolysis
three dimensional shape with a specific of sucrose to glucose and fructose.
surface configuration. This enables it to
find the correct substrate from among
the large number of diverse molecules in
the cell.
A molecule acted upon by an enzyme
is called a substrate. Enzymes are
specific and act on specific substrates
and each enzyme catalyzes only one
reaction. Enzyme consists of a protein
portion, named apoenzyme and a non
protein component, named cofactor
(Figure 4.11).
Apoenzyme + Cofactor Holoenzyme

(Protein (Non (Active
portion) protein) enzyme)
(Inactive (Inactive Figure 4.12: Mechanism of product
form) form) formation from substrate through Enzyme
Apoenzyme is the inactive form of

the enzyme which gets activated after
binding with a cofactor. Coenzymes
are small organic molecules that can be
loosely or bound to an apoenzyme and
they transport chemical group from one
enzyme to another.
Cofactor is a chemical compound or
metallic ion that is required for enzyme
Figure 4.11: Structure of enzyme activity. Example: NAD+ is derived from
62

vitamin B. Some cofactors are metal ions 4.10.3 Microbial Enzymes
including iron (Fe), copper (Cu), magnesium Many microbes synthesize and excrete
(Mg), manganese (Mn), Zinc (Zn), calcium large quantities of enzymes into the
(Ca) and cobalt. If the cofactor is tightly or surrounding medium. Using this feature
firmly attached to the apoenzyme it is called of these tiny organisms many enzymes like
a prosthetic group. The prosthetic group Amylase, Cellulase, Catalase, Protease,
may be organic [such as vitamin, sugar, and and Lipase are produced commercially.
lipid] or inorganic [such as metal ion] but is Microbial enzymes are extensively used
not composed of amino acids. in food processing, preservation, washing
The complete enzyme consisting of the powder preparation, leather industry, and
apoenzyme and its cofactor is called the paper industry and in scientific research
holoenzyme. (Table 4.4).
Table 4.4: Industrial application of microbial enzymes

Industries Enzymes Microbial Sources Application
Pharmaceutical •• Glucose •• Penicillium •• To detect free glucose level
industry oxidase notatum in diabetic patients
•• Streptokinase •• Streptococci •• Anti coagulants
•• Protease •• Clostridium spp •• C onversion of fibrinogen to
•• coagulase •• Staphylococcus fibrin
aureus
Dairy Industry •• Catalase •• Aspergillus niger •• Remove Hydrogen peroxide
•• Lactase •• Lactobacillus spp in milk (detoxification)
•• Increase sweetness in milk
Baking •• Amylase •• Bacillus subtilis •• Increase bread shelf life
Industry •• Lipase •• Candida Lipolytica •• Enhances flour quality and
dough stability
Polymer •• Lipase •• Candida spp •• Polyester preparation
Industry •• Peroxidase •• Pseudomonas spp •• Formation of cross links
Leather •• Protease •• Bacillus spp •• Unbarring of hides,
Industry Lipase •• Aspergillus spp degreasing and softening of
leather
Textile •• Cutinase •• Pseudomonas spp •• Cotton Scouring
Industry •• Collagenase •• Clostridium •• Wool Finishing
•• Laccase histolyticum •• Bleach termination
•• Fabric dyeing
Recombinant •• DNase •• Escherichia coli •• Nuclease enzyme that break
DNA •• Ligase •• Actinomycetes phosphodiester bond of
technology DNA or RNA
•• Joins the nick in DNA
fragments
63

An irreversible inhibitor inactivates
Infobits an enzyme by bonding covalently to
a particular group at the active site.
Idoenella sakaiensis is a bacterium
A reversible inhibitor inactivates an
capable of breaking down PET plastics.
enzyme by non covalent, more easily
The bacterium first uses PETase to
reversible interactions. Competitive
break down the PET plastic. This has
inhibitor is any compound that bears a
potential importance in the recycling
structural resemblance to a particular
process of PET plastics.
substrate for binding at the active site of
an enzyme. Non competitive inhibitors
Lipase is used in the do not compete with the substrate for
determination of the enzyme’s active site; instead, they
triglyceride and blood interact with another part of the enzyme.
cholesterol level. Uncompetitive inhibitors bind only to
Lipase producing microorganism the enzyme substrate complex without
have been found in industrial wastes, binding to the free enzyme (Figure 4.13).
vegetable oil processing factories,
a. C ompetitive b. Non-competitive
diary plants and soil contaminated Inhibition Inhibition
with oil.
Substract
Substract
4.10.4 Enzyme Regulation Inhibitor
Inhibitors: An enzyme inhibitor is a

molecule that binds to an enzyme and
decreases its enzyme catalyzing activity
(Flowchart 4.1). This adverse affect of Inhibitor
inhibitors on the rate of enzymatically

catalyzed reactions are called inhibition. Figure 4.13: Competitive and
non-competitive inhibition
Inhibition
Administration of
the enzyme DNase I
Reversible Irreversible Allosteric
to the lungs of cystic
Competitive fibrosis patients
decrease the viscosity of the mucus
Uncompetitive and the breathing is made easier.
Mixed
Feedback inhibition
Non-competitive
In Feedback inhibition, the final product
allosterically inhibits the enzyme that
Flowchart 4.1: Types of Inhibition
catalyses the first stage in the series
64

of reactions. This process is used to Biochemical pathway that functions in
regulate the synthesis of amino acids both anabolism and catabolism are called
(Flowchart 4.2). Example: Threonine amphibolic pathways, meaning that they
deaminase is the first enzyme the are dual purpose. The energy of catabolic
conversion Threonine to Isoleucine. reactions is used to drive anabolic reactions.
Isoleucine inhibits Threonine deaminase The energy for chemical reactions is
through feedback inhibition. stored in ATP. The chemical reactions are
Substrate catalyzed by different enzymes. Enzymes
catalyze chemical reactions by lowering
Product Enzyme 1 the activation energy. Most of the cells
energy is produced from the oxidation of
Intermediate 1 carbohydrates. During respiration organic
molecules are oxidized. Energy is generated
Enzyme 2 from the ETC. In aerobic respiration, O2
Feedback Inhibition
Intermediate 2 function as the final election acceptor. In

anacrobic respiration, the final electron
Enzyme 3 acceptor is an inorganic molecule NO2-,
SO42- other than O2.
Intermediate 3
Complete oxidation of glucose
Enzyme 4 molecule takes place in 3 sequential
reactions.
Intermediate 4 ¾¾ Glycolysis occurring in cytoplasm
Product ¾¾ Krebs cycle occurring is mito
chondrial matrix
Flowchart 4.2: Feedback Inhibition

E TC (Oxidative Phosphorylation)
4.10.5 Uses of Microbial Enzymes occurring is inner mitochondrial matrix.
In aerobic prokaryotes, 38 ATP molecules
Microbial enzymes are
can be produced from complete oxidation
•• helpful to save energy and prevent of a glucose molecule in glycolyins,
pollution krebscycle, and ETC. In eucaryotes
•• highly specific 36 ATP molecules are produced from
•• be immobilized and reused complete oxidation of a glucose molecule.
•• inexpensive and more stable In incomplete oxidation of glucose
molecules will revolt in fermentation,
•• easily extracted and purified
O2 in anaerobic condition. Various
•• genetically manipulated to yield higher commercial products are produced from
quality pyruvic acid. Lipid can be catabolised by
lipase which hydrolyze lipid into glycerol
Summary and fatty acid. Then fetly acids are
The sum of all chemical reactions within a catabolised by Beta oxidation. Proteins
living organism is known as Metabolism. can be catabolised by Deamination and
65

Transamination process into amino 5. The correct sequence of anaerobic
acids. Carbohydrate, Fat, Protons can all reactions in yeast is
be the source of electrons and protons Cytoplasm
a. Glucose pyruvate
for respiration. Microbial enzymes are Mitochondria Ethanol + CO
2
extensively used in food processing,
Cytoplasm
preservation, paper industry and in b. Glucose pyruvate
Cytoplasm
scientific research. Lactic acid
Cytoplasm
c. Glucose pyruvate +
Evaluation
Energy Mitochondria CO2 + H2O
Multiple choice questions Cytoplasm
d. Glucose pyruvate
1. High energy transfer compounds are Cytoplasm Ethanol + CO2
capable of
6. For every one molecule of sugar glucose
a. Accepting large which is oxidized molecule
amount of free of pyruvic acid are produced.
energy a. 1 b. 2 c. 3 d. 4
b. Transferring large 7. Assertion (A. : In substrate level
amount of free phosphorylation ATP is generated
energy when a high energy phosphate
c. Measuring free energy is directly transferred from
d. None of the above a phosphorylated compound
2. In an aerobic respiration the terminal (substrate. to ADP.
2 ADP 2 ATP
electron acceptor is
a. oxygen Reason (R) : P
hosphoenol pyruvic
b. nitrogen acid 2 pyruvic acid
c. hydrogen a. A is true, A is supported by R
d. nitrate b. A is false, but R is not supported by A
3. Utilizable energy or energy is available c. Both A and R are false
to do work is termed as
d. A is true, R is false
a. free energy
8. Which one of the following is correct
b. U
tilisable energy
Apoenzyme + Cofactor
c. Kinetic energy = Holoenzyme
d. Thermal energy
Haloenzyme + Coenzyme
4. The reactant in glycolynis is = Apoenzyme
a. Pyruvic acid Apoenzyme + Holoenzyme
b. Citric acid = Coenzyme
c. glucose Coenzyme + Cofactor
d. Glucose-6-phosphate = Holoenzyme
66

9. Match the correct order given 8. Mention the classification of enzymes
below based on chemical reaction.
a. Catalase - 1. Detect Blood 9. Define fermentation.
glucose level 10. Write about the types of fermentation
b. Glucose - 2. Break down of with few examples.
oxidase H 2O 2
11. Mention the importance of the
c. Protease - 3. Clot the plasma
enzymes.
d. Coagulase - 4. Leather
12. Write short note on the component of
manufacture
the enzyme.
10. Statement A: Oxidation of glucose
13. Explain the enzyme regulation
to pyruvic acid yield only 4 ATP by mechanism.
substrate level phosphorylation.
14. Explain EMP pathway or glycolytic
S tatement B: The total ATP which is pathway.
produced through TCA is 24.
15. Describe TCA cycle.
a. Statement A is true, B is false 16. Explain electron transport chain.
b. Both A and B are true 17. These are three mechanism for the
c. A is false, B is true phosphorylation of ADP to ATP.
d. Both A and B are false Write the name of the mechanism in
the following reaction given below.
Answer the following 1 An electron liberated from ?
1. Define metabolism chlorophyll by light, is passed
2. Write the difference between down an ETC
catabolism and Anabolism. 2 Cytochrome c passes two ?
electrons to cyt a
3. ATP is an energy storage compound,
3 Phosphoenol pyruvic acid ?
where does it get this energy from?
4. What is holoenzyme?
5. What is Active site? pyruvic acid
6. Explain the structure of ATP.
7. Write about the types of 18. Name the stages of aerobic respiration?
phosphorylation.
67

Chapter
5 Food Microbiology
5.1 Food Microbiology

Learning Objectives
The field of food
After studying this chapter the microbiology is very
students will be able to, broad, encompassing the
• Know the sources of microorganisms study of microorganisms
in food which have both beneficial
and deleterious effects on
• Understand the factors that
the quality and safety of raw and processed
influence growth of microorganisms
foods. The primary tool of microbiologists
in food
is the ability to identify and quantitate food-
• Learn about the food spoilage
borne microorganisms. Microorganisms in
• Appreciate the food preservation food include bacteria, molds, yeasts, algae,
methods viruses, parasitic worms and protozoans.
• Learn and compare the food Microorganisms are associated with
poisoning and food intoxication the food we eat in a variety of ways.
• Classify the food borne diseases They may influence the quality of our
• Understand the microbial standards food. Naturally occurring foods such as
and grading of milk fruits and vegetables normally contain
• Know the fermented milk products some microorganisms and may be
like cheese, yoghurt and curd contaminated with additional organisms
during handling and processing. Food
can serve as a medium for the growth of
Chapter Outline microorganism, and microbial growth may
cause the food to undergo decomposition
5.1 Food Microbiology and spoilage. Food may also carry
5.2 Food Spoilage pathogenic microorganisms which when
5.3 Food Borne Disease ingested can cause disease. When food
5.4 Food Preservation Methods with microorganisms that produce toxic
substances is ingested, it results in food
5.5 Dairy Microbiology
poisoning. Apart from the pathogenic
5.6 Cheese microorganisms, some microorganisms
5.7 Yogurt are used in the preparation and
5.8 Curd preservation of food products.
68

5.1.2 Sources of Microorganism in
FSSAI: Food Safety
Food
and Standards
Authority of India The primary sources of microorganisms
(FSSAI) is an in food include,
autonomous body established under the 1. Soil and water
ministry of health and family welfare, 2. Plant and plants products
Government of India. FSSAI maintains
3. Food utensils
the food quality levels in order to ensure
safety and provides satisfaction to every 4. Intestinal tract of human and animals
consumer. 5. Food handlers
6. Animal hides and skins
7. Air and dust
5.1.1 Classification of Foods
Foods may be classified as 5.1.3 Factors that Influence Growth of
a. Fresh foods Microorganisms in Food
These are foods which have not been Many factors influence the growth of the
preserved and not spoiled yet. For example; microorganisms in food. Some of the
vegetables, fruits and meat spoil immediately factors are intrinsic and some others are
after harvesting or slaughtering. extrinsic.
b. Preserved foods 1. Intrinsic factors
Foods are preserved by adding salt, sugar, The intrinsic factors include pH, moisture
acetic acids and ascorbic acids. Example: content, oxidation – reduction potential,
Jam, Pickles. In this way their shelf life is nutrient status, antimicrobial constituents
improved. and biological structures.
c. Canned foods a. pH: Every microorganisms has a
In canning, food products are processed and minimal or maximal, and an optimal
sealed in the air tight containers. It provides pH for its growth. Microbial cells are
longer shelf life ranging from one to five significantly affected by the pH of
years. Example: Baked beans, Olives. food because they apparently have no
mechanism for adjusting their internal
d. Processed foods
pH. In general, yeasts and molds are more
During food processing, original nature acid tolerant than bacteria. Foods with
of food is changed or altered. It is done low pH values (below 4.5) are usually not
by Freezing, Canning, Baking and Drying. readily spoiled by bacteria and are more
Example: Breakfast cereals, Cakes, Biscuits susceptible to spoilage by yeast and molds.
and Bread. Most of the microorganisms grow best at
e. Fermented food products pH value around 7.0.
These foods are subjected to fermentation b. Moisture content: The preservation
by the action of microorganisms. Example: of food by drying is a direct consequence
Kefir, Cheese. of removal of moisture, without which
69

microorganisms do not grow. The water have been shown to have antimicrobial
requirement of microorganism is defined activity. Some species contain essential
in terms of the water activity (aw) in the oils that possess antimicrobial activity.
environment. Water activity is defined as Among these are allicin in garlic, eugenol
the ratio of the water vapour pressure of in cloves and cinnamon.
food substrate to the vapour pressure of 2. Extrinsic factors
pure water at the same temperature. The
These include those properties of the
water activity of most fresh food is above
storage environment that affect both the
0.99. The minimum value of aw for the
foods and microorganisms present in them.
growth of the microorganisms in foods
Storage temperature, pH, presence and
should be around 0.86.
concentration of gases in the environment
c. Oxidation reduction (O/R) potential are some of the extrinsic factors that affect
The oxygen tension or partial pressure the growth of microorganisms.
of oxygen around a food and the O-R
potential or reducing and oxidizing
power of the food itself influence the type Food Corporation
of organisms which can grow and the of India: FCI is
changes produced in the food. The O-R an organization
potential of the food is determined by, created and run
i. The O-R potential of the original food. by Government of India. FCI is a
statutory body established through
ii. The poising capacity, (the resistance to
Food Corporation Act, 1964 to meet
change in potential, of the food.)
the following objectives of food policy.
iii. The oxygen tension of the atmosphere Effective price support operations
about the food and for safeguarding the interests of the
iv. The access which the atmosphere has farmers.
to the food.
d. Nutrient Content
5.2 Food Spoilage
The kinds and proportions of nutrients in
Spoilage of food can be defined as any
the food are all important in determining
visible or invisible change which can
what organism is most likely to grow.
make food or product derived from food
Consideration must be given to (i) foods
unfit for human consumption. Spoilage
for energy (ii) foods for growth and
of food not only causes health hazard
(iii) accessory food substances or vitamins
to the consumer but also causes great
which may be necessary for energy or
economic losses. Spoilage leads to loss of
growth.
nutrients from food and cause change in
e. Antimicrobial constituents original flavor and texture. It is estimated
The stability of foods against attack by that about 25% of total food produced is
microorganism is due to the presence of spoilt due to microbial activities despite a
certain naturally occurring substances that range of preservation methods available.
70

Food spoilage is considered as a complex ii. Semi – perishable foods
phenomenon where by a combination of This class of foods if properly stored can
microbial and bio-chemical activities take be used for a longer duration. These foods
place. Due to such activities various types
include processed cereals, pulses and their
of metabolites are formed which aid in
products like flour, semolina, parched rice
spoilage (Figure 5.1).
and popcorn. Shelf life of these products
depends on the storage temperature and
moisture in the air. Foods like potato,
onion, nuts, frozen foods and certain
canned foods can be stored for a week to
a couple of months at room temperature
without any undesirable changes in the
products.
iii. Non – perishable foods
These foods remain stable for long
period unless handled improperly. Non-
perishable foods include sugar, jaggery,
hydrogenated fat, vegetable oil, ghee,
whole grains, dhals, whole nuts and
processed foods like dry salted fish/meat,
papads, canned foods, jams and murabbas.
These foods do not spoil unless they are
handled carelessly.
5.2.1 Causes of Food Spoilage

Food and water may be infected by germs.
When flies carrying germs sit on our food,
they pass on the germs to our food. There
are various factors which are responsible
Figure 5.1: Food spoilage
for food spoilage such as.
The ease with which foods are spoiled •• Microorganism
depends upon factors described. The
•• Insects
foods are thus, divided into different
classes. They are: •• Rough handling
i. Perishable foods •• Transport
These foods are readily spoiled; require •• improper storage
special preservation and storage condition •• enzyme activity (Chemical reaction)
for use. This includes, foods such as dairy
•• unhygienic conditions
products, eggs, poultry, meat, fish, fruits
and vegetable. These foods get spoiled physical changes, such as those caused by
easily by natural enzymes. freezing, burning, drying pressure.
71

Signs of food spoilage include difference caused by or thought to be caused by the
in appearance from the fresh food such as consumption of food or water. The term
a change in colour, a change in texture and “food poisoning” as applied to diseases
an unpleasant odour or taste. caused by microorganisms is used very
loosely to include both illness caused
HOTS by the ingestion of toxins elaborated by
the organisms and those resulting from
1. Why do concentrated citrus juices infection of the host through the intestinal
prevent spoilage problems? tract. A further classification of food borne
2. Name a few organisms responsible disease is shown in flowchart 5.1.
for food spoilage. All these food – borne diseases are
associated with poor hygienic practices.
Whether by water or food transmission,
5.3 Food Borne Disease the fecal – oral route is maintained, with
Food borne disease has been defined by the food providing the vital link between
the world health organization (WHO) “as hosts. Fomites, such as sink faucets,
a disease of an infectious or toxic nature drinking cups, and cutting boards, also
Food Poisoning
Food intoxication Food infection
Bacterial Food Fungal Food

Algal Food poisoning
Poisoning (Bacterial poisoning
(phycointoxication)
food intoxication) (mycointoxications)
Botulism
Mushroom poisoning Fish poisoning
Example: Clostridium
Example: Amanita Example: Gymnodynium
botulinum
Bacterial diaseases:
Staphylococcal – Viral disease:
Example: Shigellosis
poisoning Example: Polio, Hepatitis
(Bacillary dysentry)
Example: Staphylococcus A& E Gastro enteritis
Escherichia Cholera
aureus viruses
Brucellosis
Flowchart 5.1: Types of Food – Poisoning

72

play a role in the maintenance of fecal – 5.3.2 Food Poisoning
oral route of contamination.
Food borne intoxication (or) food
There are two primary types of food poisonings is caused by ingesting food
related diseases: food – borne infections containing toxins formed by bacteria
and food intoxications or food poisoning. which resulted from the bacterial growth
in the food item. Food poisoning refers
HOTS to the toxicity introduced into food by
microorganisms and their products.
Name some of the uncooked foods that Microbial growth in food products
have been implicated in food borne also can results in food intoxication.
disease transmission? Intoxication produces symptoms shortly
after the food is consumed because growth
5.3.1 Food Borne Infection of the disease – causing microorganism is
not required. Toxins produced in the food
Food borne infection involves the ingestion can be associated with microbial cells or
of the pathogen followed by growth in the can be released from the cells.
host, including tissue invasion and/or the
Food poisoning is caused by various
release of toxins. The major diseases of this
factors as follows.
type are summarized in table (5.1).
Table 5.1: Major Food – Borne Infectious Diseases

Incubation period
S.No Disease Organism Major Foods Involved
and characteristics
1. Salmonellosis Salmonella 8–48 hr Meat, poultry, Fish,
typhimurium Enterotoxin and eggs, dairy product.
S. enteritidis cytotoxins
2. Campylobacteriosis Campylobacter Usually 2–10. Most Milk, or, poultry
jejuni toxin heat labile product, water.
3. Listeriosis L. monocytogenes Varying periods. Meat products,
Related to meningitis especially pork and
and abortion milk.
newborns and the
elderly.
4. Escherichia coli Escherichia coli, 24–72 hrs Entero Cooked ground beef,
(Diarrhea and includes serotype toxigenic Positive raw milk
colitis) 0157:H7 and negative strains :
hemorrhagic colitis
5. Shigellosis Shigella sonnei, S. 24–72 hrs Egg products,
flexneri puddings
6. Vibrio Vibrio 16–48 hr Seafood, shellfish
parahamolyticus parahaemolyticus
73

1. Microorganism of plant food products. 3. Salting
2. Microorganism of Animal food Add salt to preserve pickles and fish.
products. 4. Sweetening
3. Microorganism of processed food. Sugar act as a preservative
4. Standard chemicals added to the food. when added in large
5. Excess use of preservatives in food. quantities. For example,
food can be stored for a
6. Presence of higher population of
long time in the form of
Microorganism in food.
jams, jellies and murabbas
7. Toxin produced by various types of (Figure 5.2) by adding sugar.
Microorganism.
5.4 Food Preservation Methods

Foods can be preserved by a variety
of methods. It is vital to eliminate or
reduce the populations of spoilage and
disease – causing microorganisms and to
maintain the microbiological quality of a
food with proper storage and packaging.
Contamination often occurs after a
package or can is opened and just before
the food is served. This can proved an ideal Figure 5.2: Murabbas
opportunity for growth and transmission of 5. Drying
pathogens, if care is not taken. Preservation In this method, the food items are dried in
of food is the process by which food is stored sun to stop the growth of bacteria in them.
by special methods. Cooked or uncooked Certain foods, like raw mangoes, fishes,
food can be preserved in different ways to potato chips and papads are preserved by
be used later Table 5.2. Some methods of this method.
preservation are:
6. Canning
1. Freezing
In this method, air is removed from food
Food kept in a refrigerator remains fresh and put in airtight cans so that germs
for some day. Germs do not grow easily do not grow on them. Food items like
in cool places we preserve food items, like vegetables, seafood, and dairy product are
milk, fruit, vegetable and cooked food by preserved through this method.
keeping them in a refrigerator. Advantages of food preservation
2. Boiling •• Germs do not grow easily in preserved
By this method, we can preserve food for food and make it safe to eat.
a short period of time. Germs in milk •• Preservation enables us to enjoy
are killed by pasteurization. It is done seasonal fruits like strawberries and
by boiling milk for sometimes and then mangoes even during the off season.
cooling it quickly.
74

Table 5.2: Basic Approaches to Food preservation
S.No Approach Examples of process
1. Removal of Avoidance of microbial contamination, physical filtration,
microorganisms centrifugation.
2. Low temperature Refrigeration, Freezing
3. High temperature Partial or complete heat inactivation of microorganisms
(pasteurization and canning
4. Reduced water Water removal, as with Lyophilization or freeze drying use of
availability spray dryers or heating drums decreasing water availability by
addition of solutes such as salt or sugar.
5. Chemical – based Addition of specific inhibitory compounds (Example: organic
preservation acids, nitrates, sulfur dioxide)
6. Radiation Use of ionizing (gamma rays) and non ionizing (UV) radiation
7. Microbial product – The addition of substances such as bacteriocins to foods to
based inhibition control food – borne pathogens
Disadvantages
Infobits
•• Excess salt and sugar are used in the
preservation of food which is not good “Typhoid Fever and Canned Meat”
for health.
Minor errors in canning have led to major
•• Some methods of food preservation typhoid outbreaks. In 1964 canned beef
may lead to loss of nutrients. produced in South America was cooled,
Methods of food preservation after sterilization with non chlorinated
water. The vacuum created when the
Principles of Food preservation cans were cooled drew Salmonella typhi
In accomplishing the preservation of foods into some of the cans, which were not
by the various methods, the following completely sealed. This contaminated
principles are involved. product was later sliced in an Aberdeen,
Scotland, Food store and the meat slicer
1. Prevention or delay of microbial became a continuing contamination
decomposition. source the result was a major epidemic
a.
By keeping out microorganism that involved 400 people. The Salmonella
(asepsis) typhi was a South American strain
and eventually the contamination was
b.
By removal of microorganism.
traced to the contaminated water used
Example: Filtration to cool the cans. This emphasizes the
c.
By hindering the growth and importance of careful food processing
activity of microorganism Example: and handling to
Low temperature, drying, anaerobic control the spread
conditions or chemicals. of disease during
food production
d. By killing the microorganism and preparation.
Example: Heat or radiation
75

2. Prevention or delay of self –
decomposition of the food.
Cheese
a. By destruction or inactivation of Milk Pasteurized
food enzymes Example: Blanching
, aw
powder milk
H
T, p
b. By prevention or delay of purely T
chemical reactions Example: T, pH
Prevention of oxidation by means Evaporated
Yoghurt Raw milk T
milk
of antioxidants.
T, a
T
3. Prevention of damage because of T, a
insects, animals, mechanical causes, etc. Dried milk Cream
Sweetened
5.5 Diary Microbiology condensed
milk
The area of dairy microbiology is large and
diverse. The bacteria in dairy products Flowchart 5.2: Various products obtained
may cause disease or spoilage. Some from raw milk
bacteria may be specifically added to milk
for fermentation to produce products like
good quantity of nutrients and high water
yoghurts and cheese (Figure 5.3).
content, milk an excellent nutrient for
the microbial growth. The main source
of microorganisms under interior eats,
surrounding environment and manual
milking process, make the source of
contamination (Flowchart 5.2).
pH–Hydrogen ion concentration
T–Elevated temperature
H–Reduced water pressure
aw–water activity
5.5.2 Composition and Properties

Milk is considered to be the “Most nearly
perfect” food for man and hence is one
Figure 5.3: Yoghurts and cheese
of the most important ingredients of the
diet. It is an extremely complex mixture
5.5.1 MILK
and usually contains (Table 5.3).
Milk is the fluid, secreted by mammals for
the nourishment of their young ones. It is in 5.5.3 Sources of Microorganisms in Milk
liquid form without having any colostrum.
¾¾ Three sources contribute to the
The milk contains water, fat, protein and
microorganism found in milk the
lactose. About 80–85% of the protein is
udder interior, the teat exterior and
casein. Due to moderate pH (6.4–6.6),
76

Table 5.3: Complex mixture teats have been reported to contribute up
to 105 cfu ml-1 in the milk. Contamination
Approximate
S.No Composition from bedding and manure can be source
percentage
of human pathogens such as E.Coli,
1. Liquid (Water) 87%
Campylobacter, Salmonella, Bacillus
2. Solids 13% spp. and Clostridia spp.
3. Fat 4%
¾¾ Milk – handling equipment such as
4. Protein 3.3%
teat cups, pipe work, milk holders and
5. Lactose 5%
storage tanks is the principal source
(Milk Sugar)
of the microorganisms found in raw
6. Ash content 0.7%
milk. Micrococcus and Enterococcus.
(Vitamins and
minerals)
5.5.4 Microbiological Standard and
Grading of Milk
its immediate surroundings, and the
In India, raw milk is graded by Bureau of
milking and milk handling equipment.
Indian standards (BIS) 1977. The Indian
¾¾ Bacteria that get on to the outside standard institute (ISI) has prescribed
of the teat may be able to invade the microbiological standard for quality of milk.
opening and hence the udder interior.
¾¾ Coliforms count in raw milk is
The organisms most commonly
satisfactory if, coliforms are absent in
isolated are micrococcus, streptococci
1:100 dilution.
and the diptheroid Corynebacterium
bovis. Aseptically taken milk from a ¾¾ Coliforms count in pasteurized milk is
healthy cow normally contains low satisfactory is coliforms are absent in
number of organisms, typically fewer 1: 10 dilution (Table 5.4).
than 102–103 cfu ml-1 Grading of milk
¾¾ The udder exterior and its immediate The quality of milk is judged by certain
environment can be contaminated standards and it is known as grading milk.
with organisms from the cow’s general Grading of milk is based upon regulations
environment. Heavily contaminated pertaining to production, processing and
Table 5.4: Microbiological Standard and Grading of Milk

S.No Product Temperature Bacterial count/ml Chemical and others
1. Grade A Cooled to 50ºF Individual producer Antibiotics should be
raw milk for and maintained milk should not exceed less than 0.05 unit/ml
pasteurization there at until 100,000/ml prior to
processed combining with other
produce of milk
2. Grade A Cooled to 45ºF Milk and Milk products Phosphates less than
pasteurized or less 20,000/ml coliforms limit 1mg/ml
milk products not exceeding 10/ml
77

distribution. This includes sanitation,
pasteurization, holding conditions and
microbiological standards. The U.S public
health secrine publication “Milk ordinance
and code” shows the following chemical,
bacteriological and temperature standards
for grade A milk and milk products.
5.5.5 Methylene Blue dye Reduction

Test (MBRT)
Methylene blue dye reduction test
commonly known as MBRT test is used as a
quick method to access the microbiological
quality of raw and pasteurized milk. This
test is based on the fact that the blue colour
of the dye solution added to the milk get
decolorized when the oxygen present in Figure 5.4: Methylene Blue dye Reduction
the milk get exhausted due to microbial Test
activity. The sooner the de colorization,
more inferior is the bacteriological quality Decolourization is considered complete
of milk assumed to be MBRT test may be when only a faint blue ring (about 5mm)
utilized for grading of milk which may persists at the top (Figure 5.4).
be useful for the milk processor to take a Recording of Results – During
decision on further processing of milk. incubation, observe colour changes as
Procedure follows:
The test has to be done under sterile a. If any sample is decolourized on
conditions. Take 10ml milk sample in sterile incubation for 30 minutes, record the
MBRT test tube. Add 1 ml Methylene Blue reduction time as MBRT – 30 minutes.
dye solution (dye concentration 0.005%). b. Record such readings as, reduction
Stopper the tubes with sterilized rubber times in whole hours. For example, if the
stopper and carefully place them in a test colour disappears between 0.5 and 1.5
tube stand dipped in a serological water hour readings, record the result as MBRT
bath maintained at 37 1°C, records this time – 1 hour, similarly, if between 1.5 and 2.5
as the beginning of the incubation period. hours as MBRT-2 hour and so on.
Table 5.5: Microbiological Quality of Milk

S.No Grade Methylene blue reductase test in hrs Total plate count/ml
1. Very good 5 and above Not exceeding 0.2 million
2. Good 3–4 Between 0.2 to1.0 million
3. Fair 1–2 Between 1–5 million
4. Very poor 0.5 over 5 million
78

Infobits
Major categories and Examples of Fermented Milk Products

Category Typical Examples
i. Lactic Fermentations Buttermilk cultured buttermilk Langofil,
Mesophilic Tetmjolkymer
Thermophilic Yogurt, Laban, Zabadi Bulgarian buttermilk
Therapeutic Bifighurt, Acid ophilur milk, Yakult
ii. Yeast-lactic fermentations Kefir, Koumiss, Acidophilus-yeast milk
iii. Mold – lactic Villi
fermentations
c. Immediately after each, reading, remove They are two groups of cheese, fresh
and record all the decolourized samples and cheese and ripened cheese. The fresh
then gently invert the remaining tubes if the cheese are made up of mild coagulated by
decolourization has not yet begun (Table 5.5). acid or high heat example. cottage cheese,
while ripened cheese are made through
5.6 Cheese lactic acid bacterial fermentation and
There are about 2000 varieties of cheese coagulated by an enzyme preparation.
made from mammalian milk. Cheese is The curd is removed and salted and whey
thought to have originated in south western is separated. The salted curd is held in
Asia some 8000 years ago. The Romans controlled environment. During this
encouraged technical improvements process, various physical and chemical
and stimulated the development of new changes occur to give a characteristic
varieties during their invasion in Europe flavour and texture. So the mammalian
between 60 B.C and A.D. 300. The cheese origin of milk influences the flavour and
name is derived from Latin name caseus aroma of a natural ripened cheese.
(Figure 5.5). Microbiology of cheese
A large number of microorganisms
plays a role in the ripening process. On
the first day of cheese making process,
the microbial number in the starting
material ranges from one to two billion.
Therefore, the production declines
because of insufficient oxygen, high
acidity and the presence of inhibitory
compounds that are produced as the
Figure 5.5: Cheese cheese ripens. It is mainly the action of
79

a. Preparing the milk
b. Forming a curd.
c. Cutting
d. Cooking
e. Separating the whey
f. Salting the residue
g. Applying microbes
h. Pressing the curd
i. Ripening the young cheese
Types of Cheese
Figure 5.6: Swiss cheese
Cheese can be divided
among different categories
their cellular enzymes on lactose, fat and or types, according to their
proteins that creates the ripened cheese firmness. There are various
flavour. The gas forming culture of system for classifying
Propionibacterium shermanii is essential cheese and there are
for giving swiss cheese its eye, or holes variations within each system (Table 5.6).
and flavour (Figure 5.6).
The specificity of cheese depends upon 5.7 Yoghurt or Bulgarian Milk
the varieties of microorganisms used. The Yoghurt is derived from a Turkish word
process of cheese making, involves nine ‘Jugurt’ which is the most popular fermented
steps: milk in the world now – a – days. It is made
Table 5.6: Types of Cheese

Soft cheese
Soft, Smooth and creamy texture. Soft cheese is not pressed or cooked during
the manufacturing process. Example: Camembert
Semi-soft cheese
A little more firm and compact than soft cheese, the semi-soft category
contains the largest variety of cheese. Example: Havarti
Firm cheese
Cheese in the category is considered to be an “all purpose” cheese. Cheese is
pressed to remove as much whey as possible after the curdling process which
creates a firm cheese. Example: Cheddar
80

Hard cheese
Hard cheese has a moisture content of less than 50% due to the cheese being
the cheese to lose some of its moisture content and have a stronger flavour.
Example: Romano
Blue cheese
Cow, sheep or goats milk with a blue or green-blue mold. The mold is derived
from spores from Penicillium roqueforti, Penicillium glaucum or other being
injected into the cheese curds. People who are allergic to penicillin are not
advised to eat blue cheese. Example: Roquefort
Fresh, un ripened or infant cheese

Fresh cheese is not ripened, aged or fermented during the manufacturing
process or at any point during the lifespan of the cheese. Fresh cheese has a
very short shelf life. Example: Cottage cheese, Cream cheese.
light or lite cheese

Light cheese is made by reducing the amount of butterfat which makes the
cheese rubbery in texture and much less flavourful than full fat versions of
cheese. Light cheese has a high moisture content which makes it have a shorter
shelf life. Example: Cheese with 7% Milk Fat, Cheddar which 19% Milk fat.
Processed cheese
This cheese is created by melting together blend of grated cheese, milk, milk
solids or water, food colouring and seasonings. Example: Processed cheese
shies, cheese spreads “swokies”.
from milk, skimmed milk or flavoured culture is added to it. Heating improves
milk. For the preparation of yoghurt, the the milk by inactivating immunoglobulins,
milk should be free from contamination. remove excessive oxygen to produce micro
The solid content (not fat should be aerophilic environment which support the
between 11–15% which can be obtained by growth of starter culture. Besides, heating
adding skin or whole milk powder in fresh also induce the interactions between whey
milk that normally contains 8% solids. the or serum proteins and casein which increase
product can be further improved by adding yoghurt viscosity. The milk is now cooled to
small amount of modified gums which bind 40–43ºC so as to allow fermentation using
water and impart thickening to the product. starter organisms such as Streptococcus
At this stage the size of the fat particles in salivarius sub sp. thermophilus and
the milk should be around 2µm because Lactobacillus delbruckii sub sp. bulgaricus
this improves the milk’s viscosity, product’s together at a level of 2% by volume (106–
stability and milk appear form. The milk is 107 cfu/ml). It is to be carried out for about
then heated at 80–90ºC for 30 min., starter 4h during which lactose is converted into
81

lactic acid, pH decreases to a level of 6.3
– 6.5 to 4.6 – 4.7. The flavour in yoghurt
is due to acetaldehyde which should be
present at 23 – 41 mg/kg (Figure 5.7).
Figure 5.7: Yoghurt
Figure 5.8: Curd
Kefir: Kefir is in lemon juice or vinegar and then draining

fact, fermented milk, off the liquid portion called whey milk
produced by a mixed that has been left to sour (raw) milk alone
lactic acid bacteria or pasteurized milk with added lactic acid
and alcoholic yeast. The microflora bacteria or yeast (Example: Lactobacillus
responsible is not spread uniformly acidophilus) will also naturally produce
throughout the milk but is supplemented curds and sour milk cheese is produced
as discrete kefir grains. The Kefiran i.e. this way. The increased acidity causes the
large layers of polysaccharide material milk protein (casein) to tangle into solid
folds upon to produce a cauliflower masses or curds in cow’s milk, 80% of the
like Florets produce Kefir. The capsular protein and caseins (Figure 5.8).
homo fermentative Lactobacillus Uses
kefiranolaciens produces Kefiran.
•• Enhances healthy digestion
Lactobacillus Kefir contributes the
required effervescence in the product. •• improves immunity
•• For stronger bones and teeth
•• Helps to lose weight
•• Beauty benefits of curd – for healthy
and Radiant skin, prevent premature
wrinkles remove dark spots and
dandruff.
Summary
5.8 Curd Micro organisms are associated, in a variety
Curd is a dairy product obtained by of ways with all of the food we eat. They
curdling or coagulating milk with rennet may influence the quality, availability and
or an edible acidity substance such as quantity of our food naturally occurring
82

foods such as fruits and vegetables Evaluation
normally contain same micro organisms
and may be contaminated with additional
organisms during handling. Many factors 1. The primary sources
that influence the growth of the micro of micro organisms
organisms in food some of the factors are in food include
intrinsic and some others are extrinsic
factors. Spoilage of food can be defined i. Soil and water
as any visible or invisible change which ii. Food utensils
can make food or product from food iii. Food handlers
unacceptable for human consumption iv. Air and dust
spoilage of food not only causes health
a. (i) and (ii)
hazard to the consumer but also cause
large economic losses. Food poisoning b. (ii), (iii)
refers to the toxicity introduced into food c. (i), (ii), (iii) and (iv)
by micro organism and their product. Food d. None of the above
intoxication or food poisoning results from 2. The micro organisms grow best at PH
ingestion of foods containing performed value around
microbial toxins. Foods can be preserved by
a. 4.0 b. 7.0 c. 3.4 d. 9.2
a variety of methods. It is vital to eliminate
or reduce the populations of spoilage and 3. The aw of most fresh food is above
disease causing micro organisms and to
maintain the micro biological quality of a a. 0.99 b. 0.88 c. 0.77 d. 0.66
food with proper storage and packaging. 4. The minimum value of aw for the
Food borne disease has been defined growth of the micro organisms in
by the world health organization (WHO) foods should be around
as a disease of an infectious or toxic nature a. 0.99 b. 0.86 c. 0.78 d. 0.50
caused by or thought to be caused by 5. Choose mismatched pair:
the consumption of food or water. Food Asepsis – Keeping out of Micro
borne infection involves the ingestion of organisms
the pathogen followed by growth in the Filteration – Removal of Micro
host, including tissue invasion the release organisms
Heat (or)
of toxins. The area of diary microbiology
Radiation – Killing the Micro
is large and diverse. The bacteria in diary organisms
products may cause disease or spoilage. Prevention of
Some bacteria may be specifically added Damage – Blanching
to milk for fermentation to produce
products like yoghurt and cheese.
83

6. Milk is contain % of the 15. Classify the Food intoxication?
case in protein. 16. List out the methods of food
a. 90–95% b. 80–85% preservation?
c. 60–65% d. 50–100%
17. What are the principles of food
Answer the following preservation?
1. What is food spoilage? 18. Classify the food poisoning?

2. What is perishable food? 19. Describe the Food-Borne
intoxications.
3. Define food poisoning.
4. Define food intoxication. 20. Write about bacterial food borne
disease?
5. What is food borne infection?
21. Write about the composition of milk.
6. List out the sources of micro organism
in food. 22. What are the sources of milk.
7. Tabulate the major causes of food 23. Write about MBRT.
spoilage.
8. Explain food poisoning. Student Activity
9. What are the advantage and To study the growth of fungus

disadvantages of food spoilage. Take a piece of bread. Make it moist
10. Write about the bacterial food and keep it in a warm corner of the
infection. room for 3–4 days observe it after 3–4
11. Explain – Milk. days. Record your observation.
12. Tabulate – the micro biological To understand the principle of food
quality of milk. preservation
13. What are the factors that influence Take two apples. Keep one apple in
growth of micro organisms in food? the fridge and one outside for 2–3
14. Write about the causes of food days. Record your observation.
spoilage.
84

Chapter
6 Industrial Microbiology
6.3 Strain Improvement

Learning Objectives
6.4 Preservation of Industrially
After studying this chapter the Important Microorganisms
6.5 Fermentors
• Know the concepts involved in
6.6 Industrial Production of Penicillin
industrial microbiology and the
production of industrially microbiology 6.7 Industrial Production of Wine
production by microorganisms. 6.8 Industrial Production of Single Cell
• Understand the primary and Protein
secondary screening process. 6.9 Industrial Production of Citric Acid
• Gain knowledge in the field of strain
6.10 Immobilization
improvement of microorganisms.
• Describe the structure components Industrial microbiology
and function of a fermentor. is a branch of science
• Known the principles behind that deals with the study
fermentation medium, fermentation and uses of various
process, upstream processing and microorganisms that
downstream processing. are responsible for the
• Know the values of microorganism production of many products which has
used in the production of penicillin industrial and economic applications. Man
citric acid, wine and single cell protein. has been using many microorganisms for
• Analyze the basics behind the production of foods, (bread, cheese,
immobilization of microorganisms yoghurt, pickles)–beverages (beer, wine)
for many centuries. The birth of industrial
microbiology largely began with the
Chapter Outline studies of Pasteur on fermentation. The
term Fermentation originates from a
6.1 Industrially Important Micro Latin verb “Fervere” which literally means
Organisms and their Products to boil. In alcohol production, CO2 (gas
6.2 Screening of Industrially Important bubbles) Figure 6.1 are formed during
Microorganism boiling of liquid.
85

acids, vitamins, enzymes, organic acids
and nitrogenous bases are produced by
wide variety of microorganisms. These
primary metabolites are essential for
the growth of microorganisms and they
are produce during Logarithmic phase.
Secondary metabolites do not play a role
in development, growth and reproduction
of microorganisms. They are produce at
the end of growth phase near stationary
Figure 6.1: Bubble formation in grape
phase. They usually accumulate during
juice fermentation
the period of nutrient limitation or waste
6.1 Industrially Important Microbes product accumulation that follows the
and their Products exponential phase. These compounds have
no direct relationship to the synthesis of cell
Microorganisms have the powerful capacity to
materials and normal growth. They are the
produce numerous products, during their life
end products of the primary metabolism.
cycle. Flowchart 6.1 shows the production of
Products such as steroids, alkaloids,
valuable metabolic products during the growth
antibiotics are secondary metabolites.
of microorganisms on a suitable medium
under controlled environmental conditions. Excessive production of the primary
Microbial products are often classified as and secondary metabolites produced by the
primary and secondary metabolites. microorganisms are useful in the large scale
Primary metabolites consist of in industrial production. Unlike primary
compounds related to the synthesis of metabolites, secondary metabolites are
microbial cells in the growth phase. produced in small quantities and their
Primary metabolites such as amino extraction is difficult (Figure 6.2).
Nutrient
Primary Secondary
metabolites metabolites
Metabolic Essential
products metabolites Antibiotics Bio organic
Ethanol Amino acids Alkaloids Steroids
Acetone Nucleotides Gibberlins Amino Acids
Latic acid. Vitamins Pigments Ascorbic acid
Flowchart 6.1: Various metabolites produced in Industrial fermentation
86

• recombinant products through
the DNA recombinant technology.
(Table 6.1 shows some industrially
important microorganisms)
The nutritional
yeast is called food
yeast. The yeast cells
are killed during
manufacturing, and not alive in the
final product. It is used in cooking; it
has a cheesy, nutty or savory flavour.
Yeast S. cerevisiae is used as food
yeast. It is a vegan food, available in
both fortified (with some vitamins)
and unfortified form.
Figure 6.2: Production of primary and
The industrial production of
secondary metabolites in the growth cycle
commercial products is carried out
of microorganism
by fermentation process. The term
Some industrially important products fermentation is defined scientifically in
are, a strict sense as a biological process that
• microbial cells (living or dead), occurs in the absence oxygen (anaerobic).
microbial biomass and components In industrial sense any process mediated
of microbial cells, by or involving microorganisms in
• microbial metabolites, which a product of economic value is
• intracellular or extracellular obtained is called fermentation. The term
enzymes, Industrial fermentation also means large
• modified compounds that has been scale cultivation of microorganisms even
microbiologically transformed, and though most of them are aerobic.
Table 6.1: Industrially important microorganisms

Product Microorganisms Uses
Vitamin B12 Streptomyces Vitamin supplements
Lactic acid Lactobacillus delbrueckii Chemical reagents
Citric acid Aspergillus niger Food preservative
Acetic acid Acetobacter Vinegar, solvent
Ethanol Saccharomyces Chemical reagents drinks
Penicillin Pencillium chrysogenum Antibiotic
87

There are many microbiological processes 4. It should be easily cultivated on large scale.
that occur in the presence of air (aerobically) 5. The strains should be able to protect
yielding incomplete oxidation products. themselves from contamination.
Examples: i) the formation of acetic acid The strain should be in pure culture, free
(vinegar) from alcohol by vinegar bacteria from other microorganisms including
ii) citric acid from sugar by certain Bacteriophages. These characters are
molds such as Aspergillus niger. These screened for the production of desirable
microbial processes are often referred to products from microorganisms.
as fermentations, although they do not
decompose in the absence of air. Isolation of industrially important
microorganisms
Infobits Success of fermentation depends upon
the isolation of microorganism. The
The German Eduard Buchner, winner
microorganisms are isolated from their
of the 1907 Nobel Prize in chemistry,
natural habitats like soil, lakes, river mud or
determined that fermentation was even in unusual habitats or environments
actually caused by a yeast secretion that he such as extreme cold, high altitude,
termed zymase. The experiment for which deserts, and deep sea and petroleum
Buchner won the Nobel Prize consisted fields and are tested directly for the
of producing a cell–free extract of yeast product formation and isolated or it can
cells and showing that this “press juice” be genetically modified. Different types of
could ferment sugar. This finding dealt yet microorganisms are isolated by different
another blow to vitalism by demonstrating methods. Different microbes with desired
for the first time that fermentation could activity are isolated using various culture
occur outside living cells. techniques. Screening includes primary
screening and secondary screening.
6.2 Screening of Industrially Primary screening: The elementary
Important Microorganism steps that are performed to select the desired
The next step after isolation of organisms and eliminate the undesirable
microorganisms is the selection or organisms are termed as primary screening.
screening. For the successful fermentation Methods such as crowded plate technique,
process, selection of microorganisms is the
auxanography and enrichment culture
prime important step.
technique are some of the techniques used
The microbes used in the industrial in primary screening are used in primary
microbiology should have following screening. For screening of antibiotic
characters. producing organisms crowded plate
1. The strain should be a high–yielding technique is described here,
strain.
2. The strain should have stable biochemical Crowded plate technique
and genetical characteristics. 1. It is used for the screening of antibiotic
3. It should not produce undesirable producing organisms.
substances. 2. Soil is serially diluted
88

3. The serially diluted sample is spread 8. The formation of inhibitory zones
on the nutrient agar plates around certain colonies indicates their
4. The plates are incubated and the agar plate antibiotic sensitivity
having 300 to 400 colonies are observed 9. The diameter of the zones of inhibition
for antibiotic producing activity is measured in millimeters. Crowded
5. The ability of a colony to exhibit plate technique is depicted in the
antibiotic activity is indicated by the diagram (Figure 6.3).
presence of a zone of growth inhibition Enrichment isolation
surrounding the colony
The process of enrichment provides a
6. The technique is improved by using suitable condition to support the growth
test organism of microorganisms. It allows the growth
7. The antibiotic produced by the of the specific microbe while inhibiting
organisms in the soil may inhibit the the other non-target microbe. The growth
growth of test organism of target microorganisms is enriched
Figure 6.3: Crowded plate technique

89

by providing sole carbon source. For 3. Secondary screening should yield the
screening microorganisms degrading types of information which are needed
the compound, different inhibitors are in order to evaluate the true potential
employed which have the ability to block of a micro organism industrially
a specific metabolic pathway of the non– usage.
target microbe, pH and temperature are 4. Secondary screening may be qualitative
also adjusted favoring the growth of desired and quantitative in its approach.
microorganisms. Soil Calcium carbonate
5. It is done by using paper, thin layer or
enrichment technique is used for isolation
other chromatographic techniques.
of secondary metabolite producing
microorganisms (actinomycetes). 6. The product’s physical, clinical, and
biological properties of the product
Secondary screening are determined.
It is very useful in sorting out
7. It detects gross genetic instability in
microorganisms that have real commercial
microbial cultures.
value from many isolates obtained during
primary screening. 8. It gives information about the number
of products produced in a single
HOTS fermentation.
9. It determines the optimum conditions
Why microorganisms are exploited for growth or accumulation of a
more than plant and animal cells for product associated with particular
production of commercial products? culture.
10. It gives information about the different
1. As primary screening allows components of the fermentation
the deflection and isolation of medium.
microorganisms which posses,
11. It helps in providing information
potentially interesting industrial
regarding the product yield potential
applications. It is further followed
of different isolates.
by secondary screening, to check the
capabilities and gain information 12. It reveals whether microorganisms are
about these organisms. capable of a chemical change or of even
destroying their fermentation product.
2. Through primary screening only few
or many microorganism that produce There are various methods employed
a industrially important product, are for secondary screening which includes
isolated. The information about the test conducting on petridish containing
product that formed is very less. So, solid media or by using flasks or small
through secondary screening, further fermentors containing liquid media, giant
sorting out is performed. In this colony technique, and filtration method
method, only microorganisms with real liquid medium method (using Erlenmeyer
commercial value are selecting and those flask). Here giant colony technique’s
it lacks the potential are discarded. explained in detail.
90

Giant Colony Technique the test organism. The growth of the test
The Streptomyces culture is inoculated organism has been inhibited by antibiotic
onto the central areas of petriplates in the vicinity of the Streptomycete is
then measured in millimeters. These
containing a nutritious agar medium or
Streptomycetes that have produced
they are streaked in a narrow band across
antibiotics with observable microbial
the centre of plates. The plates are then
inhibition spectrum are retained for
incubated until growth and possibly,
further testing (Figure 6.4).
sporulation have occurred. Strains of
micro organisms to be tested for possible 6.3 Strain Improvement
sensitivity to the antibiotics (the test
Improvement of the production strain(s)
organisms) are then streaked from the
offers the great opportunities for cost
edges of the plates up to but not touching reduction without significant capital outlay
the Streptomycete growth. The plates are in industries. Moreover, success in making
further incubated to allow the growth of and keeping a fermentation industry
competitive depends greatly on continuous
improvement of the production strain(s).
Improvement usually resides in increased
yields of the desired metabolite. The
science and technology of manipulating
and improving microbial strains, in order
to enhance their metabolic capacities for
biotechnological applications, are referred
to as strain improvement.
Need for strain improvement

Microbes exist in the nature produce
certain compounds of biological interest.
However the industrial application of
producing those compounds by natural
strains is not an economical one so, wild
strains are changed by the changing
their gene pattern or by regulating their
enzymes production. As a result, the
specific product is produced in excess.
Knowledge of the function of
enzymes, rate limiting steps in pathways,
and environmental factors controlling
synthesis further helps in designing
Figure 6.4: Giant Colony Technique screening strategies.
91

Attributes of Improved strains 6. Shorten fermentation times.
1. Assimilate inexpensive and complex 7. Overproduce natural products or
raw materials efficiently. bioactive molecules not synthesized
2. Alter product ratios and eliminate naturally for example insulin.
impurities or by products in 8. Excrete the product to facilitate
downstream processing. product recovery.
3. Reduce demand on utilities during Generally wild strains of microorganisms
fermentation (air, cooling water, or produce low quantities of commercially
power). important metabolites. So, genetic
4. Provide cellular morphology in a form improvements have to be made and
suitable for product separation. new strains need to be developed for
5. Create tolerance to high product any substantial increase in the product
concentration. formation in a cost effective manner.
Protoplast fusion is defined as fusion of two different protoplasts. A cell

wall less nature of plant, bacteria, fungal is called protoplast. It is re moved
by either mechanical or enzymatic means. Protoplast has nucleus other
protoplasmic contents which are surrounded by cytoplasmic membrane.
92

actinomycetes (a form of fungi-like
HOTS
bacteria) for very long period of time.
The microorganisms can be preserved by
An organism is isolated from soil, which desiccating on sand, silica gel, or paper
is a very low yielding one. How will you strips.
enhance the production activity?
b. Agar Slopes
The following techniques at practical Microorganisms are grown on agar slopes
genomic level help to improve the in test tubes and stored at 5 to −20 °C for
microbial strain. They are: six months. If the surface area for growth
1. Selection of mutants is covered with mineral oil the m icro-
2. Recombination organisms can be stored for one year.
3. Regulation c. Liquid Nitrogen

4. Genetic engineering This is the most commonly used technique
5. Protoplast fusion to store micro-organisms for a long period.
Storage takes place at temperatures of less
6.4 Preservation of Industrially than -196 °C and even less in vapour phase.
Important Micro Organisms Micro organisms are made stationary
and suspended in a cryoprotective agent
The selected microorganism of industrial
before storing in liquid nitrogen.
interest must be preserved in its original
form for any further use and research. d. Drying
There are different methods for microbial This method is especially used for
preservation. Suitable methods are sporulating microorganisms (organisms
selected based on the: that produce spores). They are sterilized,
a. Type of micro-organism inoculated, and incubated to allow
b. Effect of the preservation method on microbial growth, then dried at room
the viability of micro-organism temperature. The resultant dry soil is
stored at 4° to 5 °C.
c.
Frequency at which the cultures are
withdrawn e. Lyophilization
d. Size of the microbial population to be This process is also known as freeze-
preserved drying. The microbial culture is first
e. Availability of resources dried under vacuum, filled in ampoules
f.
C ost of the preservation method. (glass vessels) then frozen. This is a most
Followings are some of the methods of convenient technique, since it is cheap to
microbial preservation: store and easy to ship. The disadvantage
is that it is difficult to open the freeze
a. Desiccation dried ampoules; also, several subcultures
This involves removal of water from the have to be done to restore the original
culture. Desiccation is used to preserve characteristics of the microorganisms.
93

6.5 Fermentors 6.5.1 Basic Design of a Fermenter
The main function of a fermenter is to The materials used for construction of
provide a suitable environment in which fermenter withstand repeated steam
an organism can efficiently produce a sterilization and are nontoxic. The reaction
vessel is designed to withstand vacuum
target product. Most of them are designed
or else it may collapse while cooling. The
to maintain high biomass concentrations,
internal surface is smooth and corrosion
which are essential for many fermentation resistant. Either stainless steel or glass is
processes. Fermentor design, quality of used for construction.
construction, mode of operation and the
Conventional bioreactors are
level of sophistication largely depend upon cylindrical vessels with dome top and
the production organism, the optimal bottom (Figure 6.5).
operating conditions required for target It is surrounded by a jacket and
product formation, product value and sparger at the bottom through which air
the scale of production. The performance is introduced. The agitator (for mixing
of any fermenter depends on many of cells and medium) shaft is connected
factors, but the key physical and chemical to a motor at the bottom. (Figure 6.6) It
parameters that must be controlled are has ports for pH, temperature, dissolved
agitation rate, oxygen transfer, pH, and Oxygen sensors for regulation. Antifoam
temperature and foam production. agents like animal vegetable oil, lard oil,
corn oil and soya bean oil are used to
HOTS control the foam. Modern fermentors
are usually integrated with computers
What will happen if antifoam agents are for efficient process monitoring and data
not used in the Fermentation process? acquisition. Parts of the fermenter and
their functions are given in Table 6.2.
Figure 6.5: Design of a fermenter

94

Figure 6.6: Components of Fermentor
Table 6.2: Components and their uses:

S. no Parts of fermenter Functions
1 Impeller (agitator) To stir the media continuously and hence prevent cells from
setting down and distribute oxygen throughout the medium.
Impellor speed decreases as the size of the fermenter increases
2 Sparger (aerator) Introduce sterile oxygen to the media in case of aerobic
fermentation process
3 Baffles (vortex breaker) Disrupt vortex and provide better mixing
4 Inlet Air filter Filter air before it enter the fermenter
5 Exhaust Air filter Trap and prevent contaminants from escaping
6 Rota meter Measure flow rate of Air or liquid
7 Pressure gauge Measure pressure inside the fermenter
8 Temperature probe Measure and monitor change in temperature of the medium
during the process
9 Cooling jacket To maintain the temperature of the medium throughout the
process
10 pH probe Measure and monitor pH of the medium
11 Dissolve oxygen probe Measure dissolve oxygen in the fermenter
12 Level probe Measure the level of medium
13 Foam probe Detect the presence of the foam
14 Sampling point To obtain samples during the process
15 Valves Regulation and control the flow liquids and gases
95

Table 6.3 shows common substances used
here are different
T in the industrial fermentation process.
types of fermentor
Waste products from other industrial
used in industrially
processes such as molasses, ligno cellulosic
micro biology which
waste, and corn steep liquor are generally
includes.
used as substrates for industrial fermentation.
1. Stirred tank bioreactor
Apart from carbon and nitrogen
2. Tower bioreactors sources, some other components like
3. Air lift bioreactors minerals, vitamins, growth factors are
4. Packed–bed bioreactors also used in Industrial fermentations.
5. Fluidized bed bioreactors Minerals
6. Photo bioreactors Normally, sufficient quantities of cobalt,
copper, iron, manganese, molybdenum,
and zinc are present in the water supplies,
6.5.2 Media Used in the Industrial
Productions and as impurities in other media
ingredients. For example, corn steep
Fermentation Medium liquor contains a wide range of minerals
Most fermentation requires liquid media, that will usually satisfy the minor and
often referred to as broth, although some trace mineral needs.
solid-substrate fermentations are also
operated. Fermentation media must satisfy Vitamins and growth factors
all the nutritional requirements of the Many bacteria can synthesize all necessary
microorganism and fulfill the technical
vitamins from basic elements. For other
objectives of the process. Animal fats and
bacteria, filamentous fungi and yeasts,
plant oils are also incorporated into some
they must be added as supplements to
media, often as supplements to the main
carbon source. the fermentation medium. Most natural
carbon and nitrogen sources also contain
Medium used for large scale production
at least some of the required vitamins as
should have the following characteristics.
minor contaminants. Other necessary
1. It should be cheap and easily growth factors, amino acids, nucleotides,
available. fatty acids and sterols, are added either in
2. It should maximize the growth of pure form or, for economic reasons, as less
the microorganism productivity and expensive plant and animal extracts.
the rate of formation of the desired
product. Precursors
3. It should minimize the formation of Some fermentation must be supplemented
undesired products. with specific precursors, notably for
It should contain carbon source, nitrogen secondary metabolite production.
source, energy source, micro nutrients When required, they are often added in
required for the industrial production. controlled quantities and in a relatively
96

Table 6.3: Some common substrates used in the industrial fermentation process:
Carbon source
Molasses It is a byproduct of sugar industry. It is a cheap source of
carbohydrates It also contains nitrogenous substances, vitamins,
trace elements. (Example:) sugar cane, beetroot molasses
Malt extract It is an aqueous extract of malted barley
starch, dextrin cellulose They can be metabolized by microorganism. They used for
the industrial production of alcohol
Whey It is a byproduct of dairy industry used in the production of
alcohol, SCP, vitamin B12, lactic acid, gibberllic acid
Methanol ethanol Methanol in the cheapest substrate. It is utilized only by few
bacteria yeast. Methanol is used for SCP. Ethanol is used for
acetic acid production
Hydro molasses It is a byproduct in glucose production from corn
Sulphate waste liquor It is a spent sulfite liquor form the paper pulping industry.
It is used in the production of ethanol by Saccharomyces
cerevisiae, and in the growth of Torula utilis as a feed
Nitrogen sources
Inorganic: Ammonium It is a cheap source of nitrogen
salts and ammonia
Urea (Organic) It is a good and cheap source of organic source
Corn steep liquor It is formed during starch production from corn. It is rich in
(Organic) several amino acids
Yeast extract It is rich in amino acids, peptides vitamins
Soy meal It is a left out residue on preparing soybean oil from soybean
seeds. It is used in antibiotic production
Peptones The proteins hydrolysates are called as peptones. The source
of peptones includes meat, cotton seeds and sunflower seeds
pure form. Examples: Phenyl acetic acid engineering, mathematics and computer
or phenylacetamide added as side chain technology. A typical operation involves both
precursors in penicillin production. upstream processing (USP) and downstream
processing (DSP) stages (Figure 6.7).
6.5.3 Large Scale Production
Basic Steps of Industrial Fermentation 6.5.4 Upstream Processing
Successful development of a fermentation It is the first step in which biomolecules
process and fermentors requires major like bacteria or other cells are grown in a
contributions from a wide range of other fermentor. Upstream processing involves
disciplines, particularly biochemistry, inoculation development, scale up,
genetics, molecular biology, chemistry, medium preparation and sterilization of
chemical engineering and process media and fermentation process.
97

inoculums is generally 1–10% of the total
volume of the medium.
In general, fermentation/ bioprocess
techniques are developed in stages starting
from a laboratory and finally leading
to an industry. The phenomenon of
developing industrial fermentation process
in stages is referred to as scale–up. Scale–
up is necessary for implementing new
fermentation technique developed using
mutant organisms.
The very purpose of scale–up is to
develop optimal environmental and
Figure 6.7: An overview of Fermentation operating conditions at different levels for
process a successful fermentation industry where
conditions like substrate concentration
Inoculum development agitation and mixing, aeration, power
It is a preparation of a population of micro consumption and rate of Oxygen transfer
organisms from a stock dormant culture are studied. In a conventional scale–up, a
to a state useful for inoculating a final fermentation technique is developed in 3–4
production fermentor. stages. The initial stage involves a screening
process using Petri dishes or Erlenmeyer
It is a critical stage in fermentation
flasks followed by a pilot project to
process. determine the optimal operating conditions
It is a stepwise sequence employing for a fermentation process with a capacity
increasing volume of media. of 5–200 litres. The final stage involves the
Inoculum media usually balanced for rapid transfer of technology developed in the
cell growth and not for product formation. laboratory to industry.(Figure 6.8)
It has to be continuously noted that a
Inoculum scale up fermentation process that works well at the
It is the preparations of the seed culture in
amounts sufficient to be used in the larger
fermenter vessel. It involves growing
the microorganisms obtained from the
pure stock culture in several consecutive
fermenter. By doing this, the time
required for the growth of microbes in the
fermenter is cut down, so that the rate of
productivity is increased. The seed culture
obtained is then used for inoculation in Figure 6.8: Scale up in industrial
fermentation medium. The size of the fermentation
98

laboratory scale may work poorly or may i. Batch Fermentation
not work at all on industrial scale. Therefore The medium and culture are initially fed
it is not always possible to blindly apply the into the vessel and it is then closed. After
laboratory conditions of a fermentation that, no components are added apart from
technique developed to industry.
Oxygen. The pH is adjusted during the
At the laboratory scale, one is course of process by adding either acid
interested in the maximum yield of the or alkali. The fermentation is allowed to
product for unit time. At the industry run for a predetermined period of time
level, besides the product yield, minimal
and the product is harvested at the end.
operating cost is another important factor
Foaming is controlled by adding antifoam
for consideration.
agents such as palm oil or soybeans oil.
Preparation and sterilization of media Heat generated is regulated by providing
According to the specific industrial water circulation system around the vessel
production basic components needed to for heat exchange.
carry out fermentation are selected as per
the required volume. ii. Continuous fermentation
Medium components should be This is an open system. It involves the
free from contamination. So all the removal of culture medium continuously
medium components employed in the and replacement of them with a fresh
fermentation process are sterilized. sterile medium in a bioreactor.
Sterilization is mostly carried out by In this method, homogenously mixing
applying heat and to lesser extent other reactors which include chemo stat and
physical methods, chemical methods turbid stat bioreactors are used. Examples:
(disinfectants) and radiation (using UV production of antibiotics, organic solvents,
rays, γ rays). Batch Sterilization is carried beer, ethanol and SCP.
out at 121°C (20 to 60 mins) where as
continuous sterilization is done at 140°C iii. Fed batch system
for (30 to 120 secs). Much energy is wasted It is a combination of both batch and
on batch sterilization on compared with continuous systems. In this, additional
continuous sterilization nearly 80 to nutrients are added to the fermentors
90% of energy saved during this process. as the fermentation is in progress. This
Air and heat sensitive components are extends the time of operation, but the
sterilizied by membrane filters. products are harvested at the end of the
production cycle as in batch fermenter.
Fermentation Process
It involves the propagation of the HOTS
microorganism and the production of the
desired product. Fermentation process is
Why does industry prefer continous
divided depending on the feeding strategy
culture?
of the culture and medium as follows.
i. Batch Fermentation Followed by the fermentation, production,
ii. Continuous Fermentation products are harvested or separated by
iii. Fed batch Fermentation downstream processing.
99

6.5.5 Downstream Processing
The various processes used for the
actual recovery of useful products from
fermentation or any other industrial
processes are called downstream processing.
The cost of downstream processing
(DSP) is often more than 50% of the
manufacturing cost, and there is product
loss at each step of DSP. Therefore, the DSP
should be efficient, involve as few steps as
possible and be cost-effective. Methods Figure 6.9: Structure of Penicillium notatum
involved in the downstream processing are
outlined in the flowchart (6.2). Table 6.4 so the spore suspensions are maintained in
shows Difference between upstream and a dormant state to prevent contamination.
downstream processing. Most penicillin form filamentous broth
Table 6.4: Difference between upstream and hence is difficult to mix and it hinders
(usp) and downstream (dsp) processing oxygen transfer due to their high viscosity.
This is avoided by using bubble columns
USP DSP
air lift reactors which agitates the medium
USP overall DSP depends upon
makes the selection of cost– providing even oxygen distribution.
procurement and effective media
maintenance of Infobits
inoculum
USP involves DSP concentrates on In later (1939) using (Flemings’ work)
in strain media optimization Howard Florey and Ernst Chain managed
improvement for maximum to purify penicillin in a powdered
to enhance and productivity yield form. In 1941, they successfully treated
yield and profit a human. In 1943, they produced
It is a continuous For DSP, penicillin on a large scale. This helped
development of fermentation immensely to treat causalities during the
selected strains conditions are 2nd World War ww1 that had bacterial
to increase the optimized for the
inflations due to their wounds.
economic yield growth of micro
organism or the
production of a Penicillin has a basic structure 6-amino
desired product penicillanic acid 6-(APA). It consists
of a thiazolidine ring with a condensed
6.6 Penicillin Production β-lactum ring. It carries a variable side
Penicillin is a broad spectrum antibiotic. chain in position 6. Natural penicillins
Penicillin is first obtained from the mould, are produced in a fermentation process
Penicillium notatum (Figure 6.9). without adding any side chain precursors.
Penicillium chrysogenum is a high yielding If a side chain precursor is added to the
strain, used for the commercial production broth, desired penicillin is produced and
of penicillin. This strain is highly unstable, it is called bio-synthetic penicillin.
100

Desired Product in Culture Broth
Intracellular Product Extracellular Product Whole Organism
Cell Disruption
(Physical, Chemical,
Enzymatic Methods)
Broth With Solids And Liquid
Physical Chemical Enzymatic

Method Method method
Ultrasonication Alkalies Lysozyme Solid –Liquid

Osmotic shock Organic Glucanase Separation (Flotation,
Heat shock solvents Mannarase Flocctulation, Filtration,
Homogenizatio Protease Centrifugation
Detergents
nimpingement
Grinding with
glass beads Concentration
(evaporation,
liquid-liquid extraction,
membrane filtration,
Purification by precipitation,
chromatography adsorption)
(gel-filtration,
ion-exchange, affinity,
hydrophobic interaction)
Formulation
(drying, freeze-drying, Final product
crystallization)
Flowchart 6.2: Downstream processing methods

101

Semi synthetic penicillin is one in which, 1. Using dry spores to seed the
both fermentation and chemical approach fermentation medium.
are used to produce useful pencillins. 2. Making suspension along with non toxic
It can be taken orally and active against wetting agent like Sodium lauryl sulphate
gram negative bacteria. (eg) Amphicilin. and inoculating germinated organism
Nowadays, semi synthetic pencillins 3. Using pellet inocula obtained by the
makeup the bulk of the penicillin market. germination of spores
The lyophilized spores (or) spores
The main objective in well sporulated frozen agar slant are
of producing semi suspended in water or in a dilute solution
synthetic penicillin of a nontoxic wetting agent.
is to generate
compounds with improved properties. (1:10,000 sodium lauryl sulphonate)
(eg) acid stability, Resistance to enzymic ↓
degradation, broader spectrum of Spores are then added to a bottles
activity. Here side chain is removed to containing wheat bran solution
form (6- APA) via immobilization in a It is incubated for 5-7 days at 24°C for
column of penicillin acylase. Penicilln heavy sporulation.
G is converted to (6-APA) and phenyl ↓
acetic. Then it is chemically to acylated The resulting spores are then transferred
produce Semi Synthetic Pencillin. to production tank
New kinds of synthetic penicillin ↓
can also be produced which are readily The micro organism in the inoculum
absorbed by the intestine compared to tank is checked for contamination.
natural penicillin. Example: Phenithicilin.
Production process
The initial strain of Penicillium
chrysogenum (NRRL, 1951) was low The production tanks are inoculated
yielding strain and so it is was treated with with a mycelial growth.
mutagenic agents such as X-rays, UV right ↓
and some other repeated methods to get a Production medium contains
high yielding strain Q-176. following medium components.
Production methods
Carbon source as Lactose, Nitrogen
Penicillin production is done by one of
source as Ammonium sulphate, Acetate
the following.
or Lactate (Corn steep liquor is the cheap
1. Surface culture and easy source of nitrogen)
2. Submerged fermentation process Mineral sources as K, P (Potassium di
hydrogen phosphate), Mg, S (Magnesium
Inoculum Production sulphate), Zn, Cu(Copper sulphate)
Inoculation methods (Corn steep liquor supply some of these
To inoculate fermentation medium one of minerals)
the following methods can be employed.
102

Precursor (Example: phenyl acetic acid) Third phase: In this phase, the
is added to the medium concentration of antibiotics decreases in
↓ the medium. Autolysis of mycelium starts,
Antifoam agent (Example: corn or liberating Ammonia leading to slight rise
soybean oil) is added before sterilization in pH.
Recovery
The sufficient aeration and agitation is
given and are incubated at 25°C to 26°C After penicillin fermentation, the broth
for 3 to 5 days at PH range of 7 to 7.5 is filtered on rotary vacuum filter
↓
Penicillin Production
Mycelium is separated
Process of penicillin production occurs in ↓
three phases: To the broth sulphuric acid or
First phase: Growth of mycelium phosphoric acid is added
occurs in this phase where the yield of ↓
antibiotic is low. The pH increases due to Penicillin is converted into anionic
the release of NH3. form
Second phase: In this phase, intense ↓
synthesis of penicillin occurs due to rapid It is extracted in counter current
consumption of Lactose and Ammonium solvent extractor, by using organic
nitrogen. The mycelial mass increases and solvent, amyl acetate, methyl isobutyl
the pH remain unchanged (Figure 6.10). (ketone)
Figure 6.10: Production of Penicillin

103

↓
Aqu-aori is the
It is then back extracted with water
concept that oceans

from the organic solvent by adding
and other bodies of
potassium or sodium hydroxide
water, might impart
↓
unique characteristics on the aging
Shifts between water and solvent aid in
process of submerged wine in water.
the purification of penicillin
The ocean provides a unique environ-
↓
ment with cold temperatures, constant
The resulting sodium or potassium
pressure, and little to no light and con-
penicillin is then crystallized
↓ stant motion.
Then it is washed and dried and used
for commercial purpose (anthocyanin). During the preparation
of red wine, all the anthocyanin pigments
6.7 Industrial Production of Wine are solubilized by the extract. Pink wine
An alcoholic distilled beverage is is obtained from either pink grapes or
produced by concentrating alcohol from red grapes in which fermentation last
fermentation by distillation. Beer or ale is for only 12 to 36 hour and only less
produced by the fermentation of malted amount of anthocyanin pigments are
grains. Wine is prepared from grapes solubilized. White wine is prepared from
belonging to species Vitis vinefera. It is the white grapes or from the red grapes
also produced from other fruits like peach, in which pigment involved in colouring
pear, dandelion and honey. Generally wine is removed.
contains 16% of alcohol. Wine production Generally yeasts are the natural
from crushed grapes is called enology. microbiota of grapes
The various forms of wine are listed below
Both wild yeast and cultivated yeast
in the table 6.5.
are involved in the wine fermentation.
Red wine is extracted from the skin Natural yeast is not potable because they
of red grapes containing red pigment do not produce much wine and are less
Table 6.5: Shows different varieties of wine

Red wine It has red pigments
White wine It does not contain red pigments
Rose wine It has less red pigments
Dry wine It has more alcohol content
Sweet wine It has more sugar content
Fortified wine It is fortified with other alcoholic beverage
Sparkling wine It has considerable amount of CO2
Still wine It does not contain carbon dioxide
Distilled wine Brandy (alcohol content 21%)
Table wine It has low alcohol and sugar content
104

alcohol tolerant and produce undesirable Must is inoculated with Saccharomyces
compounds, affecting the quality of the ellipsoideus (2.5%) and selected
wine. fermentation is carried from 50 to
The cultivated wine yeast, 50000 gallons at 20 to 24°C
Saccharomyces ellipsoideus, is used for ↓
commercial production. Figure 6.11 Oak, cement, stone glass lined metal are
shows steps involved in wine production used as fermentor
↓
Temperature and time required for
Infobits fermentation White wine: 10–21°C,
7–12 days; Red wine: 24–27°C, 3–5 days
Saccharomyces is called Brewing ↓
Yeasts, or Baker’s Yeast. The brewing In red wine production, after three to five
strains can be classified into two days of fermentation, sufficient tanin and
groups. The ale strains (Saccharomyces colour is extracted from the pomace and the
cerevisiae) and the lager strains wine is drawn off for further fermentation
(Saccharomyces pastorianus or ↓
Saccharomyces carlsbergenris). Racking improves flavour and aroma,
The ale strains are top fermenting where wine is separated from the sediment
strains. Lager strains are hybrid containing yeast cells as precipitate form
strains of Saccharomyces cerevisiae ↓
and Saccharomyces eubayanus and The wine is subjected to aging at lower
are often referred to as bottom temperature. Ageing process is typically
fermenting. (Saccharomyces yeasts can much longer for red wine than white
form symbiotic matrices with bacteria wine
and are used to produce Kombucha, ↓
Kefir, and Ginger beer). Wines are clarified in a process called
fining. Fining is done by filtration
through casein, tannin, diatomaceous
Steps involved in Wine production earth or bentonite clay, asbestos,
Grapes are stemmed, cleaned and membrane filters or centrifugation
crushed ↓
↓ The wine produced is placed in casks,
Sodium or Potassium Meta – bisulphate tank and bottles
is added to check the undesirable After wine production, cork should be
microorganism used for preventing the entry of air into
↓ the bottles. The presence of air allows the
Must (crushed grapes) is treated with growth of vinegar bacteria that convert the
Sulfur dioxide to kill the wild yeasts and ethanol to acetic acid. The final alcohol
bacteria or sometimes pasteurized to content of wine varies depending upon
destroy the natural microbiota the sugar content of the grapes, length of
↓ the fermentation and type of strain used.
105

6.8 Industrial Production of Single
Cell Protein
Single cell protein refers to the microbial
cells or total protein extracted from pure
microbial cell culture (monoculture) which
can be used as protein supplement for
humans or animals. During ancient times,
the tribes in the Central African Republic
used a spiral shaped Cyanobacterium named
Spirulina platensis as food. They collected it
as mats from the bottom of seasonally dried
up ponds and shallow waters around Lake
Chad and dried them in the sun and made
small cakes called “Dihe”.
During the World war II, when there
were shortage in proteins and vitamins
in the diet, the Germans produced yeasts
and a mould named Geotrichum candidum
was used as food.
The term Single Cell Protein
was coined by C.L Wilson (1966) at
Massachusetts Institute of Technology
Figure 6.11: Steps involved in wine (MIT), to represent the cells of algae,
production bacteria, yeasts and fungi, grown for
their protein contents. The name was
Infobits introduced by Prof. Scrimshow of MIT
in 1967. The organisms like Pseudomonas
Zymology: facilis, P. flava, Chlorella, Anabaena,
Zymology also known as “Zymurgy” Spirulina, Chlamydomonas, and Agaricus
(from the Greek–means, the workings are commonly used for SCP production.
of fermentation) is an applied science Large scale production of SCP is shown in
which studies the biochemical process the Figure 6.12
of fermentation and its practical uses.
There are several methods available for
It includes selection of fermenting
SCP production. In the Japanese method,
yeast and bacteria, species and
flat tray is used with artificial sunlight
their use in brewing, wine making,
algae are cultivated in shallow ponds
fermenting milk and other fermented
with mechanical stirrers or in deeper
foods. The wine yeast Zymurgist one
ponds (not more than 20–30 cm deep)
who studies or practices zymurgy; a
with circulation pumps. Optimum, light
knowledgeable brewer.
is an important parameter for maximum
106

Infobits
Some Commercial Products of Yeast

Sl. No. Product Micro Organism Uses
1. Baker’s yeast, beer, Saccharomyces cerevisiae Baking industry brewing
wine, ale, bread industry
2. Soy sauce Saccharomyces rouxii Food Condiment
3. Sour French bread Candida milleri Baking
4. Commercial alcohol Saccharomyces cerevisiae Fuel, Solvent
(ethanol) Kluyveromyces fragilis
5. Riboflavin Eremotherium ashbyi Vitamin supplement
6. Microbial protein Candida utilis Microbial protein from
Saccharomyces lipolytica petroleum products.
Animal food supplement
(single cell protein) from
paper-pulp waste
Pruteen was the 1st

commercial SCP used
as animal feed additive
with 72% of protein.
Pruteen was produced from bacteria
named Methylophilus mehylotrophus
cultured on methanol.
In India, National Botanical
Research Institute (NBRI) and the
Central Food Technological Research
Institute (CFTRI) are involved in the
production of SCP.
In CPFTRI, SCP is produced from
algae cultured on sewage.
and optimum pH is varied according to

Figure 6.12: Large scale production of SCP the strain and intensity of light. Example:
Spirulina is cultivated at 25–35ºC with
growth of SCP. Scenedesmus sp. grows pH 9.5. Table 6.6 shows different types of
20 times faster in optimum light than in microorganisms and substrates used for
natural conditions. Optimum temperature SCP production.
107

Steps involved in SCP production Table 6.6: List of microorganisms and
Provision of carbon source with added substrates used for SCP production
nitrogen, CO2, ammonia, trace minerals Microorganisms Substrates
for growth Bacteria
↓ Pseudomonas sp. Alkanes
Prevention of contamination by using Methylomonas sp. Methanol
sterilized medium and fermentation Yeast
equipments Candida utilis Sulfite liquor
↓ Lactobacillus Whey
Selected microorganism is inoculated in bulgaricus
a pure form Fungi
↓ Aspergillus niger Molasses
Adequate aeration and cooling is Trichoderma viridae Straw, starch
provided Algae
↓ Spirulina maxima Carbon di oxide
Microbial biomass is harvested Scenedesmus acutus Carbon di oxide
and recovered by flocculation or Actinomycetes
centrifugation flocculants Nocardia Alkanes
↓ Mushroom
Harvested algae are dewatered and Agaricus bisporus Compost, rice
dried on open sand beds Volvariella volvacea straw
↓ Cotton straw
Processing biomass and enhancing it During the cultivation of SCP, care
for use and storage must be taken to prevent and control the
contamination by other micro organisms,
Advantages of using microorganisms for which produce mycoxins or cyanotoxins.
SCP production: This is controlled by using the fungus
1. Microorganisms grow at a very rapid Scytaliclium acidophilum which grows at
rate under optimal culture conditions. a low PH. It allows the hydrolysation of
paper wastes to a sugar medium and also
2. The quality and quantity of protein creates aseptic condition at low cost.
content in microorganisms is better
compared to higher plants and 6.9 Industrial Production of Citric Acid
animals.
Citric acid is obtained from citrus fruits;
3. A wide range of raw materials which pineapple etc., and after the development
are otherwise wasted, can be fruitfully of microbial fermentation, citric acid
used for SCP production production becomes very cheap, easy and
4. The culture conditions and the cost effective. 70% of citric acid produced is
fermentation processes are very simple. used in food and beverage industry. Many
microbial strains such as fungi Aspergillus
5. Microorganisms can be easily handled flavus, Aspergillus niger and Trichoderma
and subjected to genetic manipulations. viridae, yeast Hansenulla polymorpha
108

and Candida lipolytica are generally are The medium is kept at 28–30ºC with
involved in the production of citric acid. relative humidity 40–60% and aerated
Citric acid production can be carried with purified air for 8–12 days
out in the following three methods. ↓
Citric acid produced is determined by
a. Koji process or solid state fermentation
checking the pH or the total acid content
b. Liquid surface culture of the broth.
c. Submerged fermentation ↓
Fermented liquid is drained off and
Media used in citric acid production processed further for the recovery of
Citric acid production is carried out by using citric acid
carbohydrates and n–alkenes. Generally
beet molasses, cane molasses, sucrose, Infobits
commercial glucose and starch hydrolysate
are used as carbohydrate sources. The Influence of trace metals in citric acid
carbohydrate material is diluted and mixed production:
with a nitrogen source (ammonium salts Citric acid production is highly
or urea) and the pH and temperature are influenced by the trace metals.
adjusted according to the process. Particularly, iron and manganese in
excess amount affect the citric acid
Inoculum development
production. They affect the cellular
Fungal strains that are used for production
morphology and change pellets
are stored in soil or silica gel in the form
to filamentous growth (i.e.,) from
of spores. Spores are suspended in a freshly
productive form to unproductive
prepared sterile water containing between
80 and after a period of growth, it can be form.
used as inoculum for large scale production.
Recovery
Steps involved in citric acid production
The mycelial mat is pressed.
Production Medium
↓
Sucrose, beet molasses, used as carbon
source need pretreatment, as it contains Milk of lime is added so calcium citrate is
excessive amount of trace metals. So formed.
ferrocyanide or ferricyanide is added to the ↓
production medium before sterilization.
Inorganic salts, carbon, hydrogen, oxygen Again sulphuric acid is added, so calcium
trace metals. Nitrogen, potassium, sulphate is formed.
phosphorus, sulphur and magnesium ↓
are taken in Aluminum or stainless steel
The remaining citric acid solution
shallow pans or tray (5–20 cm deep).
is filtered and washed. Finally the
↓
impure solution of citric acid subjected
Inoculated with spores of A. niger by
blowing over the strains of Aspergillus to treatment with activated carbon
niger for fermentation and finally pure form of citric acid is
↓ collected.
109

Uses albumin polystyrene, Calcium alginate
It is used as a Acidulant in food, (Jams, polyacrylamide, collagen carrageenan and
Preserved fruits, Fruit drinks) and polyurethane, inorganic materials (brick,
pharmaceutical industries. rand, glass, and ceramics, magnetic) are
used for immobilization.
1. It is mainly used in food and beverage
industry (Jams, preserved fruits, fruit The linkage is mediated by ionic
drinks) bonds, physical absorption or bio specific
binding.
2. It is used is pharmaceuticals, and other
industrial processes The immobilization methods can be
classified into four categories
3. Citrate and citrate esters are used as
plasticizers i. Carrier–binding
4. It is used as a chelating and sequestering ii. Cross–linking
agent (Tanning of animal skins) iii. Entrapping
Generally citric acid obtained from iv. Combining
citrus fruits, pineapple etc., After the Among all these methods entrapping is
development of microbial fermentation, discussed in brief.
citric acid production becomes very cheap
and easy cost effective. Entrapping
The enzymes, cells are not directly attached
6.10 Immobilization to the support surface, but simply trapped
It is technique used for the physical or inside the polymer matrix. Entrapping
chemical fixation of plant, animal cells, is carried out by mixing the biocatalyst
organelles, enzymes or other proteins into a monomer solution followed by a
(monoclonal antibodies) onto a solid polymerization. It is done by change in
matrix or retained by a membrane, in temperature or by chemical reactions.
order to increase their stability and make The diagrammatic representation of
possible their repeated or continued use. entrapping methodology is shown in
Figure 6.13.
The immobilized enzyme is defined
as the enzyme physically confined or Advantages of immobilization
localized in a certain defined region 1. Immobilized growing cells serve as self
of space with retention of its catalytic proliferating and self regenerating bio
activity which can be used repeatedly and catalyst
continuously.
The selection of appropriate carrier
and immobilization procedure is very
essential procedure is very essential for
the immobilization technique.
Various types of materials like
cellulose, dextran, agarose, gelatin, Figure 6.13: Immobilization (Entrapping)
110

2. They are stable protein, are produced in large scale by
3. They are used either repeatedly in using microorganisms.
a series of batch wise reactions or Immobilization is need for the physical
continuously in flow systems. or chemical fixation of plant, animal cells,
organelles, enzymes or other proteins into
HOTS a solid matrix or retained by a membrane.
Immobilized cells can be used for repeated
The technique of immobilized enzymes doses.
may increase the use of enzymes in
industry for product modification. Evaluation
Why? Give reason. Multiple choice questions
1. T
he term fermentation
Summary originates from a
Latin verb
Industrial microbiology is a branch, of
microbiology that deals with the study a. Wear
and uses of various microorganisms. The b. Fervere
birth of industrial microbiology largely c. Severe
began with the studies of Pasteur on
d. Cheer
fermentation. Various products of both
primary and secondary metabolites are 2. metabolites are
produced by different microorganisms. produced in small quantities during
Both primary and secondary screening industrial production.
is involved in the isolation of industrially a. Secondary metabolites
important microorganisms. The isolated b. Primary metabolites
strains are modified for higher yield
c. Tertiary metabolites
through various procedures (example)
protoplast fusion. Thus strain improvement d. Neutral metabolites
improves the fermentation efficiency. 3. enrichment
Fermentation is carried under a suitable technique for isolation of secondary
parameters, in a controlled environment metabolite producing Actinomycetes.
is called Fermentor. Fermentation process a. Soil-Sodium Carbonate
involves both cup stream processing and b. Soil-Calcium Carbonate
downstream processing. In cup stream
c. Soil-Strontium Carbonate
processing, inoculum preparation scale up,
preparation of medium and sterilization d. Soil-Potassium Carbonate
of media, are carried out. 4. The microbes used in the industrial
Down stream processing, includes the microbiology has these qualities
separation and purification of products. Statement A: The strain should have
The industrially important products like stable biochemical and genetical
penicillin, wine and citric acid, single cell characteristics.
111

Statement B: It is a high yielding C. Citric acid 3. Scenedesmus
strain. D. Single cell 4. Saccharomyces
a. Statement A alone is true protein cerevisiae
b. Statement B alone is true a. 4 1 2 3
b. 3 2 1 4
c. Statement A and B are true
c. 2 4 1 3
d. Both A and B are false
d. 2 3 4 1
5. Match the following:
9. are produced at the end of
A. Lactic acid 1. Penicillium
the growth phase or stationary phase.
chrysogenum
a. Tertiary b. Secondary
B. Citric acid 2. Lactobacillus
delbrukeii c. Primary d. All the above
C. Penicillin 3. Saccharomyces 10. Assertion (A): Red wine is prepared

from red grapes.
D. Ethanol 4. Aspergillus niger
Reason (R): Anthocyanin is
a. 4 2 1 3
responsible for pigmentation.
b. 2 4 1 3
a. The statement A is not supported by
c. 1 3 4 2 Statement R
d. 2 4 3 1 b. Statement A alone is correct
6. is an example for c. Statement R alone is correct
primary screening.
. The statement A is supported by
d
a. Photography Statement R
b. Cinematography
c. Auxanography Answer the following
d. Telegraphy 1. Define Fermentation?

7. Strain improvement is the technology of 2. What is bioreactor?
(Assertion) Manipulating and improving 3. What is racking?
microbial strains. (Reason) It is done by 4. Define fining?
Recombination and Protoplast fusion. 5. What are primary metabolites? Give
a. Statement (A) is not supported by (R) one example?
b. Statement A is supported by (R) 6. What are secondary metabolites?
Give example?
c. Statement (A) alone correct
7. What are the characteristics of
d. Statement (B) alone is correct
microbes in industrial microbiology?
8. Define primary screening with
A. Penicillin 1. Aspergillus niger example.
B. Wine 2. Penicillium 9. Define secondary screening with
chrysogenum example.
112

10. Crowded plate technique. Explain. 23. Define semi synthetic penicillin?
11. Giant colony technique–Explain in 24. What are the different types of wine?
detail. 25. How will you prepare white wine
12. Write salient features of secondary from red grapes?
screening. 26. Explain the steps in wine production?
13. What is strain development? What 27. List the steps involved in SCP
are the attributes of improved strains? production.
14. List any five methods of preservation 28. What are the disadvantages of SCP?
of micro organisms. 29. Explain the steps involved in citric
15. Explain the components of fermentor. acid production.
16. What is cup stream processing? 30. What are the uses of citric acid.
17. Define downstream processing? 31. Define Immobilization.
18. Difference between upstream and 32. What are the materials used for
downstream processing. Immobilization?
19. Write the steps in downstream
processing? Student Activity
20. Write the components of fermentor 1. Ask the students to prepare wine
and its function. by using the grapes available in the
21. Explain the various types of super market.
fermentation median components 2. Fermentor design and their
used in Industrial microbiology? components.

22. Explain penicillin production any 3. Wine production

two process.
113

Chapter
7 Medical Bacteriology
7.9 Salmonella Typhi

Learning Objectives
7.10 Vibrio Cholerae
7.11 Mycobacterium Tuberculosis
7.12 Treponema Pallidum
• Understand the importance of
Medical bacteriology. 7.13 Leptospira Interrogans
• Describe the pathogenesis of
Medical Bacteriology
various bacterial diseases such as
is the subset of Medical
skin, respiratory infection, food
microbiology, which
poisoning, diarrhea and dysentery
deals with the study of
diseases, STD and zoonotic diseases.
bacterial pathogens. It
• Know the collection of appropriate includes the pathogenesis,
clinical specimens and laboratory diagnosis, treatment and prevention of
diagnosis of various bacterial various bacterial diseases. Robert Koch is
infections. considered as the Father of Bacteriology.
• Specify how bacterial infections
are prevented and treated with 7.1 Pathogenic Attributes
appropriate antibiotics.
The host-parasite relationship is
determined by the interaction between
Chapter Outline host factors and the infecting pathogens.
Pathogenicity refers to the ability of a
7.1 Pathogenic Attributes pathogen to produce disease. Virulence
7.2 Routes of Entry is the ability of the pathogen to cause
disease.
7.3 Staphylococcus Aureus
Adhesion, invasiveness (Streptococcal
7.4 Streptococcus Pyogenes infections), Bacterial toxins (endotoxins
7.5 Neisseria Meningitidis and exotoxins), capsule enzymes
7.6 Corynebacterium Diphtheriae (proteases, collagenase, coagulase and
other enzymes). These are already
7.7 Clostridium Tetani explained in the XI Standard text book.
7.8 Shigella Dysenteriae
114

7.2 Route of Entry 7.3.1 Morphology
To establish an infection, pathogen must •• Staphylococci are gram positive
first enter the host. Normal defense spherical cocci, (0.8µm–1.0µm in
mechanisms and barriers (For example diameter) arranged characteristically
Skin, mucus, ciliated epithelium, lysozyme) in grape like clusters (Figure 7.1).
make it difficult for the pathogen to enter •• They are non-motile and non-sporing
the body. and few strains are capsulated.
Sometimes these barriers are break
through for example cut in the skin, The grape like
wound, tumor, ulcer which provides portal cluster formation in
of entry for the bacteria. Some bacterial Staphylococcus aureus
pathogens have the means to overcome is due to cell division
the barriers through various virulence occurring in three perpendicular
factors and invade the body. planes, with daughter cells tending to
Certain bacteria are infective when be remaining in close proximity.
introduced through optimal route. The
various route of entry of pathogens, 7.3.2 Cultural Characteristics
which are cut or abrasion or wound
(skin), Ingestion, Inhalation, arthropod •• They are aerobes and facultative
bite, sexual transmission and congenital anaerobes, optimal temperature is
transmission. These are already explained 37°C and optimum pH is 7.4–7.6.
in the XI Standard text book. The various •• They grow on the following media
bacterial pathogens, its pathogenesis and shows the characteristic colony
clinical symptoms, laboratory diagnosis, morphology (Table 7.1 & Figure 7.2).
control, prophylaxis and treatment with Table 7.1: Staphylococci aureus colony
appropriate antibiotics are discussed below. morphology on various media
7.3 Staphylococcus Aureus (Pyogenic Media Colony Morphology

Cocci) Nutrient Agar Colonies are circular,
smooth, convex, opaque
The genus Staphylococcus is included in
and produces golden
the family Micrococcaceae. Staphylococcus
yellow pigment (most
is a normal flora of skin and mucous
strains).
membranes, but it accounts for human
infections, which is known as staph Blood Agar Beta haemolysis
infection. The name Staphylococcus was Mannitol salt It is a selective medium
derived from a Greek word, ‘staphyle’ Agar (MSA) for S. aureus produces
means bunch of grapes and ‘kokkos’ yellow colored colonies
means berry. Staphylococcus aureus is a due to fermentation of
pathogenic species that causes pyogenic mannitol
infections in human.
115

Figure 7.1: Gram staining of
Staphylococcus aureus
7.3.3 Virulence Factors

1. Peptidoglycan → It is a polysaccharide
Figure 7.2: Colony morphology of
polymer. It activates complement and Staphylococcus aureus on MSA
induces the release of inflammatory
cytokines.
5. Enzymes: S. aureus produces several
2. Teichoic acid → it facilitates adhesion enzymes, which are related to virulence
of cocci to the host cell surface. of the bacteria.
3. Protein A → It is chemotactic, a. C oagulase – It clots human plasma
antiphagocytic, anticomplementary and converts fibrinogen into fibrin.
and induce platelet injury.
b. Staphylokinase – It has fibrinolytic
4. Toxins: activity.
a. Hemolysins – It is an exotoxin, those c.
Hyaluronidase – It hydrolyzes
lysis red blood cells. They are of four hyaluronic acid of connective tissue,
types namely α-lysin, β-lysin, γ- thus facilitates the spread of the
lysin and delta lysin. pathogens to adjacent cells.
b.
Leucocidin – It damages PMNL Other enzymes – S. aureus also
d.
(polymorphonuclear leucocytes) produces lipase, nucleases and
and macrophages. proteases
c. Enterotoxin – It is responsible for
manifestations of Staphylococcus 7.3.4 Pathogenicity
food poisoning. S. aureus is an opportunistic pathogen
d. Exfoliative toxin – This toxin causes which causes infection most commonly
epidermal splitting resulting in at sites of lowered host resistance.
blistering diseases. (Example: damaged skin)
e.
Toxic shock syndrome toxin – Mode of Transmission: Staphylococcus
TSST is responsible for toxic shock infections are transmitted by the following
syndrome. ways.
116

pus), styes (a painful swelling of hair
HOTS
follicle at eyelids), carbuncles (painful
cluster of boils of the skin), Impetigo (skin
Why many hospitalized patients are infection with vesicles, pustules which
at increased risk for opportunistic ruptures), pemphigus neonatorum (an
infection? auto immune diseases that affect skin and
mucous membranes)
Direct Contact Deep infections: It includes
Osteomyelitis (inflammation of bones),
Staphylococcus Fomites
tonsillitis (inflammation of tonsils),
Infections (inanimate objects)
pharyngitis (inflammation of pharynx)
Airborne droplets. sinusitis (inflammation of sinuses),
(Sneezing or periostitis (inflammation of membrane
coughing) covering bones), bronchopneumonia
Staphylococcal diseases may be (inflammation of lungs), empyema
classified as (collection of pus in the body cavity),
septicemia (blood poisoning caused
Cutaneous by bacteria and its toxins), meningitis
infections (inflammation of meninge), endocarditis
Deep (inflammation of endocardium), breast
infections and renal abscess.
Food Food Poisoning: Staphylococcal food

poisoning poisoning may follow 2–6 hours after
the ingestion of contaminated food
Staphylococcal Nosocomial (preformed enterotoxin). It leads to
infection infections nausea, vomiting and diarrhea.
Skin Nosocomial infection: S. aureus is
exfoliative a leading cause of hospital acquired
diseases infections. It is the primary cause of lower
respiratory tract (LRT) infections and
Toxic shock
surgical site infections and the second
syndrome
leading cause of nosocomial bacteremia,
(TSS)
pneumonia, and Cardiovascular
It includes the following infections, which infections.
are as follows: Exfoliative diseases: These diseases
Cutaneous infections: Wound (injury), are produced due to the production of
burn infections (tissue injury caused by epidermolytic toxin. The toxin separates
heat), pustules (A small elevated skin the outer layer of epidermis from the
lesions containing pus), furuncles (boil underlying tissues leading to blistering
forms around a hair follicle and contains disease. The most dramatic manifestation
117

of this toxin is scalded skin syndrome.
The patient develops painful rash which
slough off and skin surface resembles
scalding.
HOTS
Why most infections acquired through

the skin are non-communicable
diseases?
Toxic shock syndrome toxin: It is caused

by TSST-1 and characterized by high
fever, hypotension (low blood pressure), Figure 7.3: Skin infections caused by
Staphylococcus aureus
vomiting, diarrhea and erythematous rash.
TSS became widely known in association Direct Microscopy: Gram stained
with the use of vaginal tampons by smears of clinical specimens is done,
menstruating women but it occurs in where gram positive cocci in clusters
other situations also. were observed.
7.3.5 Laboratory Diagnosis Culture: The collected specimen is

inoculated on selective media-MSA
Specimens: The clinical specimens are and the media incubated at 37°C for
collected according to the nature of 18–24 hours. Next day culture plates
Staphylococcal infections, which is given are examined for bacterial colonies,
in the (Table 7.2). which are identified by gram staining,
Table 7.2: Clinical specimen collected colony morphology and biochemical
for Staphylococcal infections tests such as
a. Catalase test: The genus Staphylococci are
Infections Clinical Specimens
catalase positive. This test distinguishes
Suppurative Pus
Staphylococcus from Streptococcus
lesions
(catalase negative).
Respiratory Sputum
b.
C oagulase test: This test helps in
infections
differentiating a pathogenic strain from
Septicemia Blood non-pathogenic strain. S. aureus is
Meningitis CSF coagulasepositive (Figure 7.3).
Food poisoning Faeces, food or vomitus 7.3.6 Treatment
Specimens should be transported Benzyl penicillin is the most effective
immediately to the laboratory and antibiotic. Cloxacillin is used against beta
processed. lactamase. Producing strains (β-lactamase
118

is produced by few strains of S. aureus
which cleaves β – lactam ring of penicillin).
Vancomycin is used against MRSA
(Methicillin Resistant Staphylococcus
aureus) strains.
Topical applications: For mild
superficial lesions, topical applications
of bacitracin or chlorhexidine is
recommended.
Control measures: Proper sterilization
of medical instruments must be done.
Intake of antibiotics must be taken under
proper medical advice. The detection of
source & carriers among hospital staff,
their isolation and treatment should be Figure 7.4: Gram staining of
practiced. Streptococcus pyogenes
7.4 Streptococcus Pyogenes (Flesh •• They are non – motile, non –

eating Bacteria) sporing. Some strains are capsulated
(Figure 7.4).
The genus Streptococcus includes a
large and varied group of bacteria. They
7.4.2 Cultural Characteristics
inhabit various sites, notably the upper
respiratory tract. However, some species of •• They are aerobe and facultative
which Streptococcus pyogenes is the most anaerobe. Optimum temperature is
important and are highly pathogenic. The 37°C and pH is 7.4 to 7.6
name Streptococcus is derived from Greek •• They grow only in media enriched with
word ‘Streptos’ which means twisted or blood or serum. It is cultivated on blood
coiled. agar. On blood agar, the colonies are
small, circular, semitransparent, low
7.4.1 Morphology convex, with an area of clear hemolysis
•• They are Gram positive, spherical around colonies (Figure 7.5).
or oval cocci and arranged in chains •• Crystal violet blood agar – a selective
(0.6µm–1µm) medium for Streptococcus pyogenes.
Year Scientist Contributions

1874 The odor Billroth Streptococci were discovered
1884 Friedrich Julius The cocci were isolated from human lesions
Rosenbach and gave the name Streptococcus pyogenes
119

which helps in attachment to the host
cell. Middle layer of cell wall consists of
Group Specific C – Carbohydrate that is
used for Lancefield grouping. Inner layer
of cell wall is made up of peptidoglycan
which has pyrogenic and thrombolytic
activity.
Toxins and Enzymes: Streptococcus
pyogenes produces several exotoxins and
enzymes which contribute to its virulence.
Toxins and Hemolysins: Streptococci
produces two types of hemolysins which
are Streptolysin O and Streptolysin S.
Figure 7.5: Colony morphology of Erythrogenic toxin: (Pyrogenic exotoxin)
Streptococcus pyogenes on blood agar – The induction of fever is the primary
effect of this toxin and it is responsible for
7.4.3 Antigenic Structure the rash of scarlet fever.
Capsule: It inhibits phagocytosis Enzymes: The various enzyme of
Cell wall: The outer layer of cell wall Streptococcus pyogeneswhich exhibits
consists of protein and lipoteichoic acid virulence activity are listed in Table 7.3.
Infobits
Classification
The aerobic and facultative anaerobic Streptococcus are classified based on haemolytic
properties. Three types of haemolytic reactions are observed on blood agar medium,
which are:
Aerobic and Facultative Anaerobic Streptococci

(Based on Haemolytic properties)
Alpha Beta Gamma

haemolytic haemolytic haemolytic
Incomplete Complete No
haemolytic haemolytic haemolytic
Based on serological grouping of carbohydrate C antigen of Beta haemolytic
organisms, they are classified into 20 Lancefield groups (A to H & K to V)
Streptococcus pyogenes is beta haemolytic organism which is included in Group A.
120

Table 7.3: Enzymes of Streptococcus pyogenes and its virulence nature
Enzymes Virulence nature
streptokinase (fibrinolysin) It promotes the lysis of human fibrin clot by catalyzing
the conversion of plasminogen into plasmin. It
facilitates the spread of infection by breaking down the
fibrin barrier around the lesions.
Deoxyribonucleases It liquefy the highly viscous DNA that accumulate in
thick pusand responsible for thin serous character of
streptococcal exudates
Hyaluronidase It breaks down hyaluronic acid of the tissues and
favors spread of streptococcal lesion along intercellular
spaces.
Other enzymes NADase, lipase, amylase, esterase, phosphates and
other enzymes
Streptococcal diseases may be broadly

Streptokinase: It is classified, and it is shown in flowchart 7.1
given intravenously
for the treatment
Streptococcal diseases
of early myocardial
infarction and other thromboembolic
disorders. Streptococcus equisimilis is
the source of streptokinase used for Suppurative Non-suppurative
thrombolytic therapy in patients. diseases complication
•• Respiratory •• Acute Rheumatic
7.4.4 Pathogenesis tract infection fever
Streptococcus pyogenes is intrinsically •• Skin Infections •• Acute glomerulo-
a much more dangerous pathogen •• Streptococcal nephritis
than Staphylococcus aureus and has a toxic shock
much greater tendency to spread in the syndrome
tissues. •• Deep infections
Mode of transmission: Streptococcal •• Genital
infections are transmitted by the following infections
ways:
Flowchart 7.1: Classification of
Direct Contact Streptococcal diseases
Transmission of Fomites Suppurative Infections
Streptococcal
infection Airborne droplets 1. Respiratory tract infection
a. Streptococcal sore throat: Sore throat
Carriers
(acute tonsillitis and pharyngitis) is the
121

most common streptococcal diseases. 3. Streptococcal toxic shock syndrome
Tonsillitis is more common in older Streptococcal pyrogenic exotoxin leads
children and adults. The pathogen may to streptococcal toxic shock syndrome
spread from throat to the surrounding (TSS). It is a condition in which the entire
tissues leading to suppurative (pus – organ system collapses, leading to death.
formation) complication such as cervical
adenitis (inflammation of a lymph node 4. Genital infections
in the neck) otitis media (inflammation Streptococcus pyogenes is an important
of middle ear), quinsy (ulcers of tonsils) cause of puerperal sepsis or child bed fever
Ludwig’s angina (purulent inflammation (infection occur when bacteria infect the
around the sub maxillary glands) uterus following child birth)
mastoiditis (inflammation of mastoid
process). 5. Deep infection
b. Scarlet fever: The disease consists Streptococcus pyogenes may cause
of combination of sore throat and a pyaemia (blood poisoning characterized
generalized erythematous (redness of skin by pus forming pathogens in the blood)
or mucous membranes) rash. septicemia (A condition in which bacteria
circulate and actively multiply in the
2. Skin infections bloodstream) abscess in internal organs
a. Erysipeals: It is an acute spreading such as brain, lung, liver and kidney.
lesion. The skin shows massive brawny
oedema with erythema it is seen in elderly Non – Suppurative Complication
persons or elders.
Streptococcus pyogenes infections are
b. Impetigo: (Streptococcal pyoderma) sometimes followed by two important
It is a skin infection that occurs most non – suppurative complications which
often in young children. It consists of are, acute rheumatic fever and acute
superficial blisters that break down and glomerulonephritis. These complications
eroded areas whose surface is covered occur 1–4 weeks after the acute infection
with pus. It is the main cause leading to and it is believed to be the result of
acute glomerulonephritis in children. hypersensitivity to some streptococcal
c. Necrotizing fasciitis: It is an invasive, components.
infection characterized by inflammation
1. Rheumatic fever
and necrosis of the skin, subcutaneous fat
and fascia. It is a life-threatening infection. It is often preceded by sore throat and
most serious complication of haemolytic
streptococcal infection. The mechanism
The strain which
by which Streptococci produce rheumatic
cause necrotizing
fever is still not clear. A common cross
fasciitis to have been
named as “Flesh – reacting antigen exist in some group
eating bacteria or” killer bacteria. A streptococci and heart, therefore,
antibodies produced in response to the
122

streptococcal infection could cross react test to differentiate Streptococci
with myocardial and heart valve tissue, fromStaphylococci.
causing cellular destruction. Serology: Serological tests are done for
2. Acute glomerulonephritis rheumatic fever and glomerulonephritis.
It is established by demonstrating high
It is often preceded by the skin infection.
levels of antibody to streptococci toxins.
It is caused by only a few “nephritogenic
The standard test is antistreptolysin
types (strains)”. It develops because some
Otitration. ASOtitres higher than 200
components of glomerular basement
units are indicative of prior Streptococcal
membrane are antigenically similar to the cell
infection.
membranes of nephritogenic streptococci.
The antibodies Formed against Streptococci
7.4.6 Treatment and Prophylaxis
cross react with glomerular basement
membrane and damage. Some patients •• Penicillin G is the drug of choice.
develop chronic glomerulonephritis with •• In patients allergic to penicillin,
ultimate kidney failure. erythromycin or cephalexin is used.
•• Antibiotics have no effect on
HOTS established glomerulonephritis and
rheumatic fever.
Why are some staphylococcal skin •• Prophylaxis is indicated only in the
infections similar to streptococcal skin prevention of rheumatic fever, it
infection? prevents streptococcal reinjection and
further damage to the heart.
7.4.5 Laboratory Diagnosis •• Penicillin is given for a long period
Specimens: Clinical specimens are in children who have developed early
collected according to the site of lesion. signs of rheumatic fever.
Throat swab, pus or blood is obtained for
culture and serum for serology. Myth: Eating choc-
olate encourages the
Direct Microscopy: Gram stained smears
development of acne.
of clinical specimens is done, where Gram
positive cocci in chains were observed. It Fact: It is the oils
is indicative of streptococcal infection. and fats in many chocolate products,
and not chocolate itself, that promote
Culture: The clinical specimen is
sebum production and subsequent
inoculated on blood agar medium and
acne. Chocolate in low-fat chocolate
incubated at 37° C for 18–24 hours. After
milk and in fat-free chocolate candies
incubation period, blood agar medium
does not encourage acne. Acne suffers
was observed for zone of beta – haemolysis
do not need to give up chocolate,
around colonies.
they need to reduce their lipid
Catalase test: Streptococci are catalase consumption.
negative which is an important
123

7.5 Neisseria Meningitides What is Meningitis?
Normal meninges
(Meningococcus)
The genus Neisseria is included in
the family Neisseriaceae (Figure 7.6).
It contains two important pathogens
Neisseria meningitidis and Neisseria Infected meninges
gonorrhoeae, both the species are strict
human pathogens. N. meningitides causes
meningococcal meningitis (formerly
known as cerebrospinal fever).
The word Meningitis is derived from
Greek word ‘meninx’ means membrane Figure 7.7: Pathogenesis of Meningitis
and ‘itis’ means inflammation. It is an
Table 7.4: Colony morphology of
inflammation of meanings of brain or
Neisseria Meningitides on media
spinal cord. Bacterial meningitis is a much
more severe disease than viral meningitis. Name of the Colony
media Morphology
7.5.1 Morphology
Chocolate agar Colonies are large,
They are Gram negative diplococci colorless to grey
(0.6µm–0.8µm in size) arranged typically opaque colonies.
in pairs, with adjacent sides flattened. Mueller Hinton Colonies are small,
They are non – motile, capsulated agar round, convex grey,
(Fresh isolates). translucent with
Cocci are generally intracellular when entire edges. The
isolated from lesions (Figure 7.7). colonies are butyrous
in consistency and
7.5.2 Cultural Characteristic easily emulsified.
They are strict aerobes, but growth
humidity. The optimum temperature is
is facilitated by 5–10% CO2 and high
35°C–36°C and optimum pH is 7.4–7.6.
They are fastidious pathogens, growth
occurs on media enriched with blood
or serum. They grow on the following
media and show the characteristic colony
morphology (Table 7.4).
7.5.3 Pathogenesis
N. meningitidis is the causative agent
of meningococcal meningitis, also
Figure 7.6: Gram staining of Neisseria known as pyogenic or septic meningitis.
Meningitides Infection is most common in children
124

and young adults. Meningococci are strict The meningococci gain entry in the
human pathogens. Human nasopharynx nasopharynx and attaches to the epithelial
is the reservoir of N.meningitidis. cells with pili. They are engulfed by epithelial
The pathogenesis is dicussed in the cells of mucosa and penetratesintonearby
flowchart 7.2. blood vessels, thereby damaging the
epithelium and causes pharyngitis.
Source of infection – Airborne droplets
Route of entry – Nasopharynx
Site of infection – Meninges Cocci spread from the nasopharynx to
meninge by travelling along the perineural
Incubation period – 3 days sheath of the olfactory nerve, through the
cribriform plate to the subarachnoid space
7.5.4 Laboratory Diagnosis or through blood stream.
Specimens: CSF, blood, nasopharyngeal
scrapings from petechiae lesions are
Pathogen entering the blood vessels rapidly
the specimens collected from pyogenic
permeates the meninges and produce
meningitis patients. meningitis (most complication in children).
Direct Microscopy: CSF is centrifuged, It is marked by the following clinical
and smear is prepared from the deposit manifestations, which are fever, sore
for gram staining. Meningococci are throat, headache, stiff neck, and vomiting
convulsions (fits).
Gram negative diplococci, present mainly
inside polymorphs and many pus cells are
also seen.
The pathogen sheds endotoxin into the
Culture: The centrifuged deposit of CSF generalized circulation, which damages the
is inoculated on chocolate agar. The blood vessels and leads to vascular collapse,
plate is incubated at 36°C under 5–10% hemorrhage, petechiae lesion (a small red or
CO2 for 18–24 hours. After incubation purple spot caused by bleeding into the skin).
period, meningococcusis identified by
gram staining, colony morphology and
biochemical reactions. N. meningitides is A few develop sudden meningococcemia
(water house – friderichsen syndrome)
catalase and oxidase positive (Figure 7.8).
characterized by shock, disseminated
intravascular coagulation and multisystem
failure.
It has a violet onset, with fever, chills,

shock and coma. Generalized intravascular
clotting, cardiac failure, damage to adrenal
glands and death occurs within a few hours.
Flowchart 7.2: Pathogenesis of Neisseria

Figure 7.8: Oxidase test Meningitides
125

7.5.5 Treatment and Prophylaxis bacterium Non – motile, non – sporing
Penicillin – G is the drug of choice. In and non – capsulated (Figure 7.9a & b).
penicillin allergic cases, chloramphenicol •• The bacilli are arranged in a
is recommended. characteristic fashion in angular
•• Monovalent and polyvalent vaccines fashion resembling the letters V or L.
(capsular polysaccharide) induce good This has been called Chinese letter or
immunity in older children and adults. cuneiform arrangement (Figure 7.10).
•• Conjugate vaccines are used for •• They are club shaped due to the
children below the age of 2 years. presence of metachromatic granules at
one or both ends. These granules are
7.6 Corynebacterium Diphtheriae composed of polymetaphosphates and
represent energy storage depots.
Several species of the genus
Corynebacterium are normal flora of skin,
upper respiratory tract (URT), urogenital
and intestinal tract. The most important
member of the genus is C. diphtheriae the
causative agent of diphtheria, a localized
inflammation of the throat with greyish
white pseudomembrane and a generalized
toxemia due to the secretion and
dissemination of a highly potent toxin.
The name Corynebacterium diphtheria
is derived from Greek word ‘Coryne’ –
“Club shaped swellings” or“Knotted rod”
‘Diphthera’ – Leather.
7.6.1 Morphology
•• They are Gram positive slender rods, Figure 7.10: Gram staining of
pleomorphic club shape or coryneform Corynebacterium tetani
Figure 7.9: (a) Gram staining of Corynebacterium diphtheriae (b) Albert’s staining
showing metachromatic granules
126

7.6.2 Cultural Characteristics a. Fragment A (24,000 Dalton) – It has all
•• They are aerobic and facultative enzymatic activity.
anaerobe. Optimum temperature is b.
Fragment B (38,000 Dalton) – It is
37°C and pH 7.2. responsible for binding the toxin to the
•• They grow on the following media target cells.
and show the characteristic colony Mode of Action
morphology (Table 7.5).
The toxin acts by inhibiting protein
synthesis, specifically fragment A inhibits
Table 7.5: Colony Morphology of
Corynebacterium diphtheriae on cultural polypeptide chain elongation in the
media presence of NAD by inactivating the
elongation factor (EF – 2) the toxin has
Media Colony Morphology special affinity for myocardium, adrenal
Loeffler’s They grow on this gland and nerve endings.
Serum slope medium very rapidly.
Colonies appear after 7.6.3 Pathogenicity
6–8 hours of incubation.
Source of infection – Airborne droplets
The colonies are small,
Route of entry – Upper respiratory tract
circular white or creamy
and glistening Incubation period – 3–4 days
Tellurite Grey or black colonies. Site of infection – Faucial (nasal, otitis,
Blood Agar Based on colony conjunctival, laryngeal, genital) diphtheria
morphology on tellurite is most commonly seen in children of
medium, three main 2–10years.
biotypes – Gravis, Faucial diphtheria is the most
Intermedius and Mitis. common type. The infection is confined
Toxin to humans only. The toxin has both local
(flowchart 7.3) as well as systemic effects.
•• The pathogenicity is due to production
of a very powerful exotoxin by virulent Systemic effects
strains of diphtheria bacilli. The toxin diffuses into the blood stream
•• The toxigenicity of diphtheria bacillus and causes toxemia. It has got affinity for
depends on the presence of a tox+ cardiac muscle, adrenal and nerve endings.
gene which can be transferred from It acts on the cells of these tissues.
one bacterium to another by lysogenic
bacteriophages, of which beta phage is 7.6.4 Clinical Manifestations
the most important. 1. Laryngeal obstruction, asphyxia (it is a
Properties condition of severe deficient supply of
oxygen, causing suffocation).
The diphtheria toxin is a heat – labile
protein and has a molecular weight of about 2. Diphtheritic myocarditis (inflammation
62,000 Dalton. It consists of two fragments. of heart muscle), polyneuropathy
127

The bacilli enters through URT and it Culture: The swabis inoculated on
remains confined to the site of entry where Loeffler’s serum slope, after overnight
they multiply and produces diphtheria toxin incubation at 37°C, the plates were
observed for characteristic colonies,
which are identified by gram staining.
The toxin causes local necrotic changes along 7.6.6 Prophylaxis

with superficial inflammatory reaction.
The necrosed epithelium together with Diphtheria can be controlled by
fibrinous exudate, leucocytes, erthrocytes immunization. Three methods of
and bacteria consitute the greyish immunization are available (Table 7.6).
psedudomembrane, which is a characteristic
feature of diphtheritic infection. 7.6.7 Treatment
The specific treatment for diphtheria
consists of administration of antitoxin
The membrane extends from oropharynx with dose of 20,000–100, 000 units of ADS
to larynx and into trachae. The intramuscularly and antibiotic therapy
mechanical obstruction of the trachae by using penicillin.
psedudomembrane can cause suffocation
and death. 7.7 Clostridium Tetani
Flowchart 7.3: Localized effect of The genus Clostridium consists of
diphtheria toxin anaerobic, spore forming Gram positive
bacilli. The spores are wider than the
(damage of multiple peripheral nerves), bacterial bodies, giving the bacillus
paralysis of palatine (the top part of a swollen appearance resembling a
the inside of the mouth) and ciliary spindle. The name Clostridium is derived
muscles. from the word ‘kluster’ (a spindle).
3. Degenerative changes in adrenal Most species are saprophytes found in
glands, kidney and liver may occur. soil, water and decomposing plant and
animal matter. Some of the pathogens
7.6.5 Laboratory Diagnosis
are normal flora of intestinal tract of
Specimen: Two swabs from the lesions human and animals.
are collected. One swab is used for smear The genus Clostridium includes
preparationand other swab for inoculation bacteria that causes 3 major diseases of
on culturemedia. human – Tetanus, gas gangrene and food
Direct microscopy: Smears are stained poisoning. Clostridium pathogenicity is
with both Gram stain and Albert stain. mainly due to production of a powerful
a. Gram Staining – Gram positive slender exotoxin.
rods were observed. Clostridium of medical importance
b.
Albert staining – Club shaped with may be classified based on diseases they
metachromatic granules were observed. produce, which is given the Table (7.7).
128

Table 7.6: Immunization for diphtheria
Immunization
Active Passive Combined
DPT (Triplevaccine) 500–1000 units of Antidiph- It consists of administration
3 doses of 0.5ml each are theritic serum, (ADS) is ad- first dose of adsorbed toxoid
given intramuscular route ministered subcutaneously. on one arm, while ADS is
at an interval of 4–6 weeks given on another arm.
after birth. Booster dose of
DPT are given at 18 months
and at the age of 5.
Table 7.7: Clostridium sp. causing Table 7.8: Colony characteristics of

pathogenic diseases. Clostridium tetani
Organisms Diseases Media Colony Morphology
Clostridium tetani Tetanus Blood agar They produce α – hemolysis
Clostridium perfringens Gas gangrene which subsequently develop
Clostridium botulinum Food poisoning into β – hemolysis (due
to tetanolysis) it produces
7.7.1 Morphology swarming growth.
They are Gram positive spore forming Cooked Growth occurs as turbidity
rods. The spores are spherical and meat broth with gas formation. The
terminal in position giving a drumstick (CMB) meat is not digested but
appearance. They are motile and non – becomes black on prolonged
capsulated. incubation
7.7.2 Culture Characteristics

Tetanolysis
•• They are obligate anaerobes, optimum •• Heat labile and oxygen labile toxin.
temperature is 37°C and pH is 7.4.
•• It lysis erythrocytes and also acts as
•• It grows on ordinary media, but growth neurotoxin.
is enhanced by addition of blood
and serum. Clostridia tetani grows Tetanospasmin
on the following media and show the •• It is heat labile and oxygen stable
characteristic colony morphology powerful neurotoxin.
(Table 7.8). •• It is protein in nature. consisting of
a large polypeptide chain (93,000
7.7.3 Toxins
Dalton) and a smaller polypeptide
Clostridium tetani produces two distinct chain (52,000 Dalton) joined by a
toxins namely, disulphide bond.
a. Tetanolysis (haemolysin) •• Mode of Action: Tetanospasminis a
b. Tetanospasmin (neurotoxin) neurotoxin, which blocks the release of
129

Myth: Rust causes
tetanus if introduced
into a wound – for
example, by stepping
on a rusty nail.
Fact: Rusty nails are more likely to be
Figure 7.11: Tetanus – opisthotonos contaminated with tetanus endospores
because they have been exposed to soil
inhibitory neurotransmitters (glycine and dust longer than new, rust free nails.
and gamma – amino butyric acid) across Any object that causes a wound, rusty
the synaptic junction. The toxin acts or not, can inoculate the tissue with the
presynaptically, the abolition of spinal bacterial endospores of C. tetani. Rust
inhibition causes uncontrolled spread itself neither causes tetanus nor makes
of impulses in CNS (Central Nerves it worse.
System). This results in muscle rigidity
and spasms (due to the simultaneous Microscopy: Gram staining shows Gram
contraction of agonists and antagonists, positive bacilli with drumstick appearance.
in the absence of reciprocal inhibition Culture: The clinical specimen is
Figure 7.11). inoculated on blood agar and incubated
at 37°C for 24–48 hours under anaerobic
7.7.4 Pathogenesis conditions. The colonies are confirmed
Clostridium tetani is the causative by gram staining, where it shows gram
organism of tetanus or lock jaw disease. positive bacilli with drumstick appearance.
pathogenesis of Clostridium tetani was
discussed in detail in flowchart 7.4. 7.7.7 Treatment
Source of infection – Soil, dust, faeces. Tetanus patients are treated in special
isolated units, to protect them from noise
Route of entry – Through wound
and light which may provoke convulsions.
Incubation period – 6–12 days The spasm can be controlled by diazepam
7.7.5 Clinical Feature

Clostridium difficile is
It includes, pain and tingling at the the causative agent of
site of wound, Lock jaw ortrismus (It antibiotic associated
is reduced opening of the jaws), Risus colitis. It is an acute
sardonicus (mouth kept slightly open), colitis with or without
Dysphasia (impairment of the ability to pseudo membrane formation. It is an
speak or to understand language) and important complication in patients
acute asphyxia. on oral antibiotic therapy. Many
antibiotics have been incriminated
7.7.6 Laboratory Diagnosis but lincomycin and clindamycin
are particularly prone to cause
Specimens: Wound swab, exudates or pseudomembranous colitis.
tissue from wound.
130

Table 7.9: Immunization for tetanus
Active immunization Passive immunization Combined prophylaxis
a. Tetanus toxoid (TT) Antitetanus serum (ATS) Tetanus toxoid in one
b. DPT Human Antitetanus arm and ATS or HTIG in
immunoglobulins (HIIG) another arm.
Tetanus develops by the contamination of (0.1–0.2 mg/kg) injection. Antibiotic

wound with Clostridum tetani spores. The therapy with penicillin or metronidazole
spores germinate in reduced oxygen tension should be done for a week or more.
(anaerobic environment).
7.7.8 Prophylaxis
It is done by the following methods, which
The vegetative cell grows and produces a are as follows.
potent neurotoxin called tetanospasmin. a. Surgical prophylaxis: It aims at
The toxin is absorbed from the site of its removal of foreign body, blood clots
production and enters into the blood stream. and damaged tissue in order to prevent
It ascends to central nervous systems (CNS) anaerobic conditions favorable for the
through motor nerves. germination of spores.
b. Immunoprophylaxis: Tetanus is a
preventable disease. Immune prophylaxis
The toxin blocks synaptic inhibition in the is of 3 types, which is given in the
spinal cord. It acts presynaptically. (Table 7.9).
Infobits
The toxin affects most of the voluntuary Clostridial Toxins As Therapeutic

muscles in the body, causing muscle rigidity Agents
and spasma due to uncontrolled contractions.
Botulinum toxin is the most poisonous
substance known, is being used for the
treatment of specific neuromuscular
The first symptoms appears in head and
disorders characterized by involuntary
neck because of shorter length of cranial muscle contraction. Since approval of
nerves. Masseter muscles are first affected Botulinum toxin (botox) by the FDA
causing ‘Lock Jaw’. in 1989 for 3 disorders – Strabismus
(crossing of the eyes), blepharospasm
(spasmodic contraction of eye
muscles) and hemifacial spasm
In severe cases progressive spasm of the back (contraction of one side of the face).
or extensor muscles produces opisthotonos In 2000, dermatologists and
(extreme arching of the back) usually death plastic surgeons began using Botox to
occurs due to respiratory paralysis. eradicate wrinkles caused by repeated
muscle contractions as we laugh,
Flowchart 7.4: Pathogenesis of smile or frown.
Clostridium tetani
131

7.8 Shigella Dysenteriae (Dysentery
Bacillus)
The genus Shigellaare exclusively
parasites of human intestine and other
primates. Shigella dysenteriae is the
causative agent of bacillary dysentery or
shigellosis in humans. It is a diarrheal
illness which is characterized by frequent
passage of bloodstained mucopurulent
stools. The four important species of the Figure 7.12: Gram staining of Shigella
genus Shigella are: Shigella dysenteriae,
Shigella flexneri, Shigella sonnei and
Shigella boydii.
7.8.1 Morphology
Shigella are short, Gram negative rods
(0.5µm× 1–3 µm in size). They are non –
motile, non – sporing and non – capsulated
(Figure 7.12).

•• They are aerobes and facultative
anaerobes. Optimum temperature is
37°C and optimum pH – 7.4.
•• They can be grown on the following Shigella on SS agar
media and show the characteristic
colony morphology (Table 7.10 & 7.8.3 Toxins
Figure 7.13). Shigella dysenteriae produces toxins,
which is of 3 types, namely, endotoxin,
Table 7.10: Colony morphology of exotoxin and verocytotoxin. The mode of
Shigella action of these toxins is illustrated in the
Colony Table 7.11.
Media
Morphology
7.8.4 Pathogenesis
Nutrient Agar Colonies are circu-
lar, convex smooth The pathogenic mechanism of Shigella
and translucent dysenteriaeis discussed below in
flowchart 7.5.
MacConkey Agar Colourless colonies
Source of Infection – Patient or carriers
SS – Agar Colourless colonies
Route of entry – faecal – oral route
132

Table 7.11: Various toxins of Sh. dysenteriae causes bacillary dysentery.
Shigelladysenteriae
The pathogen enters into the host by the
ingestion of contaminated food.
Toxins Mode of Action
Endotoxin It is released after autolysis,
it has irritating effect on The bacilli reaches large intestines and
intestinal wall which causes adheres to the epithelial cells of villi. It
diarrhea and subsequently multiples inside the cell and penetrates
intestinal ulcers. into lamina propria.
Exotoxin It is a powerful toxin and
acts as Enterotoxin as well
as neurotoxin. As the pathogen multiples it produces
As Enterotoxin – It induces toxins, it stimulates an inflammatory
reaction and causes extensive tissue
fluid accumulation
destruction. It leads to necrosis of
As Neurotoxin – It surface epithelial cells.
damages the endothelial
cells of small blood vessels
of CNS which results in The necrotic epithelia, become soft and
polyneuritis and coma friable and are sloughed off leaving
Vero It acts on Vero cells behind transverse superficial ulcers.
cytotoxin
Abdominal cramps and pain are caused

Site of infection – Large intestine by the disruption of the muscular
Incubation Period – Less than 48 hours function of the intestine
(1–7 days)
Mode of transmission – Food, finger,
The degeneration of intestinal villi and
faeces and flies
local erosion causes bleeding, heavy
mucous secretion resulting in bacillary
7.8.5 Clinical Manifestations
dysentery
•• Frequent passage of loose, scanty
faeces containing blood and mucus. Flowchart 7.5: Pathogenesis of Shigella
dysenteriae
•• Abdominal cramps and tenesmus
(straining to defecate). 7.8.6 Laboratory Diagnosis
•• Fever and vomiting. Specimens: Fresh stool is collected.
•• Hemolytic uremic syndrome (It is a Direct Microscopy: Saline and Lugol’s
condition caused by the abnormal iodine preparation of faeces show large
destruction of red blood cells). number of pus cells, and erythrocytes.
133

Culture: For inoculation, it is best to use
mucus flakes (if present in the specimen)
on MacConkey agar and SS agar. After
overnight incubation at 37°C, the plates
are observed for characteristic colonies,
which is confirmed by Grams staining
and biochemical reactions.
7.8.7 Treatment and Prevention

•• Uncomplicated shigellosis is a self –
limiting condition that usually recovers
spontaneously.
•• In acute cases, oral rehydration therapy
(ORT) is done. Figure 7.14: Gram staining of Salmonella
•• In all severe cases, the choice of typhi
antibiotic should be based on the 7.9.2 Cultural Characteristics
sensitivity of prevailing strain.
They are aerobic and facultative anaerobe,
•• Many strains are sensitive to Nalidixic
optimum temperature - 37°C and pH is
acid and Norfloxacin.
7–7.5.
•• Improving personal and environmental
They grow on the following media and
sanitation.
show the following characteristic colony
•• The detection and treatment of patients morphology (Table 7.12).
and carriers.
Table 7.12: Colony morphology of
Salmonella typhi
7.9 Salmonella Typhi (Eberthella
Typhi) Media Colony Morphology
The genus Salmonella consists of bacilli Nutrient Colonies are large, circular,
that parasites the intestines of vertebrates Agar smooth, translucent
and human beings. It causes Enteric fever, MacConkey Colourless colonies (non
which includes Typhoid and Paratyphoid Agar – lactose fermenters)
fever. The most important species of the SS – Agar Colourless colonies with
genus is Salmonella typhi which causes black centered.
typhoid fever.
7.9.3 Pathogenicity
7.9.1 Morphology Salmonella typhi causes typhoid fever
Salmonellae are Gram – negative rods (1– and its pathogenesis is discussed in
3µm× 0.5 µm in size). They are motile with flowchart 7.6.
peritrichous flagella, non – capsulated and Source of infection – food, feces, fingers,
non – sporulated (Figure 7.14). flies
134

Route of entry – faecal oralroute 7.9.4 Clinical Manifestations
(ingestion) •• The illness is usually gradual,
Incubation period – 7–14 days with headache, malaise (feeling of
discomfort), an2orexia (loss of appetite),
The infection is acquired by ingestion of coated tongue, abdominal discomfort
contaminated food and water with either constipation or diarrhea.
•• Hepatosplenomegaly (enlargement of
liver and spleen), step ladder pyrexia
The bacilli reaches the small intestine and (continuous fever) and rose – spots
attach to the epithelial cells of intestinal villi
(during 2nd or 3rd week).
and penetrate to the lamina propria and
submucosa
Specimens: Blood, stool and urine are the
These bacilli are phagocytosed by neutophils clinical samples collected from typhoid
and macrophages. They resist intracellular patients. The selection of relevant
killing and multiply within these cells. specimen depends upon duration of
illness, which is very important for
diagnosis (Table 7.13 & Figure 7.15).
The pathogen enters the mesenteric lymph Table 7.13: Specimen collection for
nodes, multiply there and enters blood typhoid.
stream through the thoracic duct
Duration Specimen %
of disease examination Positivity
1 Week
st
Blood culture 90
A transient bacteraemia follows and internal
2 Week
nd
Blood culture 75
organs like liver, gall bladder, spleen, bone
marrow, lungs, lymph nodes and kidneys Faeces culture 50
are infected Widal test Low titer
3 Week
rd
Widal test 80–100
Blood culture 60
The bacilli multiply abundantly in the Faeces culture 80
gall bladder (bile juice) and are dischared
continuously into the intestine involving
peyer’s patches and ileum.
It becomes inflamed, necrosed and slough

off, leaving behind typhoid ulcers. These
ulcers may lead to two major complication,
which are Intestinal perforation and
haemorrhage.
Flowchart 7.6: Pathogenesis of Figure 7.15: Colony morphology of

Salmonella typhi Salmonella typhi on SS agar
135

The bacteriological diagnosis of enteric the end of 1st week and rise sharply during
fever consists of the following methods, the 3rd week of enteric fever.
which are:
•• Isolation of the bacilli 7.9.6 Prophylaxis
•• Demonstration of antibodies Various types of vaccine and their doses

are given in Table 7.15.
Isolation of the bacilli
Table 7.15: Various types of vaccine and
The typhoid bacilli are isolatedfrom the their doses.
following clinical specimens which are
tabulated (Table 7.14). Vaccine Doses
Demonstration of Antibodies: Slide – TAB – 2 doses of 0.5 ml at an interval
agglutination: The isolate is identified Vaccine of 4–6 weeks
by slide agglutination with ‘O’ and ‘H’ Typhoral 3 doses on alternate days. It
antisera. gives 65–96% protection for
Widal Test: It is an agglutination test for 3–5 years and is safe
detection of agglutinins ‘H’ and ‘O’ in
typhim – A single dose of 25µg
patients with enteric fever. Salmonella
Vi
antibodies start appearing in the serum at
Table 7.14: Isolation method of typhoid bacilli from various clinical specimens.
Specimen culture Isolation methods
Blood culture 5–10 ml of blood is collected and inoculated into
blood culture bottle containing taurocholate broth
or bile broth. After overnight incubation at 37°C the
taurocholate broth is subculture on MacConkey agar.
Pale colonies (NLF) appear on MacConkey which is
used for motility and biochemical reactions.
Clot Culture (An alternative 5 ml of blood is collected into a sterile test tube and
method to blood culture) allowed to clot. The clot is broken up with a sterile glass
rod and added to bile broth containing streptokinase,
which digests the clot and there by the bacilli are
released from the clot. Then it is subcultured on
MacConkey agar.
Faeces culture Faeces sample are inoculated directly on MacConkey’s
agar, DCA or SS agar. The plates are incubated at 37°C
for 24 hours, then characteristic colonies are observed
which is confirmed by gram staining.
Urine culture Urine samples are centrifuged, and the deposit is
inoculated into enrichment media and then on selective
media.
136

7.9.7 Treatment and Control Measures is very actively motile with a single
•• Antibacterial therapy has been very polar flagellum and the characteristic
effective in the treatment of patients. movement is called as darting motility.
In stained smears of mucus flakes from
•• Ampicillin, amoxicillin and
acute cholera patients, the Vibriois seen
cotrimoxazole are useful in the
arranged in parallel rows. This was
treatment of typhoid fever.
described by Robert Koch as “fish in
•• At present, ciprofloxacin is the drug of stream” appearance.
choice.
•• Typhoid fever can be effectively 7.10.2 Culture Characteristics
controlled by sanitary measures for Vibrio cholera is strongly aerobic. It grows
disposal of sewage, clean water supply best in alkaline media with the optimum
and supervision of food processing temperature 37°C and pH 8.2. It is non-
and handling. halophilic, therefore, cannot grow in
media with a concentration of sodium
HOTS chloride more than 7% (Figure 7.17).
Some of the media in which Vibrio
Why is proper hand washing cholerae are cultivated are tabulated below
considered the most important in Table 7.16.
element in controlling communicable Table 7.16: Colony morphology of
infections? Vibrio cholerae on various media
Media Colony morphology

7.10 Vibrio Cholerae Nutrient agar The colonies are moist,
Vibrio is one of the curved rod bacteria, translucent round disks
prominent in the Medical Bacteriology. (1–2mm in diameter)
They are present in marine environment with a bluish tinge in
and surface waters worldwide. Vibrio is transmitted light.
a member of the family Vibrionaceae.
MacConkey The colonies are
The most important member of this genus
agar colorless at first but
is Vibrio cholerae, the causative agent of
become reddish on
cholera. The term Vibrio is derived from
prolonged incubation
Vibrare (Latin word) which means “to
due to late fermentation
shake or vibrate” and the word Cholera is
of lactose.
derived from Chole (Greek word) which
means, “to bile” (Figure 7.16). Thiosulphate It is used as a selective
citrate bile medium for isolation
7.10.1 Morphology sucrose agar of Vibrios. It produces
(pH 8.6) large yellow convex
Vibrio cholerae is gram negative, curved
colonies due to sucrose
or comma shaped, (1.5um x 0.2–0.4um
fermentation.
in size) non – capsulated. The organism
137

Figure 7.16: Gram staining of Vibrio Figure 7.17: Colony morphology of
cholerae Vibrio cholerae on TCBS
Mode of Action
• • The B (binding) units of enterotoxin
get attached to the GM 1 (Ganglioside
membrane receptors I) on the surface
of jejunal epithelial cells (target
cells).
•• The A (active) subunits then enters
the target cell and dissociates into
2 fragments, A1 & A2. The A2 fragment
links biologically active A1 fragment to
the B – subunit.
Figure 7.18: Mechanism of action of
•• The A1 fragment causes prolonged
Cholera toxin
activation of cellular adenylate cyclase
7.10.3 Enterotoxin which in turn accumulates cAMP in the
target cell. This leads to outpouring of
Vibrios multiplying on the intestinal large quantities of water and electrolytes
epithelium produce an enterotoxin into small intestinal lumen. Thus,
called Cholera toxin. It is also known as resulting in profuse watery diarrhea.
Choleragen (or CT). This toxin molecule
is approximately 84,000 Dalton and Natural infection
consists of two major subunits namely A of Vibrio cholerae
and B There is only one subunit in A (1A) occurs only in human
whereas there are five subunits in B (5B) beings
(Figure 7.18).
138

7.10.4 Pathogenesis
The loss of water
The pathogenic mechanism of Vibrio during cholera is
cholerae is discussed below in flowchart 7.7. about 20–30 litres per
Source of Infection – contaminated water day.
or food
Route of entry – fecal – oral route
HOTS
Site of infection – small intestine
Incubation period – few hours to 5 days
(usually 2–3 days) Why is cholera the most severe form of
gastroenteritis?
Vibrio cholerae causes cholera, which is an
acute diarrheal disease.
7.10.5 Clinical Feature
Dehydration, anuria (absence of urine
In humans, Vibrio enter orally through
contaminated water or food. The ingested excretion), muscle cramps, hypokalemia
pathogens pass through the acid barrier of (low blood potassium) & metabolic
the stomach & multiply in the small intestine. acidosis (low serum concentration of
bicarbonates).
In the small intestine, the Vibrio penetrate 7.10.6 Laboratory Diagnosis
the mucous barrier & adhere to the microvilli
of the epithelial cells & multiply there. Specimen: Stool
Direct microscopy: It is not a reliable
method for rapid diagnosis, the
The Vibriois strictly epipathogen& do not
penetrate deep into the guts and there is no characteristic darting motility of the
bacteremia. The virulence of Vibrio cholerae vibrio can be observed under dark – field
is due to cholera toxin (the mechanism is microscope.
described earlier in the section 7.10.3 – Culture: Stool sample is directly inoculated
enterotoxinmode of action)
on MacConkey agar and TCBS agar.
The plates are examined after overnight
The toxin also inhibits intestinal absorption incubation at 37°C for typical colonies
of sodium and chloride. of Vibrio cholera, and the colonies are
identified by gram staining and oxidasetest.
The clinical manifestations & complications
7.10.7 Prophylaxis
are due to massive water and electrolyte
depletion. 1. General Measures
•• Purification of water supplies
The voided fluids areodourless and contains •• Improvement of environment
flecks of mucus, hence it is known as Rice sanitation
watery stool
•• Infected patients should be
Flowchart 7.7: Pathogenesis of Vibrio isolated, and their excreta must be
cholerae disinfected
139

2. Vaccines: Two types of oral vaccines The name Mycobacterium tuberculosis
have been tried recently: is derived form,
•• Killed oral whole cell vaccines •• Mycobacterium (Greek) – Fungus
•• Live oral vaccines like bacterium
•• Tuberculosis (Latin) – Swelling or
7.10.8 Treatment Knob
1. Oral Rehydrationtherapy: The severe
7.11.1 Morphology
dehydration & salt depletion can be
treated by oral rehydration therapy (as They are acid fast bacilli, slightly curved
recommended by WHO). rods, it may occur singly or in small clumps.
2. Antibiotics: It is of secondary They are non–motile, non–sporing, and
importance, oral tetracycline was non-capsulated.
recommended for reducing the period
of Vibrio excretion.
They are obligate aerobe, optimum
An ideal cholera temperature is 37°C and optimum pH
vaccine is yet to be is 6.4–7.0. The pathogen grows on an
found. enriched culture media – Lowenstein
Jensen medium. The colonies appear in
about 2–3 weeks. The colonies are dry,
rough, raised, irregular colonies with a
7.11 Mycobacterium Tuberculosis wrinkled surface. Initially creamy white
(Tubercle Bacillus) and becoming yellowish later (Figure 7.19).
The genus Mycobacterium is distinguished
7.11.3 Pathogenesis
by its thick, complex, lipidrich waxy cell
walls. This high lipid content (Mycolic Human tuberculosis is divisible into two
acids) imparts the characteristic of acid form, they are Primary TB and Secondary
fastness or resistance to decolorizationby
a strong acid after staining with carbol
fuchsin. Many of the Mycobacterial
species are saprophytes but several
species are highly significant human
pathogens. Mycobacterium tuberculosis is
the causative agent of tuberculosis (TB)
It is a killer disease and ranks as one of
the most serious infection diseases of the
developing countries. TB is primarily
a disease of the lungs but may spread to Figure 7.19: Acid fast staining of
other sites of the body. Mycobacterium tuberculosis
140

TB. The pathogenesis of Primary The lung lesions is frequently found in the
Tuberculosis is described in flowchart 7.8. lower lobe and called as ghon focus. The
Source of infection – Airborne droplets ghon focus together with enlarged hilar
lymph nodes is called primary complex.
Route of entry – Respiratory tract
Incubation period – 3–6 weeks.
In some persons, the lesions become dormant

Tubercle bacilli enter the host commonly and produce dense scar tissue which may
by inhalation. When bacilli are inhaled, the become calcified. Some bacilli remain in
bacilli enters into the alveolar macrophage, a dormant form as persisters which, when
where they can grow and multiply. reactivated, cause post primary TB.
Non-resident macrophage are also attracted In a minority of cases, the ghon focus
to the site of infection and these macrophages ruptures into a blood vessel. Then the
are engulfs the fubercle bacilli. The bacilli bacilli spread throughout the body with the
were carried through the lymphatic to the formation of numerous granulomas and
local hilar lymph node. known as miliary TB.
Flowchart 7.8: The pathogenesis of

Primary Tuberculosis
In the lymph nodes, cell mediated immunity
(CMI) is stimulated. The CMI response helps Secondary TB – (Post primary TB) It
to prevent the further spread of pathogen. is caused by reactivation of the primary
lesion or by exogenous reinfection.
Granulomas of secondary TB most often
Within 10 days of infection, T-lymphocytes occur in the apex of the lungs. The necrotic
produces lymphokines which activate element of the reaction causes tissue
macrophages and leads to form granuloma, destruction and the formation of large
around the foci of infection. area of caseation termed tuberculomas.
The presence of caseous necrosis and
cavities are two important clinical
The activated marophages are termed manifestations of secondary TB. The
epithelioid cells, which fuse to form cavities may rupture into blood vessels,
multinucleate giant cells. spreading the bacilli throughout the body
and break into airways, releasing the
pathogen in aerosols and sputum - called
The granuloma contains necrotic tissue as open tuberculosis (Figure 7.20).
and dead macrophage which is referred as
caseation. Granuloma formation is usually 7.11.4 Clinical Symptoms
sufficient to limit the primary infection.
It includes, cough that lasts for more than
2–3 weeks, weight loss, fever, night sweat
and loss of appetite.
141

bacilli appear as bright red bacilli against
a blue background.
Culture: The specimen is inoculated onto
LJ – medium and incubated at 37°C for
2 weeks the tubercle bacilli usually grow
in 2–8 weeks. The bacterial growth is
confirmed by Ziehl – Neelsonstaining.
1. Tuberculin Skin test

Mantoux test: This method has been
used routinely. In this test 0.1 ml of PPD
(Purified protein derivative) containing
5 TU (Tuberculin unit) is injected
intradermally on the flexor aspect of
forearm (Figure 7.21) The site is examined
after 48–72 hours and induration are
Mycobacterium tuberculosison LJ medium
measured (diameter in mm).
Positive test: Indurations of diameter d10
HOTS
mm or more is considered positive.
M. tuberculosis the world’s most deadly Negative test: Indurations of 5 mm or less

pathogen why? is negative.
2. Gene Xpert MTB

7.11.5 Laboratory Diagnosis It is an automated diagnosis test it detects
Specimen: In case of pulmonary DNA sequences specific for M. tuberculosis
tuberculosis the most usual specimen is and rifampicin resistance by PCR. Results
sputum. can be obtained within 2 hours.
Direct Microscopy: Smear is made from
7.11.6 Treatment
the sputum specimen and stained by Ziehl
– Neelson technique. It is examined under The antitubercular drugs include two
oil immersion objective lens. The acid fast types of agents which are;
Figure 7.21: Mantoux test
142

Bactericidal agents – Rifampicin
(R), Isoniazid (H), Pyrazinamide (z), 24 March is
Streptomycin. celebrated as World
Tuberculosis day
Bacteriostatic agents – Ethambutol (E).
Figure world TB day
The regimen for treating TB consists of an
intensive phase of 2 months of isoniazid,
rifampin, pyrazinamide and ethambutol, 7.12 Treponema Pallidum
followed by a continuation phase of
Treponema pallidum is included in
4 months of isonizid and Rifampin.
the family Spirochaetaceae. They are
slender spirochaetes with fine spirals
7.11.7 Prophylaxis and Control Measures
having pointed ends. Some of them are
The BCG (Bacille – Calmette – Guerin) pathogenic for humans, while others are
administered by intradermal injection of normal flora in mouth and genitalia.
the live attenuated vaccine. The immunity These pathogens are strict parasites
may last for about 10 years. and the diseases caused by Treponema
The prevention of TB can be done by are called Treponematoses. Treponema
the following general measures such as pallidum is the causative agent of syphilis.
1. Adequate nutrition. The name Treponema pallidum is derived
from Greek words, which means, Trepos
2. Practicing good hygiene (washing
– Turn Nema –Thread and Pallidum –
hands)
Pale staining.
3. Health education.
4. Cover the mouth with a tissue when 7.12.1 Morphology
you cough or sneeze. It is thin, delicate spirochete with tapering
ends, about 10µm long and 0.1–0.2 µm
Infobits wide. It has about ten regular spirals,
which are sharp and angular, at regular
Drug Resistance Tuberculosis intervals of about 1µm. They are actively
MDR-TB: Multidrug resistance motile (endoflagella), exhibiting rotation
tuberculosis refers to resistance to around the long axis, backward and
rifampian and Isoniazid. MDR-TB is forward movements and flexion of the
a global problem especially in HIV- whole body. It cannot be seen under light
patients. microscope and does not take ordinary
XDR-TB: Exensively drug resistance bacterial stains. It can be seen under
tuberculosis refer M.tuberculosis the dark ground (Figure 7.22) or phase
strains which are resistant to any contrast microscope. It can be stained by
fluoroquinolone and at lecest one silver impregnation method.
of 3 injectable second line drugs
(Kanamycin, Capreomycin and 7.12.2 Culture
Amikarin), in addition to Isoniazid
•• Pathogenic Treponema cannot be
and rifampicin.
grown in artificial culture media.
143

•• The chancre is covered by thick
exudates, very rich in spirochetes.
•• The regional lymph nodes are swollen,
discrete, rubbery and non – tender.
•• Even before the chancre appears, the
spirochetes spread from the site of
entry into the lymph and bloodstream,
so the patient may be infectious during
the late incubation period.
•• The chancre invariably heals within
10–40 days, even without treatment,
leaving a thin scar.
Figure 7.22: Dark field microscopy of
Secondary syphilis
Treponema pallidium
•• Secondary syphilis sets in 1–3
months after the primary lesion heals.
•• Treponema pallidum strains (Nichol’s During this interval, the patient is
strain) are maintained in rabbit testes. asymptomatic.
•• The secondary lesions are due to
7.12.3 Pathogenesis
widespread multiplication of the
Source of infection – Human beings spirochetes and dissemination through
(patients) the blood.
Mode of transmission – Sexual contact •• Secondary syphilis is characterized
Site of entry – Through minute abrasions/ by appearance of papular skin rashes,
cuts on skin or mucosa mucous patches in the oropharynx and
Incubation period – 10–90 days condylomata (a raised growth on the
skin resembling a wart).
•• Treponema pallidum causes venereal
•• The lesions are abundant in spirochetes
syphilis, which is acquired by sexual
and the patient is most infectious
contact. The pathogen enters the
during the secondary stage.
human body through cut on the skin
or mucosa of genital areas. •• There may be retinitis (inflammation
of the retina of the eye), meningitis,
•• The clinical disease sets in after an
periostitis, and arthritis.
incubation period of about a month.
•• Secondary lesions usually undergo
There are 3 clinical stage of venereal
spontaneous healing, in some cases
syphilis, namely – primary, secondary
taking as long as 4 or 5 years.
and tertiary syphilis.
•• After the secondary lesions disappear,
Primary syphilis there is a period of dormant known
•• A papule appears on the genital area as latent syphilis the patient does not
that ulcerates, forming a chancre of show any clinical symptoms but with
primary syphilis called hard chancre. positive serology.
144

Tertiary syphilis b.
Treponemes in tissues: It can be
•• After several years, manifestations demonstrated by silver impregnation
of tertiary syphilis appear. These method of staining.
consist of cardiovascular lesions
Serological tests
including aneurysms (enlargement of
Non – Treponemal tests – In the standard
an artery), gummata (a small rubbery
tests for syphilis includes;
granuloma that has a necrotic centre)
and meningovascular manifestations. a.
VDRL – Venereal Diseases Research
Tertiary lesions contain few spirochetes. Laboratory test.
• • In few cases, neurosyphilis such as b.
RPR – Rapid Plasma Reagain
tabesdorsalis or general paralysis (Figure 7.23).
of the insane develops. These are VDRL or RPR tests are used for
known as late tertiary or quaternary serological screening for syphilis and
syphilis. more useful for the assessment of cure
following treatment.
Congenital syphilis
In congenital syphilis, the infection is TRUST – Toluidine red
transmitted from mother to fetus by unheated serum test,
crossing the placental barrier. modified form of RPR
test.
Non – Venereal syphilis
It may occur in doctors or nurses due
Treponemal Tests: The treponemal tests
to contact with patients lesion during
includes
examination. The primary chancre occurs
usually on the fingers. a.
TPHA – Treponema pallidum
hemagglutination assay
7.12.4 Laboratory Diagnosis b. FTA –ABS – Fluorescent treponemal
The diagnosis of syphilis includes antibody absorption test.
a. Demonstration of Treponemes These two tests are used to confirm the

diagnosis.
b. Serological tests
Specimen: Exudates are collected from
the chancre. Blood (serum) is collected
for serology.
Demonstration of Treponemes
a. Dark ground microscopy: The wet film
is prepared with exudates and examined
under dark ground microscope. Under
dark field examination Treponema
pallidum appears motile spiral
organism. Figure 7.23: Rapid Plasma Reagain test
145

7.12.5 Treatment and Preventive Measure
In early syphilis
a.
Benzathine benzyl penicillin,
24 lakhs units intramuscularly in a
single dose.
b.
Alternatively, doxycycline 100 mg
twice a day orally for 15 days.
In late syphilis
Benzathine benzyl penicillin 24 lakhs
units, intramuscularly once weekly for
3 weeks.
•• Avoiding sexual contact with an
infected individual. Figure 7.24: Dark field microscopy of
•• Use of sex barriers (condoms). Leptospira interrogans
7.13 Leptospira Interrogans
Their ends are hooked and resemble
Spirochaetes of the genus Leptospira umbrella handles.
are actively motile, delicate and possess •• They are actively motile by rotatory
numerous closely wound spirals with movements.
characteristic hooked ends. Several
•• They cannot be seen under light
Leptospires are saprophytes, while many
microscope due to its thinness, best
are potential pathogens of rodents,
observed by dark fieldmicroscopy
domestic animals and humans. The genus
(Figure 7.24), phase contrast and
Leptospira consists of two important
electron microscope.
species, which are Leptospira interrogans
and Leptospira biflexa. •• They stain poorly with aniline dyes,
it may be stained with giemsa stain or
Leptospira interrrogans is the causative
silver impregnation techniques.
agent of leptospirosis, a zoonotic disease.
The word Leptospira is derived from Latin
7.13.2 Antigenic Structure
word ‘Leptos’ = fine or thin and ‘spira’ =
Coil and interrogans = Question mark Leptospires show considerable antigenic
(The shape of this spirochete accounts for cross reaction.
its name) a. Genus – Specific somatic antigen – It
is present in all members of the genus.
7.13.1 Morphology b. Surface antigens – This antigen is used
•• They are spiral bacteria (5–20µm × to classify Leptospira into serogroups
0.1µm) with numerous closely set coils. and serotypes.
146

7.13.3 Pathogenicity •• Jaundice, Albuminuria (The presence
Source of infection: Contaminated water of protein Albumin in the urine) and
purpuric hemorrhages sometimes
Route of entry: Through cuts or abrasions
occur on skin and mucosa.
on skin or mucosa
•• Aseptic meningitis is common in
Incubation period: 6–8 days
canicola infection.
•• Leptospira interrogans causes a
zoonotic disease named Leptospirosis. 7.13.4 Laboratory Diagnosis
It is transmitted to humans by direct
The diagnosis of Leptospirosis is made by
or indirect contact with water,
the following ways
contaminated by urine of carrier
animals (rat and dog). •• Direct microscopy of blood or urine
•• Leptospira enter the body through •• Isolation of pathogen by culture
cuts or abrasions on skin or through •• Serological tests.
mucous membranes of the mouth,
nose or conjunctiva. Direct Microscopy
Blood: Leptospira can be observed in the
•• After an incubation period of 6–8 days.
blood by dark – filed microscope. Blood
There is onset of febrile (related to
examination is useful in first week as
fever) illness with Leptospira in blood
Leptospira disappear from blood after
(Septicemic phase) which lasts for
8 days.
3–7 days.
•• The organisms disappear from the Urine: Leptospira may be present in
blood and invades liver, kidney, spleen, urine in the 2nd week of the disease and
meninges producing meningeal intermittently thereafterup to 6 weeks.
irritation such as headache, vomiting. Centrifuged deposit of urine may be
observed by Dark filed microscopy.
•• The pathogen persists in the internal
organs and most abundantly in the Culture: Blood (1st week) and urine
kidney. Severe Leptospirosis (Weil’s (2nd–6 week) can be cultured in Korthof ’s
disease) is associated with Fever, medium. Media are incubated at 37°C for
conjunctivitis (inflammation of 2 days and then left at room temperature
conjunctiva), albuminuria (presence for 2 weeks. Culturesare examined every
of albumin in the urine), jaundice third day for the presence of Leptospira
and hemorrhage. It is a fatal illness under DFM.
with hepatorenal (Kidney failure with Serological tests
severe liver damage).
It is very useful method of diagnosis two
Clinical manifestations types of serological tests are used, which
•• In severe cases, vomiting, headache, are,
irregular fever and intense infection of a. Screening tests: These tests are genus –
the eyes. specific and done using reactive genus
147

specific antigen (non – pathogenic antibiotics are discussed below. S. aureus
L. biflexapatoc I strain). is a leading cause of hospital acquired
Screening test includes – CFT, infections. Cloxacillin is used against
ELISA, SEL, HAT indirect IF these beta lactamase. Producing strains
tests are capable to detect IgM and IgG Streptococcus pyogenes is intrinsically
leptospiral antibodies. a much more dangerous pathogen than
Staphylococcus aureus and has a much
b. Serotype specific tests: These tests
greater tendency to spread in the tissues.
identify the infecting serovar by
Streptococcal pyrogenic exotoxin leads
demonstrating specific antibodies.
to streptococcal toxic shock syndrome
i. Macroscopic agglutination test (TSS). A common cross – reacting antigen
ii. Microscopic agglutination test exist in some group A streptococci and
heart, therefore, antibodies produced in
7.13.5 Treatment and Preventions response to the streptococcal infection
Leptospira are sensitive to penicillin and could cross react with myocardial and
tetracycline heart valve tissue, causing cellular
destruction. N. meningitidis is the
Preventive measures include rodent
causative agent of meningococcal
control, disinfection of water.
meningitis, also known as pyogenic or
septic meningitis. Clostridium tetani
Summary is the causative organism of tetanus or
Pathogenicity refers to the ability of a lock jaw disease. The four important
pathogen to produce disease. Virulence species of the genus Shigella are: Shigella
is the ability of the pathogen to cause dysenteriae, Shigella flexneri, Shigella
disease. The various bacterial pathogens, sonnei and Shigella boydii. Several
its pathogenesis clinical symptoms, Leptospires are saprophytes, while many
laboratory diagnosis, control, prophylaxis are potential pathogens of rodents,
and treatment with appropriate domestic animals and humans.
148

Evaluation 6. Shigella dysenteriae causes
Multiple choice questions a. Bacillary dysentery
1. Which of the following toxin is b. amoebic dysentery
responsible for staphylococcal c. Traveller’s diarrhoea
scalded skin syndrome? d. Colitis
a. Epidermolytic 7. The most important specimen for
toxin isolation of Salmonella typhi in the
b. Enterotoxin first week of enteric fever is
c. Coagulase a. Blood b. Urine
d. Haemolysin c. CSF d. Faeces
2. The most important bacterial cause 8. Which of the following methods can
of sore throat is be used to demonstrate T. pallidum?
a. Staphylococcus aureaus a. Silver impregnation method
b. Streptococcus pyogenes b. Dark ground microscopy
c. Haemophilus influenzae c. Immuno fluorescence staining
d. Escherichia coli d. All of the above
3. The causative agent of waterhouse – 9. Weil’s disease is caused by which of
friderichsen syndrome is the following bacteria
a. Neisseria meningitidis a. Salmonella typhi
b. Streptococcus pyogenes b. Escherichia coli
c. Treponema pallidum c. Leptospira interrogans
d. Staphylococcus aureus d. Vibrio cholerae
4. A gram-positive bacilli possessing 10. BCG Vaccine is
metachromatic granules, showing a. Live attenuated vaccine
Chinese letters arrangement are
b. Toxoid
characteristic of
c. Killed vaccine
a. Corynebacterium diphtheriae
d. None of the above
b. Mycobacterium tuberculosis
c. Bacillus anthracis Answer the following
d. Clastridium perfringens
1. Enlist the toxins and enzymes
5. Drumstick appearance of spores is a produced by Staphylococcus aureus.
characteristic feature of
2. Write a short note on non-suppurative
a. Clostridium difficle complications of streptococcus
b. Clostridium perfringens pyogenes infections.
c. Clostridium botulinum 3. Write about Meningococcal vaccines.
d. Clostridium tetani 4. Write the prophylaxis for diphtheria
149

5. Enlist the difference between bacillary 18.
Explain the pathogenesis of
dysentery and amoebic dysentery/ V. Cholerae
6. What is enteric fever? 19. Write the mode of action of
7. Write the prophylaxis for Salmonella tetanospasmin
typhi. 20. Describe the pathogenesis of lock-jaw
8. Write the mode of action of cholera disease.
toxin/ 21. What is staphylococcal toxic shock
9. Tubercubin skin test. syndrome (STSS).
10. Define chancre. 22. Describe the mode of entry of
Meningococci from nusopharymx to
11. Write the morphology of Clostridium
brain.
tetani
23. Why antibiotics are avoided in
12. Write short notes on Canicola fever.
dynsentery except in severe cases.
13. List out the antituberculosis drugs.
24. Define zoonotic bacterial disease and
14. Write the colony morphology of give two examples.
salmonella typhi shigella dynsenteriae
25. Describe the pathogenesis of primary
on ss-agar.
syphilis.
15. Discuss the pathogenesis of typhoid
26. Enlist the recent serologtical methods
fever.
used to diagnose tuberculosis.
16.
Describe the pathogenesis of
27. Write the colony morphology of
pulmonary tuber culosis.
Lowenstein – Jensen.
17. Write briefly about Lab/diagnosis of
tuberculosis.
150

Chapter
8 Medical Parasitology
8.5 Plasmodium falciparum and P. vivax

Learning Objectives
8.6 Ascaris lumbricoides
students will be able to, Medical Parasitology is
• Know the various types of parasites the branch of Medical
and hosts. Science dealing with the
study of parasites living in
• Discuss the classification of
medically important parasites. or on the body of human,
their geographical
• Discuss the pathogenesis and clinical
distribution, the diseases caused by
aspects of parasitic infections.
them, clinical features and the response
• Describe the general epidemiological generated by human against them. It is
aspects and transmission patterns also concerned with various methods of
of diseases caused by protozoa and
their diagnosis, treatment, prevention and
helminths.
their control. Parasitology is a dynamic
• Identify the methods and field, as these parasites constantly
procedures of laboratory diagnosis change their morphology, hosts, and host
of pathogenic protozoans and relationships. For this reason, Parasitology
helminths in clinical specimens. is an active field of study, which has
• Know the treatment options for raised expectations for the development
parasitic infections. of new drugs, vaccines and other control
• Implement the preventive and measures through biotechnology.
control measures of protozoans and However, these expectations are reduced
helminthic infections. by the inherent complexity of parasite
and their relationship with host, the firm
establishment of parasite and vectors in
Chapter Outline
their environments and the vast socio
8.1 Parasite and Host economic problems in the geographical
areas where parasites are most prevalent.
8.2 Entamoeba histolytica
Before learning in detail about a few
8.3 Giardia lamblia medically important parasites of human,
8.4 Leishmania donovani let us know what is a parasite?
151

8.1 Parasite and Host tissues of the host (Example: Lice). The
Parasites are living organisms, which infection by these parasites is called
depend on living host for their infestation.
nourishment and survival. They multiply Endoparasite: The parasite which lives
or undergo development in the host. Host within the host is called Endoparasite.
is defined as an organism, which harbors Invasion by the parasite is called Infection.
the parasite, provides nourishment and Most of the protozoan and helminthic
gives shelter to parasite. Host is relatively parasites causing human diseases are
larger than the parasite. endoparasites.
Endoparasites can be further classified as:
8.1.1 Association between Host and
•• Obligate parasite: This parasite is
Parasite
completely dependent on its host
The relationship between host and the and cannot survive without it.
parasite can be of the following types: Example: Hookworms.
ԂԂ Symbiosis • • Facultative parasite: This
ԂԂ Commensalism, and parasite may either live as free
ԂԂ Parasitism. living form or as a parasite when
the opportunity arises. Example:
Flowchart 8.1 describes the types of host –
Naegleria fowleri.
parasite relationship
•• Opportunistic parasite: This
8.1.2 Types and Classification of Parasite parasite is capable of producing
According to the nature of the host- parasite disease in an immune deficient host
interaction and the environmental factors, (like AIDS and cancer patients).
the parasite may be one of the following, Example: Toxoplasma gondii.
Ectoparasite: These parasites live •• Zoonotic Parasite: This parasite
on the outer surface or in the superficial primarily infects animals and is
Host – parasite relationship
Symbiosis/mutualism Commensalism Parasitism

• Both the host • Only the parasite derives • Always harm the
and parasite are benefit from the association host due to their
dependent upon without causing any association.
each other. infection to the host. • The parasite
• None of them are • It is capable of living cannot live an
harmed. independently. independent life.
Flowchart 8.1: The types of host – parasite relationship

152

transmittable to humans. Example:
Fasciola species. Common name of
medically important
•• Accidental parasite: This parasite
parasite
infect an unusual host are known
as accidental parasites. Example: Intestinal flagellates –
Echinococcus granulosus infects Giardia intestinalis
man accidentally. Oral Flagellates – Trichomonas tenax
•• Wandering or Aberrant parasites: Genital flagellates – Trichomonas vaginalis
Parasites which infect a host Blood and Tissue flagellates –
migrate to the site where it cannot Leishmania and Trypanosoma
live or develop further are called Ciliated protozoa – Balantidium coli
aberrant parasites. Example: Dog
Dog roundworm – Toxocara canis
roundworm infecting humans.
Cat roundworm – Toxocara cati
8.1.3 Types of Host
Roundworm – Ascaris lumbricoides
Definite host: The host which harbour the
Hookworm – Ancylostoma duodenale
adult parasites or where parasites undergo
sexual method of reproduction is referred Liver fluke – Fasciola hepatica
to as a definite host. The definite host may Blood fluke – Schistosoma haematobium
be a human or any other living organism. Lung fluke – Paragonimus westermani
Example: Mosquito acts as a definite host
for Plasmodium spp. in Malaria. Pork tapeworm – Taenia solium
Intermediate host: The host in which Beef tapeworm – Taenia saginata
the larval stages of the parasite live or Eyeworm – Thelazia spp
where asexual reproduction takes place Threadworm or Human pinworm –
is called the intermediate host. Example:
Enterobius vermicularis
Man acts as an intermediate host for
Plasmodium spp. in Malaria. Human whipworm – Trichuris trichiura
Reservoir host: The host which
but remains alive till they gain entry into
harbour the parasite and acts has an
important source of infection to other the definitive host or intermediate host.
susceptible hosts is known as reservoir Such a host is termed as paratenic host or
host. It is also called temporary host. transport host or carrier host.
Example: Dog is the reservoir host for
disease kala azar. 8.1.4 Classification of Medically
Natural host: The host which is Parasitology
naturally infected with a certain species of The most acceptable taxonomic
parasite, is called natural host. Example: classification of human parasites
Pig is the natural host of Balantidium coli. includes Endoparasites and Ectoparasite.
Paratenic host or transport host: Endoparasites are subclassified into
some parasites enter a host in which protozoan parasite (unicellular organisms)
they do not undergo any development and helminthic parasite (multicellular
153

organism). Parasites of medical The condition where infection is
Importance come under the Kingdom transmitted from animals to humans
called Protista and Animalia. Protista is called zoonoses.
includes the microscopic single- celled
2. Mode of transmission
eukaryotes known as protozoa. In contrast,
helminths are microscopic, multicellular A. Oral transmission: This is through
worms possessing well differentiated ingestion of contaminated food,
tissues and complex organs belonging to water, vegetables, soiled fingers or
the kingdom Animalia. Classification of fomites contaminated by faeces that
medically important parasites is given in contain the infective stage of parasite.
Flowchart 8.2. This mode of transmission is referred
to as faecal-oral route. Example: Cysts
8.1.5 Life Cycle of Parasites of Entamoeba histolytica.
B. Skin transmission: This is another
Direct life cycle
important route. The infective
The life cycle of parasite that requires only
larvae of hookworm enter the skin
single host to complete its development,
of persons walking bare footed on
is called direct life cycle. Example:
contaminated soil.
Entamoeba histolytica requires only
C. Vector transmission: It could
human host to complete its life cycle.
be a biological or a mechanical
Indirect life cycle means. Many parasitic diseases are
transmitted by insect bite. Example:
The life cycle of parasite that requires
sandfly is vector for Leishmania.
two or more species of hosts to complete
D. Direct transmission by person
its development, the life cycle is called as
to person contact. Frequently,
indirect life cycle. Example: Malarial parasite
Entamoeba, Giardia and Trichomonas
(Plasmodium spp.) requires both human are transmitted by sexual contact
host and mosquito to complete its life cycle. among homosexuals.
E. Vertical transmission: It is the
8.1.6 Transmission of Parasites transmission from mother to fetus.
It depends upon on Source or reservoir of Example: Toxoplasmosis.
infection, and mode of transmission. So far, we have learnt about the general
introduction and classification of parasites.
1. Sources of infection Now, let us learn a few important human
A. Human: Human is the source or parasites in detail.
reservoir for a majority of parasitic
Introduction to Protozoa
infection. The condition in which
the infection is transmitted from one General characteristics of protozoa:
infected human to another human is 1. They are microscopic unicellular
called anthroponoses. eukaryotes.
B. Animals: Animals act as the source of 2. The single cell has a relatively complex
infection in many parasitic diseases. internal structure and it performs
154

Medical Parasitology
Protozoa (Protozoology) Helminthes (Helminthology)

Kingdom – Protista (Unicellular) Kingdom – Animalia (Multicellular)
Amoebae Flagellates Sporozoa Ciliates

(Typically (have one or more (undergo a (Complex protozoa
amoeboid and whiplike flagella) complex life cycle bearing cilia)
use pseudopodia Intestinal and with alternating
Balantidum coli
or protoplasmic genitourinary sexual and asexual
flow to move) normal flagellates reproductive
Entamoeba phases)
• Giardia
histolytica Plasmodium
• Trichomonas
Toxoplama
• Blood
and tissue
flagellates
• Leishmania
• Trypanosoma
Nematodes (round worms) Platyhelminths (flatworms)

They are elongated and
tapered at both ends,
round in cross section and Trematodes (Flukes)
Cestodes (tape worms)
unsegmented.
have a ribbon like Typically flattened
• Wuchereria bancrofti and leaf shaped with
chain of segments
• Ascaris sp (proglottids) 2 muscular sucker
Taenia solium They lack cuticle.
T. saginata They are hermaphrodites
Fasciola (Liver fluke)
Schistosoma(blood fluke)
Flowchart 8.2: Classification of medically important parasites
155

Infobits
Parasites having direct life cycle

Protozoa Helminths
•• Giardia lamblia •• Ascaris lumbricoides
•• Trichomonas vaginalis •• Trichuris trichiura
•• Balantidium coli •• Ancyclostoma duodenale
Parasites having indirect life cycle
S.No Protozoa Definite host Intermediate host
1. Plasmodium spp. Female Anopheles mosquito Man
2. Toxoplasma gondii Cat Man
3. Cestodes Man Pig
Taenia solium
4. Trematodes Man Snail
Fasciola hepatica
5. Nematodes Man Mosquito
Wuchereria bancrofti
various complex metabolic activities

such as digestion, reproduction, Infobits
respiration and excretion.
Naegleria fowleri (Brain eating amoeba)
3. Each cell consists of nucleus and is a thermophilic, free living amoebae
cytoplasm. occasionally act as human pathogens
4. A protozoa parasite during its life producing meningoencephalitis known
cycle may exist in two stages such as as primary amoebic meningoencephali-
trophozoite and cyst. tis (PAM). Infections most often occur
when water containing Naegleria fowl-
Amoebae eri is inhaled through the nose, where it
Amoebae are structurally simple then enters the nasal and olfactory nerve
protozoans which have no fixed shape. tissue traveling to the brain. N. fowleri
The cytoplasm of amoeba is bounded by a occurs in three forms -as a cyst, tropho-
membrane and can be differentiated into zoite (amoeboid) and a biflagellate (it
an outer ectoplasm and inner endoplasm. has two flagella). The flagella form can
Pseudopodia (false foot) are formed by exist in the cerebrospinal fluid.
the amoebae by throwing out ectoplasm
followed by endoplasm. These are
employed for locomotion and engulfment
of food by phagocytosis.
Reproduction occurs by fission and
budding. Amoebae are classified as either
free living or intestinal amoebae.
156

8.2 Intestinal Amoeba – Entamoeba passed stools are ingested, they are rapidly
Histolytica destroyed in stomach and cannot initiate
infection. Therefore, the infections is not
8.2.1 Geographical Distribution
usually transmitted by trophozoites.
It is Worldwide in distribution they
are more common in the tropics than Precyst
elsewhere. It is found wherever sanitation Trophozoites undergo encystment in the
is poor. intestinal lumen. Encystment does not
occur in the tissue or in feces outside the
8.2.2 Habitat body. Precyst is smaller in size about 10 -20
Trophozoites of E.histolytica live in the µm in size. It is round or oval in shape. The
mucous and submucous layers of the large endoplasm is free of red blood cells and other
intestine of human. ingested food particles (Figure 8.1). The
nuclear structure retains the characteristics
8.2.3 Morphology of the trophozoite.
E.histolytica occurs in 3 forms as Cyst

Trophozoite, Precyst and Cyst. Precyst secretes a highly refractive cyst wall
Trophozoite: It is the growing or around it and becomes a cyst. A mature cyst
feeding stage of the parasite. It is the only is a quadrinucleate spherical body. The cyst
form present in tissues. It has no fixed begins as a uninucleate body but soon divides
shape. They vary in size from 18 to 40µ, by binary fission and develops into binucleate
average being 20 to 30µ. The cytoplasm and quadrinucleate bodies (Figure 8.1). The
is usually described as outer ectoplasm cytoplasm of the cyst is clear and hyaline
and inner endoplasm (Figure 8.1). (translucent) and the nuclear structure retain
The endoplasm contains nucleus, food the characteristic of the trophozoites.
vacuoles, erythrocytes, occasionally The mature quadrinucleate cyst,
leucocytes and tissue debris. The nucleus passed in the stool, do not undergoes
is characterised by evenly arranged any further development and remain
chromatin on the nuclear membrane alive for several months in the soil or in
and the presence of a small, compact, environment where they were deposited.
centrally located karoyosome (It is a DNA The mature quadrinucleate cysts are the
containing body, situated peripherally or infective forms of the parasite.
centrally within the nucleus). Trophozoites
exhibits active crawling or gliding motility 8.2.4 Life – Cycle of Entamoeba
by forming finger-like projections called Histolytica
Pseudopodia.
E. histolytica passes its life cycle only in
The trophozoite reproduce by binary one host, the human.
fission in every 8 hours. Trophozoites
Infective form: Mature quadrinucleate cyst.
survives upto 5 hours at 37°C and are killed
by heat, drying and chemical sterilization. Mode of transmission: Ingestion of food
Even if live trophozoites from freshly and water contaminated with cyst.
157

Figure 8.1: Trophozoite, precyst and cyst of Entamoeba histolytica
•• The cyst that are swallowed along medium, the cyst wall is damaged by
with food and water enters into the trypsin leading to excystation.
alimentary canal. The cyst wall is •• The cytoplasm gets detached from the
resistant to action of gastric juice. cyst wall and an amoeboid movement
The cyst pass through the stomach appear causing a tear in the cyst wall,
undamaged and enters the small through which quadrinucleate amoeba
intestine (Figure 8.2). is liberated. This stage is called the
•• When the cyst reaches caecum or metacyst.
lower part of the ileum, due to alkaline •• The nuclei in the metacyst immediately
undergo division to form 8 nuclei,
each of which gets surrounded by its
The amoeba infecting own cytoplasm to become 8 small
man may be classified amoebulae or metacystic trophozoites.
according to their
•• These metacystic trophozoites are
pathogenicity and
carried to the caecum and colon. They
habitat.
invade the tissues and lodge in the
A. Pathogenic submucous tissue of the large intestine
Intestinal Amoeba: Entamoeba histolytica which is their normal habitat.
B. Non pathogenic •• Trophozoite grow and multiply by
1. Mouth Amoeba: Entamoeba gingivitis binary fission. The trophozoite phase
2. Intestinal Amoeba: Entamoeba coli of the parasite is responsible for
Entamoeba nana producing the characteristic lesion of
amoebiasis.
158

Figure 8.2: Life cycle of Entamoeba histolytica
•• Some of the trophozoites in colon section, the ulcer appears like flask, with
develop into precystic forms and cysts, mouth and neck being narrow and base
which are passed in feces to repeat the being large and rounded (Figure 8.3 shows
cycle. the flask – shaped ulcer). The base of ulcer
is generally formed by the muscular coat
8.2.5 Pathogenesis and filled up by the necrotic material. The
E. histolytica causes intestinal and extra ulcers generally do not extend deeper than
intestinal amoebiasis (Flowchart 8.3). submucosal layer.
E. histolytica can live in the intestine 8.2.6 Clinical Features

without causing symptoms. But, they can
also cause severe disease. These amoebas Incubation period is highly variable, but is
may invade the wall of the intestine leading generally 4 to 5 days.
to amoebic dysentery, an illness that causes A wide spectrum, from asymptomatic
intestinal ulcers, bleeding, increased mucus infection (luminal amoebiasis), to invasive
production and diarrhoea. The ulcers are intestinal amoebiasis (dysentery, colitis,
strictly confined to the large intestine being appendicitis, toxic mega colon, amoebomas),
most numerous in the caecum and next to invasive extraintestinal amoebiasis
in the sigmoid-rectal regions. The lesions occurs. Flowchart 8.4 classifies the clinical
may be generalized or localised. A typical outcomes of infection with Entamoeba
amoebic ulcer varies from pin’s head to one histolytica. Only about 10% to 20% of people
inch or more in diameter in size. The shape who are infected with E. histolytica become
of ulcer may be round or oval. On vertical sick from the infection.
159

Infection of E. histolytica
Intestinal amoebiasis Extra intestinal amoebiasis

Infection is limited entirely to the The trophozoites migrate and
large intestine, the initial site of produce lesions in
location of the parasite. Liver – Hepatic Amoebiasis
Lungs – Pulmonary Amoebiasis
Brain – Cerebral Amoebiasis
Spleen – Splenic Abscess
Flowchart 8.3: Infection Caused by E.histolytica
Superficial Invasion of
Ulcer Blood Vessel
Deep Ulcer Perforation
Mucosa
Figure 8.3: Ulcers in intestinal amoebiasis
Symptomatic Intestinal
Infected host Extra intestinal
Asymptomatic Carrier
Flowchart 8.4: The clinical outcomes of infection with Entamoeba histolytica

160

The typical manifestation of intestinal
amoebiasis is amoebic dysentery. The
symptoms are often quite mild and
can include loose feaces, stomach
pain and stomach cramping. In acute
amoebic dysentery, the symptoms
include abdominal pain, bloody stool,
fever, tenderness, rectal tenesmus and
hepatomegaly (enlargement of liver). Figure 8.4: Amoebic liver abscess
People affected may develop anemia due
to loss of blood. On clinical and laboratory abscess (ALA) contains an odour less
ground, amoebic dysentery should be and thick chocolate brown pus called
differentiated from bacillary dysentery. anchovy sauce pus. ALA is associated
A Table 8.1 shows the difference between with an abrupt onset of high fever, right
the stools of amoebic and bacillary upper abdominal pain and tenderness.
dysentery. Anorexia (loss of appetite for food),
nausea (the sensation to vomit),
Extra intestional amoebiasis
vomiting, fatigue (extreme tiredness)
1. Hepatic amoebiasis: This is the most
and weight loss are also frequent.
common form of extra intestinal
invasive amoebiasis. Liver abscess may 2. Pulmonary Amoebiasis: It is very
be multiple or more often solitary, rare, but this may occur when direct
usually located in the upper right lobe hematogenous spread from the colon.
of the liver (Figure 8.4). Amoebic liver The patient presents with severe chest
Table 8.1: Difference between the stools of amoebic and bacillary dysentery
Amoebic dysentery Bacillary Dysentery

Macroscopic observation
Number 6–8 Over 10 motions a day
Amount Relatively large Small
Odour Offensive Odorless
Colour Dark red Bright red
Nature Blood and mucus mixed with Blood and mucus no faeces
faeces
Microscopic observation
RBC In clumps, reddish – yellow RBC in rouleaux, bright red
in colour in colour
Pus cells Scanty Numerous
Parasite Trophozoites of E. histolytica Nil
Charcot – Leyden crystals Present Nil
161

pain and have dyspnoea (shortness Cerebral Abcess
of breath). The sputum of patient is
chocolate brown. Amoebic trophozoites
may be demonstrated in the sputum.
3. Cerebral amoebiasis: The condition
is unusual. In cerebral amoebiasis, the
abscess is single, small and is located in
the cerebral hemisphere. The patient
Pulmonary
may die of rupture or involvement Amoebiasis
of cerebellam within 12–72 hours. Liver Abcess

Biopsy of the brain shows the amoebic
trophozoites. Splenic Abcess
4. Cutaneous amoebiasis: It can be

caused by perforation of an amoebic
Intestinal
abscess or surgical wound infected with Amoebiasis
amoebae. It is less frequent condition.

5. Genitourinary Amoebiasis: This
condition includes amoebiasis of the Figure 8.5: Extra-intestinal amoebiasis
kidney and genital organs. Amoebiasis
amoebic cysts by Formalin – ether is
of the genital organs is a rare condition.
the method of choice.
Lesions of amoebiasis is shown in
Figure 8.5. C. Examination of stained stool smears:
Staining by iron haematoxylin, Periodic
8.2.7 Laboratory Diagnosis Acid – Schiff (PAS) stains demonstrate the
presence of both trophozoites and cyst.
Specimens: Stool is the specimen of
Amoebic liver abscess (ALA):
choice. Other specimens collected
Demonstration of amoebic trophozoites
includes blood, rectal exudates and rectal
in the aspirated liver pus establishes the
ulcer tissue collected from the base by
diagnosis of ALA.
endoscopies.
Serology: Detection of amoebic
Methods in examination of stool antigens in the serum by Enzyme Linked
A. Direct wet mount examination of Immunosorbent Assay (ELISA).
stool: Demonstration of mature Molecular diagnosis: PCR
quadrinucleate cysts or trophozites (Polymerase chain reaction) is employed
in stool is diagnostic of intestinal to detect amoebic genome in the aspirated
amoebiasis. The wet mount of stool is liver pus for the diagnosis of ALA.
prepared in the saline, iodine or lacto Imaging methods: X – Ray magnetic
phenol cotton blue. resonance imaging (MRI) scan and
B. Examination of stool after computerized Axial Tomography (CAT)
concentration: Demonstration of Scan are the imaging methods used.
162

Treatment: Eradication of amoebae 8.3.1 Geographical Distribution
by the use of amoebicidal drugs and It is the most common protozoan pathogen
replacement of fluid and electrolyte is and is worldwide in distribution. The disease
the treatment for amoebiasis. Listed is very high in areas with low sanitation,
below the drugs used in the treatment for especially tropics and subtropics.
amoebiasis.
• Paramomycin and iodoquinol acts 8.3.2 Habitat
in the intestinal lumen but not in Giardia lamblia lives in the duodenum
tissues. and upper jejunum of human. It is the only
• Emetine, chloroquine are effective protozoan parasite found in the lumen of
in systemic infection. They act only the human small intestine.
on trophozoites.
8.3.3 Morphology
Metronidazole is the drug of choice
It exists in two forms
which acts as both luminal and tissue
amoebicides. It is low in toxicity and •• Trophozoite and
is effective against intestinal as well as •• Cyst
extra -intestinal amoebic infections.
Antoine van
8.2.8 Prevention and Control Leewenhock observed
•• Proper sanitation is the key to avoid and illustrated Giardia
amoebiasis. Washing hands with soap lamblia in his own
and water after using the bathrooms loose stool. This was the first protozoan
and before handling food. parasite of human that is recorded and
the first to be seen under a microscope.
•• Drinking safe and boiled water.
•• Avoid eating unwashed fruits and Trophozoite
vegetables. The trophozoite is in the shape of a tennis or
•• Prevention of water supplies from badminton racket. It is rounded anteriorly
faecal contamination. and pointed posteriorly. The size of the
trophozoite is 14 µ long by 7µ broad. Dorsally,
•• Early rapid detection of diseased
it is convex and ventrally, it has a concave
people and subsequent treatment
sucking disc which helps in its attachment
with amoebicidal drugs. No vaccine
to the intestinal mucosa. It is bilaterally
is available yet against amoebiasis in
symmetrical. All the organs of the body are
humans.
paired. Trophozoite of Giardia possess,
8.3 Intestinal Flagellates – Giardia •• 1 pair of nuclei
Lamblia •• 4 pairs of flagella
(Also known as Giardia duodenalis, •• Parabasal body (Blepharoplast), from
Giardia intestinalis) which the flagella arise (4 pairs)
163

•• 1 pair of axostyles, running along person – to person transmission occurs
the midline in children. Transmission occurs
•• Two sausage – shaped parabasal or through oral-anal and oral-genital route
median bodies lying transversely in sexually active homosexual males.
posterior to the sucking disc Within half an hour of ingestion, the cyst
hatches out into two trophozoites, which
•• The trophozoite is motile, with
multiply by binary fission and colonize
a slow oscillation about its long
in the duodenum. The trophozoites
axis, often resembling falling leaf
live in the duodenum and upper part
(Figure 8.6a).
of jejunum, feeding by pinocytosis.
Cyst When conditions in duodenum are
It is the infective form of the parasite. The unfavourable, encystment occurs, usually
cyst is small and oval, measuring 12 µm × 8 in large intestine. Cysts are passed in
µm and is surrounded by a hyaline cyst wall. stool and remain viable in soil and water
Its internal structure includes 2 pairs for several weeks (Figure 8.7).
of nuclei grouped at one end. A young
cyst contains 1 pair of nuclei. The axostyle 8.3.5 Pathogenicity
lies diagnonally, forming a dividing line Giardia lamblia does not invade the
within cyst wall (Figure 8.6b). tissue, but remains attached to intestinal
epithelium by means of the sucking
8.3.4 Life Cycle: Giardia Passes Life disc. It causes a disturbance of intestinal
Cycle in one Host, the Human function leading to malabsorption of fat.
Infective form: Mature cyst
Mode of transmission: Human acquires
infection by ingestion of cyst in Incubation period is variable, but is
contaminated water and food. Direct usually about 2 weeks.
a. Trophozoite b. Cyst
Figure 8.6: Trophozoite and cyst of Giardia
164

Figure 8.7: Life cycle of Giardia lamblia
The disease is asymptomatic, but in 8.3.7 Laboratory Diagnosis

some cases it may lead to abdominal Specimens: Stool and blood
cramps, flatulence, looseness of bowels,
Examination of stool sample: Giardiasis
foul smelling stool and mild steatorrhoea
can be diagnosed by identification of cysts
(passage of yellowish and greasy stools
of Giardia lamblia in the formed stools and
in which there is excess of fat). The stool
the trophozoites and cyst of the parasite in
contains excess mucus and fat but no
diarrhoeal stools.
blood and pus. Children may develop
chronic diarrhoea, malaise (discomfort), Macroscopic examination of stool: Fecal
nausea, anorexia (loss of appetite for specimens containing Giardia lamblia may
food), malabsorption of fat, vitamin A have an offensive odor. It is pale coloured
and protein. Occasionally, Giardia may with fatty substance floating in water.
colonize the gall bladder causing biliary Microscopic examination of stool:
colic and jaundice. Cysts and trophozoites can be found in
165

diarrheal stools by saline and iodine wet 8.4.1 Geographical Distribution
preparations (Figure 8.8). Leishmania species is found in the
Serodiagnosis: Immuno chromatographic Mediterranean, the Middle East, Africa
strip tests and indirect immunofluroscence and Asia including India.
(IIF) tests are readily available. For antigen
and antigen detection ELISA, Commercially 8.4.2 Habitat
available ELISA kits detects Giardia – Specific
Leishmania donovani is an obligate
antigen.
intracellular parasite of human and
Molecular methods: DNA probes and other mammalian hosts. They are always
polymerase chain reaction (PCR) have found as intracellular amastigotes in the
been used to demonstrate parasitic reticuloendothelial cells of the spleen,
genome in the stool specimen. bone marrow, liver, intestinal mucosa and
mesenteric lymph nodes of hosts.
8.3.8 Treatment
Metronidazole and Tinidazole are the 8.4.3 Morphology
drugs of choice.
The parasite exists in two forms:
8.3.9 Prevention and Control Amastigote: It is the form found in
human and other mammalian hosts.
Giardiasis can be prevented and controlled
They are found inside monocytes,
by,
polymorphonuclear leucocytes or
•• Proper disposal of human faeces, endothelial cells. They are small, round to
maintenance of food and personal oval bodies measuring 2–3µm in length
hygiene and health education. (Figure 8.8). They are also known as LD
•• After using the bathroom and before (Leishman donovan) bodies.
eating, the hands should be washed
thoroughly with soap and warm water.
Boiling of water is the best and effective
method in killing the viable cysts.
•• To reduce the risk of venereal
transmission, patients should avoid
risky sexual behavior.
•• No vaccine or effective chemo
prophylactic drug is available for
prevention of Giardiasis.
8.4 Tissue Flagellates – Leishmania

donovani
The genus is named after the scientist
Leishman, who first described the parasite Figure 8.8: Promastigote and Amastigote
in London in May 1903. form of Leishmania
166

Promastigote: This forms are found in 8.4.4 Life – Cycle of Leishmania donovani
the mid-gut of sand fly and in the culture Leishmania donovani completes its life
media. The fully developed promastigotes cycle in two different hosts. The complete
are long, slender and spindle – shaped. life cycle is given in Figure 8.9.
They measure 15µm to 25µm in length
and 1.5µm to 3.5µm in breadth. A single Host Forms
nucleus is situated at the centre. The Human and other Amastigote
kinetoplast lies near the anterior end. The mammals
flagellum is single, delicate and measures
(Example: Dogs)
15µm–28µm (Figure 8.8).
Sandfly of Genus Promastigote
Infobits Phlebotomus
There are 3 main forms of Leishmaniases Development in Human

– Visceral (also known as Kala-agar and The parasite is transmitted to human
the most serious form of the disease), and other vertebrate hosts by the bite of
cutaneous (the most common) and blood sucking female sandfly. During
mucocutaneous. The disease affects the blood meal, the sandfly deposists
some of the poorest people on earth, promastigotes on surface of the skin.
and is associated with malnutrition, These promastigotes are immediately
population displacement, poor housing, phagocytosed by fixed macrophages of
a weak immune system and lack of the host, in which they are transformed
financial resources. Leishmaniasis is
into amastigotes. The amastigotes
linked to environmental changes such as
multiply by binary fission within the
deforestation, building of dams, irrigation
macrophages. As many as 50 to 200
schemes, and urbanization.
amastigotes may be present inside the
PKDL occurs in all areas endemic
enlarged cell. These are called LD bodies.
for L.donovani but is commonest
The rupture of cell releases amastigotes
in East Africa and on the Indian
in large numbers which inturn are free
subcontinent, where up to 50%
and 10% of patients with kala-azar, to infect other cells. Free amastigotes are
respectively develop the condition. subsequently carried by circulation. These
The frequency is reported to be forms invade monocytes of the blood and
declining in India. macrophages of the spleen, liver, bone
Cutaneous Leishmaniasis marrow, lymph nodes and other tissues of
the reticuloendothelial cells.
The clinical spectrum of cutaneous
leishmaniasis ( oriental sore) is broad and Development in sandfly
may mimic that of other skin conditions, Female sandfly during a blood meal ingest
such as staphyloccal or streptococcal
free, as well as intracellular amastigotes in
infection, mycobacterial ulcer, leprosy,
the blood. In the mid gut of the sandfly,
fungal infection, cancer, sarcoidosis and
the amastigotes are transformed within
tropical ulcer.
72 hours to flagellated promastigotes.
167

Figure 8.9: Life cycle of Leishmania donovani
These promastigotes multiply by binary proliferation of reticuloendothelial cells.

fission. After a period of 6 to 9 days, these Kidney shows cloudy swelling and is
forms migrate from the midgut to the invaded by macrophages parasitized by
pharynx and buccal cavity of sandfly. Bite amastigotes.
of the infected sandfly transmits infection
to susceptible persons and the life – cycle 8.4.6 Clinical Features
is repeated. Incubation period: It is usually 3–6 months
but can be months or years.
8.4.5 Pathogenesis
Visceral Leishmaniasis is a serious
Leishmania donovani causes visceral and fatal systemic disease. In India, the
Leishmaniasis. The disease is also disease is called Kala – azar meaning
known as Dum – Dum fever, Asian fever, “black disease”.
Assam fever, or infantile splenomegaly. The disease is characterized by the
Leishmaniasis is a disease of the presence of fever, hepatosplenomegaly
reticuloendothelial system. Proliferation (Figure 8.10) (the simultaneous
and destruction of reticuloendothelial enlargement of both liver and the spleen),
cells of the internal organs are responsible hypergammaglobulinemia (a condition
for the pathological changes in visceral in which increased levels of a certain
leishmaniasis. immunoglobulin in blood serum),
Spleen, liver and lymphnodes are Leucopenia, Thrombocytopenia (deficiency
enlarged in this condition. Bone marrow of platelets in the blood), Cachexia (a
is dark red in colour and shows extensive condition that causes extreme weight loss)
168

with marked anemia, emaciation and loss
of weight. Epistaxis (bleeding from nose)
and bleeding from gums are common. In
Indian patients, the skin on the hands, feet,
abdomen, around the mouth and fore – head
becomes grayish and dark coloured. This
hypo – pigmentation of the skin is unique
in Indian patients giving the disease name
Kala – azar.
Figure 8.11: Post kala – azar dermal

leishmaniasis -PKDL
Methods of examination: This

includes, microscopy and culture
1. Direct microscopy
The amastigotes of Leishmania donovani
(known as LD bodies) can be demonstrated
in the smears of spleen, bone marrow, liver,
lymph node and peripheral blood stained
in Leishman, Giemsa or wright stains.
Splenic aspiration is the most sensitive
method to detect LD bodies. Examination
Figure 8.10: Splenomegaly of peripheral blood smear and buffy coat
Post kala – azar dermal leishmaniasis smear is more commonly used to find LD
(PKDL): It is a non – ulcerative lesion of bodies in the circulating monocytes.
the skin, which is seen after completion
2. Culture
of treatment of the kala – azar. This
condition is characterized by multiple, Promastigotes are found in the
hypopigmented, erythematous macules culture media. Tissue samples and
involving the face and trunk (Figure 8.11). aspirates are inoculated in the NNN
In Indian forms, PKDL appears after (Novy–MacNeal–Nicolle) medium for
a latent period of 2 years and may even demonstration of promastigotes.
persist as long as 20years, creating a
Laboratory diagnosis of kala – azar is
persistent human reservoir of infection.
briefly discussed in Flowchart 8.5.
Laboratory diagnosis Treatment: Pentavalent antimonials
Specimens: Aspiration from spleen, bone are the drugs of choice. Pentamidine,
marrow, lymph node, liver biopsy and Amphotericin B and Miltefosine (oral
peripheral blood. drug) are recommended.
169

Laboratory diagnosis of kala – azar
TN_GOVT_XII_Micro_Biology_CH08.indd 170
Direct evidence Indirect evidence
Animal
Demonstration
Culture (NNN) inoculation in
of LD bodies
hamster or mice
170
Serodiagnosis Molecular diagnosis Non – specific Skin test Blood picture
• DNA probe serum test Leishmanin skin Anemia
• PCR • Aldehyde test test Progressive
• Chopra’s leucopenia
antimony test
Detection of antigen Detection of antibody

• ELISA • CFT
Flowchart 8.5: Laboratory diagnosis of kala-azar
27-02-2019 14:53:05
8.4.7 Prevention and Control specific agent of malaria was discovered
Integrated insecticidal spraying (DDT and in RBC’s of a patient in 1880 by Alphonse
Malathion) to reduce sandfly population. Laveran. In 1897, Ronald Ross identified
the developing stages of malaria parasites
Reduction of reservoir by killing all
in mosquitoes in Secunderabad, India.
the infected dogs.
This led to various measures for the control
Personal prophylaxis by using anti – and possible eradication of malaria by
sandfly measures like using thick clothes, mosquito control. Both Ross (1902) and
bed nets, window mesh or insect repellants Laveran (1907) won the Nobel Prize for
and keeping the environment clean. their discoveries in malaria.
No vaccine is available against kala – azar.
Infobits
8.5 Sporozoa – Plasmodium
Three basic types of malaria
Protozoan parasites characterised by
1. Benign tertian (P. vivax and
the production of spore – like oocysts P. orale) with a fever every 2nd day
containing sporozoites were known as (Example: Monday – fever, Tuesday
sporozoa. The parasites belonging to this – no fever, Wednesday – fever).
group of protozoa do not possess any 2. Benign quartan (P. malariae)
special organs of locomotion, such as with a fever every 3rd day.
flagella or cilia. The medically important (Example: Monday; fever, Tuesday
parasite of this class that is given in the – no fever, Wednesday – no fever,
text is malaria parasites. Thursday – fever.
3. Malignant tertian (P. falciparum),
Malaria in which the cold stage is less
It is the disease condition with seasonal pronounced and the fever stage is
intermittent fevers, chills and shivering. more prolonged and severe. This
type of malaria is more dangerous
The name malaria (Mal: bad, aria: air)
because of the complications
was given in the 18th century in Italy. The
caused by capillary blockage (i.e,
convulsion, coma, acute pulmonary
The single most insufficiency and cardic failure).
important protozoan Large numbers of erythrocytes are
disease is malaria, parasitized and destroyed, which
which causes may result in dark-coloured urine.
(black water fever); intravascular
1.5 million deaths each year.
hemolysis, hemoglobinuria, and
Different species of malaria kidney failure).
parasites can develop in the same Two species of plasmodium,

mosquito and such an infected P. vivax and P. ovale, can remain
mosquito can transmit the infection in the liver, if not treated properly.
to man giving rise to cases of “mixed The organism leave the liver and
infection” the commonest being re-infect erythrocytes, causing the
P. falciparum with P. vivax. symptoms.
171

Causative agents of human malaria: 8.5.4 Life Cycle
The organisms: Four species of The malaria parasite
Plasmodium cause malaria in humans. passes its life cycle in
•• Plasmodium vivax: (Benign Tertian two different hosts and
malaria) comprises of two phase as
•• Plasmodium falciparum: (Malignant follows,
tertian malaria) Definitive host:
•• Plasmodium malaria: (Benign Female Anopheles mosquito (a sexual
Quartan malaria) phase of parasite occurs).
•• Plasmodium ovale: (Benign tertian Intermediatehost: Human (an asexual
malaria) phase of parasite occurs).
The two most common species are Thus, life cycle of malaria parasite
P. vivax and P. falciparum, WHO reports show alternation of generations- asexual
(2018) that falciparum being the most and sexual generation in two different
pathogenic of all. hosts (Figure 8.12).
8.5.1 Geographical Distribution

Malarial parasites are found in all countries. Salivary gland
Sporozoite LIVER STAGE
In India, malaria continues to be a major
public health threat. Midgut
sporozoite
merozoite
8.5.2 Habitat MOSQUITO STAGE
Schizont
Schizont
oocyst
The malarial parasites infecting man, after ookinete trophozoite
ring
passing through a developmental phase in Zygote
the parenchyma cells of the liver, reside inside BLOOD STAGE
gametocytes
the red blood corpuscles and are carried by gametes
the circulating blood to all the organs.

Figure 8.12: Lifecycle of Plasmodium spp
8.5.3 Vectors
8.5.5 Human Cycle (Asexual Phase –
Human malaria is transmitted by over
Schizogony)
60 species of female Anopheles mosquito.
Human infection occurs when the
Human malarial parasite – Plasmodium sporozoites (the infective forms of the
falciparum parasite are present in the salivary
Of all the human malaria parasites, gland of the mosquito) are injected into
P. falciparum is the most highly blood capillaries when the mosquito
pathogenic and responsible for feeds on blood after piercing the skin.
malignant tertian malaria. This is a form The malarial parasite multiplies by
of disease which runs an acute course in division and the process designated
non-immune patients and is frequently as Schizogony (schizo: to split, gone:
fatal if untreated. generation).
172

Sporozoites are minute thread-like •• These normally rupture in 6–15 days
curved organisms with tapering ends. and release thousands of merozoites
Measuring 9–12µ in length with a central into the blood stream.
elongated nucleus while, the cytoplasm •• They do not return from red blood
reveals no pigment as seen with a light cells to liver cells.
microscope. In human, schizogony occurs
Plasmodium vivax and P. ovale –
in two locations. One in the red blood
parasites in liver tissue are called
cells (erythrocytic schizogony) and other
hypnozoites.
in the liver cells (pre – or exoerythrocytic
schizogony). B. Erythrocyticstage
•• The merozoites released by
A. Pre–erythrocytic or Exoerythrocytic
pre–erythrocytic schizonts invade the
schigony
red blood cells (Parasitaemia).
•• Sporozoites do not directly
enter the RBC’s to initiate •• Merozoites are pear – shaped bodies,
erythrocyticschizogony, but undergo about 1.5 µm in length.
developmental phase in other human •• In the erythrocyte, the merozoite loses
tissues. its internal organelles and appears as
rounded body having a vacuole in the
•• This cycle lasts for about 8 days
center with the cytoplasm pushed to
in Plasmodium vivax, 6 days in
the periphery and the nucleus at one
P. falciparum and 9 days in P. ovale.
pole. These forms are called ring forms
•• This pre–erythrocytic schizogony or young trophozoites.
occurs within parenchymal cells of the
•• The parasite feeds on the hemoglobin
liver.
of the erythrocyte. They incompletely
•• The Sporozoites, which are elongated metabolize hemoglobin therefore,
spindle – shaped bodies, become hematin – globin pigment or
rounded inside the liver cells. haemozoin pigment is left behind.
•• They enlarge in size and undergo •• The malaria pigment released when
repeated nuclear division to form the parasitized cells rupture is taken
several daughter nuclei, each of which up by recticuloendothelial cells.
is surrounded by cytoplasm.
•• The ring form develops and becomes
•• This stage of the parasite is called the irregular in shape and shows amoeboid
pre–erythrocytic or exoerthrocytic motility. This is called the amoeboid
schizont or merozoites. form.
•• The heptocyte is distended by the •• When the amoeboid form reaches
enlarging schizont and the liver cell a certain stage of development, its
nucleus is pushed to the periphery. nucleus starts dividing by mitosis
•• Mature liver stage schizonts are followed by a division of cytoplasm
spherical multinucleate and contain to become mature schizonts or
2000–50,000 uninucleate merozoites. merozoites.
173

•• A mature schizont contains 8–32 •• The gametocytes do not cause any clinical
merozoites and haemozoin. The illness in the host, but are essential for
mature schizont bursts releasing the transmission of the infection.
merozoites into the circulation. •• A person who harbors the gametocytes
•• The merozoites invade fresh is referred to as a carrier or reservoir.
erythrocytes within which they
go through the same process of Infobits
development. This cycle is called
erythrocytic schizogony. Sir Ronald Ross Institute of
•• The rupture of the mature schizont Parasitology is a malaria research
releases large quantities of pyrogens. institute located in Begumpet,
This is responsible for the febrile Secunderabad, Hyderabad, India.
paroxysms characterising malaria. Established in 1955, the institute is a
division of Osmania University. The
•• In P. falciparum, erythrocytic schizog-
institute is named after Sir Ronald
ony always takes place inside the cap-
Ross, winner of Nobel Prize for
illaries and vascular regions of internal
Physiology or Medicine, 1902. Though
organs. Therefore, in these infections,
he was a surgeon by qualification,
schizonts and merozoites are usually
Ross was attracted towards research in
not seen in the peripheral blood.
tropical diseases, especially malaria.
C. Gametogony During his posting, he worked on his
•• Some of the merozoites, after a few research from a laboratory in the old
erythrocytic cycles do not develop Begumpet military hospital building.
into trophozoites and schizonts but It was in this building on 20 August
they undergo sexual differentiation to 1897 that he made the discovery of
develop into the gametocytes. the malarial parasite inside the body
of a mosquito.His study confirmed
•• Development of gametocytes takes
that mosquitoes were the carriers of
place within the internal organs and
malaria parasite.
only the mature forms appear in
circulation.
•• The mature gametocytes in 8.5.6 Mosquito Cycle (Sexual Cycle –
P. falciparum are crescent shaped. Sporogony)
•• Female gametocytes are generally •• A Female Anopheles mosquito during
more numerous and larger. its blood – meal from an infected person,
•• Male gametocytes and female sucks up both the sexual and asexual
gametocytes are called micro forms of parasite. But, only the mature
gametocytes and macro gametocytes sexual forms develop and the rest die.
respectively. •• The gametocytes are set free in the
•• Gametocyte appears in 10–12 days in midgut (stomach) of mosquito and
P. falciparum. undergo further development.
174

•• The nuclear material and cytoplasm of •• The sporozoites are distributed
the male gametocyte divides to produce through the circulating fluid into
long, actively motile, whip – like forms various organs and tissues of the
of 8 microgametes. This process is called mosquito except the ovaries.
exflagellation of male gametocytes. •• The sporozoites have a special affinity
•• The Exflagellation is completed within towards the salivary glands. The
15–30 minutes for P. falciparum. mosquito at this stage is capable of
transmitting infection to man.
•• The female gametocyte does not
divide but maturation involves by 8.5.7 Pathogenesis
condensation of nucleus to become the
In malaria, typical pathological changes
female gamete.
are seen primarily in the spleen, liver,
•• Female gamete is fertilized by one bone marrow, lungs, kidney and brain.
of the microgametes to produce Liver: The liver is enlarged. The organ
the zygote. The zygote is formed in becomes more firm and pigmented.
20–120 minutes after the blood meal. Pigments are found in parenchymal cells.
The zygote is initially is a non – motile
Spleen: The spleen is markedly
round body, but within 18–24 hours, it
enlarged. If the infection lasts over a long
gradually elongates into a vermicular
period, the spleen is usually grayish, dark
motile form. This is called the ookinete. brown or even black and is commonly
•• Ookinete penetrates the epithelial known as ‘ague cake’.
lining of stomach wall. Their anterior Bone marrow, Lungs, Kidneys and
end comes in close contact to the Brain are enlarged and pigmented. They
cell membrane by secretion of some are filled with parasitized erythrocytes.
proteolytic substances which causes Anemia is caused by destruction of large
lysis of cell membrane. Later, the number of red cells by complement
ookinete come to lie just beneath the mediated and autoimmune hemolysis. It
basement membrane. is also due to the increased clearance of
•• It becomes rounded into a sphere with both parasitied and parasitized RBCs by
an elastic membrane. This stage is the spleen.
called the oocyst. The oocyst increase
in size and undergo numerous nuclear
multiplication which develops a large The incubation period is generally 9–14
number of sickle shaped bodies known days but, it can be as short as 7 days. The
as sporozoites. most malignant form of malaria is caused
by P. falciparum hence, variable clinical
•• Number of oocysts in the stomach wall syndromes are associated with falciparum
varies from a few to over a hundred. malaria. That include,
•• Around the 10th day of infection the 1. Prodromal (initial indication of the
oocyst ruptures, releasing sporozoites onset of disease) period: Non – specific
in the body cavity of the mosquitos. symptoms such as malaise (condition of
175

general weakness or discomfort), myalgia when the level of Sodium in the blood
(severe muscle pain) headache and is too low) occur in both uncomplicated
fatigue (feeling of tiredness) are usually and severe malaria.
seen during the prodromal period.
2. Malarial paroxysm (sudden onset of 8.5.9 Complications of Severe
disease): It is the classical manifestation Falciparum Malaria
of acute malaria. It is characterised by
1. Black water fever
fever, chill and rigor (sudden feelings of
cold with shivering).The fever is caused The syndrome is the manifestation of
by rupture of red blood cells that contain repeated infections of falciparum malaria,
malarial parasites. The fever occurs which were inadequately treated with
every 48 hours in falciparum malaria. quinine. The condition is associated with
3. Anemia (A condition in which the haemoglobinaemia (excess of hemoglobin
blood does not have enough healthy in the blood plasma) and haemoglobinuria
Red Blood cells) and (excretion of free haemoglobin in the
4. Hepatosplenomegaly (simultaneously urine). The syndrome is known as
enlargement of both the liver and the black water fever due to the dark red to
spleen) brown – black appearance of the urine
The symptoms are non – specific in this condition (Figure 8.13). It is dark
with headache, pains in back and due to presence of free haemoglobin as
limbs, anorexia, nausea and a feeling of methaemoglobin or oxyhaemoglobin in
chill rather than a distinct cold phase. it. Kidney failure is the immediate cause
Hyponatremia (A condition that occurs of death.
2. Cerebral malaria
Infobits Cerebral malaria is the most common
presentation of severe malaria in adult.
Transfusion Malaria Cerebral malaria may be sudden in onset.
Malaria can be transmitted by Clinically, the condition manifests with
transfusion of blood from infected fever for 4–5 days, slowly lapsing into
donors. First reported in 1911, coma, with or without convulsions. It is
transfusion malaria is one of the most marked by a severe headache, high fever
common transfusion-transmitted even above 180°F, and changes in mental
infections today. Blood transfusion status. Death may occur within few hours.
can accidentally transmit malaria, if Algid malaria and septicemic malaria
the donor is infected with malaria. are also other serious complication of
The parasites may remain viable falciparum malaria.
in blood bank for 1–2 weeks. As 3. Pernicious malaria
this condition is induced by direct
The term pernicious malaria is referred to
infection of red cells by the merozoites.
as a series of phenomena that occur during
Pre-erythrocytic schizogony and
the course of an in treated P. falciparum
hypnozoites are absent.
infection within 1 to 3 days.
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attacks. Persistence of the erythrocytic cycle
of the parasites are called recrudescences.
In P. falciparum infections, recrudescences
are seen for 1–2 years, while in P. malariae
infection, they may last for long periods,
even upto 50 years.
Infobits
The global technical strategy for

malaria 2016-2030 was adopted by
the World health Assembly in May
2015. It provides a comprehensive
framework to guide countries in their
efforts to accelerate progress towards
Figure 8.13: Urine in Black water Malaria malaria elimination. The strategy sets
the target of reducing global malaria
4. Anaemia: An individual suffering incidence and mortality rates by at
from an attack of malaria, after a few least 90% by 2030.
paroxysms becomes temporarily anaemic.
The reduction in red blood cells is greater 8.5.11 Plasmodium vivax
in P. falciparum infection than in infection
P. vivax shows a similar life cycle in
with P. vivax and P. malariae. This is
humans and mosquitoes like that of P.
because P. falciparum invades young and
falciparum. Except in P. vivax, a latent
mature erythrocytes and the infection rate
tissue stage, the hypnozoites present in
of red blood cells is also greater.
the liver parenchyma.
HOTS Relapse in vivax malaria is caused by

these hypnozoites. Hypnozoites are the
dormant stages of the parasites. These are
Which stage is infective in Blood
single – nucleated parasites measuring
transfusion malaria?
4µm–6µm in diameter. These become
active and develop into tissue schizonts
8.5.10 Recrudescence after a short period of dormancy. This
relapse may occur at intervals up to
In P. falciparum and P. malariae infections
3 years or more after the first attack.
after the primary attack, sometimes there is
P. vivax merozoites invade only young
a period of latency, during which there is no
erythrocytes and the reticulocytes.
clinical illness. But some parasites persist in
some erythrocytes and gradually increase 8.5.12 Clinical Manifestations
in numbers. Fresh malarial attacks then
develop. It appears after a period of latency P. vivax is the most wide spread species
usually within weeks after the primary causing malaria in man. However, unlike
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Table 8.2: Comparison of course of infection – P. falciparum and P. vivax in man
Stage P. falciparum P. vivax
Pre – erythrocytic Stage lasts for 6 days. Lasts for 8 days. Each Schizont
schizogony Each Schizont produces produces about 12,000
about 40,000 merozoites approximately
approximately
Erythrocytic schizogony Each cycle lasts for 36–48 Each cycle lasts for 48 hours.
hours. First temperature First fever peak occur by 16th day
peak occurs by 12thday of infection. Primary attack lasts
of infection. Primary for 3–4 weeks.
attack last for 10–14
days.
Gemotogony Gametocytes in Gametocytes in peripheral
peripheral blood may blood may be seen on 16th day of
be seen on 21st day of infection.
infection.
Exo – erythrocytic Absent. Present. Can continue for up to
schizogony Relapses do not occur 3 years. Relapses often occur.
falciparum malaria, vivax malaria, is

less severe and death from the condition
relatively is less common. Table 8.2
describes the comparison of course of
infection in Falciparum malaria with
Vivax malaria

Diagnosis of malaria includes:
a. Parasitic diagnosis
Figure 8.14: Blood smear
b. Serodiagnosis, and
c. Molecular diagnosis
thick smears (Figure 8.14). Ring forms
Parasitic diagnosis – Demonstration of and gametocytes are most commonly seen
parasite by microscopy in the peripheral blood smear.
Specimen: Blood Thin smear
Conventional light microscopy of stained They are prepared from capillary blood of
blood smear is the gold standard for fingertip and spread over a good quality
confirmation of malaria. slide by a second slide (spreader slide) held
Two types of smears are prepared from at an angle of 30°–45° from the horizontal
the peripheral blood. They are thin and such that a tail is formed.
178

Thin smears thus prepared are air microscope. Acridine orange – stained
dried, fixed in alcohol and stained by malaria parasites appear brilliant green.
one of the Romanowsky stains such as
Serodiagnosis
Leishman, Giemsa or JSB (Jaswant singh
and Bhattacharjee) stain. It is not helpful in clinical diagnosis. It is
used mainly for epidemiological survey
Thin smears are used for:
and to identify the infected donors in
a. Detecting parasites, and transfusion malaria. The test used are
b. For determining the species of the indirect haemagglutination (IHA), Indirect
infecting parasite. fluorescent antibody (IFA) and Enzyme –
linked immunosorbent assay (ELISA) for
Thick smear
the defection of serum antibodies.
They are prepared usually with 3 drops
Rapid Antigen defection tests kits are
of blood spread over a small area of
available commercially like the dipstick,
about 10mm. The thick film is dried.
card and cassette bearing monoclonal
This smears consist of a thick layer of
antibody. These tests are based on the
dehemoglobinized (lysed) red blood cells.
detection of antigens using immune
It is not fixed in methanol.
chromatographic methods. These tests
Thick film is stained similar to thin can detect plasmodium in 15 minutes.
film. Thick smears have the advantage of
concentrating the parasites and therefore Molecular diagnosis
increase the sensitivity of diagnosis. Thick DNA probe and PCR are highly sensitive
smears are used for: methods for the diagnosis of malaria. It
a. Defecting parasites, is more sensitive than that of thick blood
b. Quantitating parasitaemia, and smear. It is highly species specific.
c. Demonstrating malarial pigments. Other tests includes the measurement
of hemoglobin, total WBC and platelet
Fluoroscence microscopy count in severe falciparum malaria, urine
The method is mainly used for mass can be tested for free hemoglobin, if black
screening in field laboratory. Fluorescent water fever is suspected. Blood urea and
dyes like acridine orange is used to stain the serum creatinine has to be monitored for
blood smears. It stains DNA as fluorescent renal failure.
green and cytoplasmic RNA as red.
8.5.14 Treatment
QBC (Quantitative Buffy coat smear)
This is a sensitive method for detection of The most commonly used drugs are
malaria parasites. In this method, blood Chloroquine, Quinine, Pyrimethamine
is collected in a capillary tube coated and Doxycycline.
with fluorescent dye and is subjected to
centrifugation. After centrifugation, the 8.5.15 Prevention and Control
Buffy coat in the centrifuged capillary The preventive measures to control
tubes is examined under a fluorescent malaria mainly depend on treatment of
179

infected individuals and reducing the
transmission of malaria. The roundworm,
Ascaris lumbricoides
The control measures include the
is the largest
use of insecticides such as DDT (Di
nematode parasite
chlorodiphenyl tri chloromethane) or
in the human intestine. An editorial
Malathion for controlling the populations
in the lancet in 1989 observed. That
of adult mosquitoes.
if all the round worms in all people
Proper use of mosquito nets, worldwide were placed end-to-end.
wearing protective clothings and use They would encircle the world
of mosquito repellants can prevent the 50 times. Soil-transmitted intestinal
mosquito bite. nematodes are called Geohelminths.
Introduction to Helminths
General characteristics of Helminthic earthworm in Latin. Male and Female
parasite: worm of Ascaris lumbricoides are shown
1. Helminths are multicellular worms. in Figure 8.15.
They are bilaterally symmetrical
animals having 3 germ layers and
belong to the kingdom Metazoa.
2. They are invertebrates characterised
by elongated, flat or round bodies.
3. Helminths develop through egg, larval
and adult stages. Flowchart 8.1 describes
the classification of helminthes.
8.6 Nematode: Ascaris Lumbricoides

8.6.1 Geographical Distribution Figure 8.15: Adult worms of Ascaris
It is the most common of human Lumbricoides
helminthes and is distributed worldwide.
•• They are large cylindrical worms
with tapering ends. The anterior end
8.6.2 Habitat
being thinner than the posterior end.
The adult worms lives in the small intestine It is the largest intestinal nematode
particularly in jejunum and in ileum. parasitizing man.
8.6.3 Morphology •• The life – span of the adult worm is

less than a year.
Adult worm
Ascaris lumbricoides resembles and Male worm
sometimes confused with the earthworm. •• The adult male worm is smaller than
Its specific name lumbricoides means female worms.
180

•• The tail – end (Posterior end) of the Fertilized Egg
male worm is curved ventrally to form a •• The fertilized eggs are produced by
hook and 2 curved copulatory spicules. fertilized females.
Female worm •• The eggs are round or oval in shape
•• The adult female worm is larger and measures 45 µm in length and
(20–40 cm) and thicker (3–6 mm) than 35µm to 50µm in breadth.
male worm. • • They are bile – stained and appear
•• The posterior end is conical and as golden brown (brownish) in
straight. The anus is in the sub terminal colour.
part and opens like a transverse slit on •• The egg is surrounded by a thick
the ventral surface. smooth shell with an outer albuminous
•• The vulva is situated mid – ventrally, coat (corticated eggs). Sometimes this
near the junction of the anterior and outer coat is lost in few eggs. Those
middle thirds of the body. This part of eggs are called as decorticated eggs
the worm is narrow and is called the (Figure 8.16).
vulvar waist. •• Each egg contains a large unsegmented
•• A single worm lays up to 200,000 eggs ovum with a clear crescentic area at
per day. each pole. The eggs float in saturated
solution of common salt.
Egg: Two types of eggs are passed in feces
by the worms.
Figure 8.16: Fertilized and Unfertilized egg of Ascaris lumbricoides

181

Unfertilized egg 8.6.4 Life – Cycle
•• The female even not fertilized by male The life – cycle of A. lumbricoides is
is capable of liberating eggs. These completed in a single host, human
unfertilized eggs are narrower, longer (Figure 8.17).
and elliptical in shape.
Infective form: Ermbryonated
•• These are heaviest of all the helminthic eggs. The fertilized egg passed in feces
eggs – It measures about 80µm × is not immediately infective. It has to
105µm in size. undergo a period of development in soil.
•• The eggs have a thinner shell with The development usually takes from
an irregular coating of albumin 10–40 days. The embryo moults twice
(Figure 8.16). during the time and becomes the infective
rhabditiform larva.
•• These eggs do not float in saturated
solution of common salt. Mode of transmission: Man acquires
the infection by ingestion of food, water
HOTS or raw vegetables contaminated with
embryonated eggs of the round worm.
What makes worm’s egg float or sink? The ingested eggs reach the duodenum
to liberate the larvae by hatching. These
Figure 8.17: Lifecycle of Ascaris lumbricoides

182

larvae then penetrate the intestinal wall worms can cause obstruction and
and are carried by the portal circulation to inflammation of intestinal tract,
the liver. They live in liver for 3 to 4 days. particularly of the terminal ileum.
Then they are carried to the right side of d.
Ectopic ascariasis (Wander lust)
the heart, then to lung. In the lung, they is due to the adult male worms.
grow and moult twice. They are restless wanderers. The
After development in the lungs, in wandering happens when the host
about 10–15 days, the larvae pierce the temperature rises above 39°C. The
lung capillaries and reach the alveoli. worm may wander up or down along
Then they are carried up the respiratory the gut. It may enter the biliary
passage to the throat and swallowed back or pancreatic duct causing acute
to the small intestine. biliary obstruction or pancreatitis. It
In the small intestine, the larvae moult may enter the liver and lead to liver
finally and develop into adults. They abscesses. The worm may go up the
become sexually mature in about 6–12 esophagus and come out through
weeks. The fertilized female start laying mouth or nose. It may crawl into
eggs which are passed in the faces to the trachea and the lung causing
repeat the cycle. respirator obstruction or lung
abscesses. Migrating downwards,
8.6.5 Pathogenesis the worm may cause obstructive
Infection of A. lumbricoides in human appendicitis. The worm may also
is known as ascariasis. The adult worm reach kidneys. “Larva migrans” is a
may produce its pathogenic effects in the term used when the larval sworms
following ways. migrate to various parts of the body.
a.
The spoliative or nutritional 8.6.6 Clinical Manifestations
effects is usually seen when the
Incubation Period is 60–70 days. Clinical
worm burden is heavy. Presence
manifestations due to adult worm vary
of enormous numbers (sometime
from asymptomatic to severe and even
exceeds 500) often interferes with
fatal infection. Clinical manifestation
proper digestion and absorption of
in ascariasis can be caused either by the
food. Ascariasis may contribute to
migrating larvae or by the adult worms.
protein – energy malnutrition and
vitamin A deficiency. Symptoms due to the migrating
larvae: leads to ascaris pneumonia and
b.
The toxic effects is due to the
larvae may enter the general circulation,
metabolites of adult worm. Ascaris
disturbances have been reported in the
allergens produce various allergic
brain, spinal cord, heart and kidneys.
manifestations such as fever,
urticaria and conjunctivitis. Symptoms due to the adult worms:
Diffuse or epigastric abdominal pain,
c.
The mechanical effects are the
abdominal cramping, abdominal swelling
most important manifestations of
(especially in children), fever, nausea,
ascariasis. In heavy infections, adult
183

vomiting and passing roundworms and
their eggs in the stool. The National
Deworming Day
8.6.7 Laboratory Diagnosis (Febuary 10th) is an
initiative of India
Specimen collected: Stool, sputum and to make every child in the country
blood. worm free. This is one of the largest
Detection of parasite public health programs reaching large
number of children during a short
Adult worm: It can be detected in stool or
period.
sputum of patient by naked eye. Pancreatic
or biliary worms can be detected by More than 836 million children
ultra-sound and endoscope. are at risk of parasitic worm infections
worldwide. According to World Health
Larvae: Larvae can be detected in
Organization 241 million children
sputum and often in gastric washings.
between the ages of 1 and 14years are
Chest X – ray may show pulmonary
at risk of parasitic intestinal worms in
infiltrates.
India, also known as Soil-Transmitted
Eggs: Detection is through Helminths (STH).
demonstration of eggs in feces. Detection
of both fertilized and unfertilized eggs
are made after staining. Eggs may be
b. Avoid eating of uncooked green
demonstrative in the bile obtained by
vegetable, food preparation and fruits
duodenal aspirates.
that may contain faecal eggs.
Blood Examination c. Treating infected persons especially
Complete blood count may show children. Deworming of school
eosinophilia in early stage of infection. children have been found effective in
control of ascariasis.
Serological tests
Ascaris antibody can be detected by IHA, Summary
IFA and ELISA
Medical Parasitology deals with the
8.6.8 Treatment study of parasites infecting humans. The
diseases caused by them and the clinical
Commonly used drugs are Albendazole manifestations produced in infected
and Mebendazole. humans. It is also concerned with various
methods of their diagnosis, treatment
8.6.9 Prevention and Control and their prevention and control. There
a. Proper health education should be are different types of parasites and hosts.
given for improved sanitation and Parasites lives on its host for its nourishment
personal hygiene. and survival. The relationship between
host and the parasite can be symbiotic,
184

commensalic or parasitic. Parasites of donovani, the unicellular tissue flagellatis
medical importance comes under the causes Leishmaniasis. Plasmodium spp., the
kingdom called Protista and Animalia. protozoan parasite which causes malaria are
Protista includes the microscopic single transmitted by female Anopheles mosquito
– celled eukaryotes known as protozoa. carring sporozoites forms of the parasite.
In contrast, helminths are macroscopic, The four species infective to humans are
multicellular worms possessing well P. falciparum, P. malariae, P. vivax and
differentiated tissues and complex organs P. ovale. Multicellular organisms and
belonging to the kingdom Animalia. intestinal worms. The helminths such as
Protozoa includes Entamoeba and Ascaris lumbricoides causes Ascariasis, an
Giardia which cause intestinal infections infection of the small intestine. Ascariasis
(dysentery and diarrhoea) Leishmania is the most common roundworm infection.
185

Evaluation c. Ascaris
Multiple choice questions d. Taenia
1. A host in which a 7. Which of the following parasitic
parasite undergoes infection can lead to malabsorption
asexual reproduction syndrome.
is . a. Amoebiasis
a. Definitive host b. Ascariasis
b. Intermediate host b. Hookworm infection
c. Reservoir host d. Giardiasis
d. perfect host 8. Flask shaped ulcers in human
intestine are related to .
2. Which of the following statement
is true concerning Entamoeba a. Giardiasis b. Amoebiasis
histolytica. c. Leishmanian d. Chaga’s disease
a. It has no cystic stage 9. This disease was first observed by
b. It is non – pathogenic Leeuwenhoek often he discovered
parasitic organisms in his stool under
c. It is not transmitted through faecal
the microscope.
– oral route
a. Chaga’s diease b. Gardiasis
d. Trophozoites live in large intestine
of human c. Malaria d. Ascariasis
3. Animals that are routinely infected 10. The common name for
with a protozoan or parasite are A. Lambricoides is round worm.
termed as . a. Round worm b. Pin worm
a. Definitive b. Intermediate c. Tape worm d. Whip worm
c. Reservoir d. Parasite
Answer the following
4. The schizonts enter which body part?
1. Which laboratory findings are
a. Blood stream b. Spleen
diagnostic for leishmaniasis?
c. Mouth d. Liver
2. How is amoebiasis diagnosed.
5. Leishmania organism are transmitted 3. Following ingestion, what is the life
to human by cycle of E.histolytica?
a. Sandflies b. tsetse files 4. What is the clinical spectrum of
c. Mosquitoes d. Reduviid bug amoebiasis?
6. The parasite is capable 5. What is the role of microscopy in the
of producing disease in an immune diagnosis of amoebiasis.
deficient host. 6. What is ALA?
a. Entamoeba histolytica 7. How Female Ascaris worm is
b. Toxoplasma gondii differentiable from male worm?
186

8. Why do some parasites need 12. With neat diagram describe the
definitive and intermediate hosts trophozoite of Giardia.
rather than just one host to complete 13. Explain the erythrocytic stage of
its life-cycle. Plasmodium falciparum.
9. What is the difference between 14. What complication arises due to
reservoir and paratenic hosts? inadequately treated patient with
10. Why is the mosquito a definitive host quinine suffering from malignant
in malaria? malaria?
11. A fecal sample was 15. Describe the life-cycle of large
subjected to Saturated roundworm which grows to a length
salt flotation from of up to 40cm that infects humans.
a 14 year old boy 16. For which parasite mosquito acts as
according to doctor’s definitive and intermediate host?
advice and check-up.
17. Fill in the blanks in the column given
The results of this test
below.
are shown under the microscope. Identify
the parasite egg and comment on it.
Human Nematode Infection

Parasite Acquired by Site in humans Transmitted
through
Ascaris Ingestion of eggs Person to person
lumbricoides
187

Chapter
9 Medical Mycology
The branch of biology

Learning Objectives that deals with the study
After studying this chapter the of fungi is known as
students will be able to, “Mycology”. The term
is derived from Greek
• Identify the pathogenic fungi most
work ‘Mykes’ means
commonly causing disease by
mushroom and ‘Logos’ means study.
using advanced techniques. Fungal
Medical Mycology is the study of fungal
infection is common in developing
epidemiology, ecology, pathogenesis,
countries so we should be aware
diagnosis and treatment in human
to prevent and treat the fungal
beings. Raymond Jacques Sabouraud
infection.
(1864–1936) is the father of Medical
• Study the taxonomy, structure Mycology.
and classification of medically
important fungi. 9.1 Classification of Fungi based on
• Study about the mycoses and its the Host Parasitic Relationship
pathogenesis, clinical feature, and
Based on the host parasitic relationship
its treatment and prophylaxis.
the fungi are grouped into three types.
• Study about the collection,
processing of the sample and its a. C ommensalism: The fungus neither
molecular diagnosis. gets benefit nor harmed by the host
parasitic relationship.
b. Mutualism: The fungus benefited from
the host parasitic relationship.
Chapter Outline
c. Parasitism: The host is harmed by the
9.1 Classification of fungi based on the fungus in host parasitic relationship.
Host parasitic Relationship Based on their wide spectrum of
9.2 Superficial Cutaneous Mycosis adaptability, fungi causing human mycoses
can be categorized into:
9.3 Subcutaneous Mycoses
a. Pathogenic fungi: The ability of the
9.4 Systemic Mycosis fungi to adapt to skin flora and cause
9.5 Opportunistic Mycosis infection.
188

b. Opportunistic fungi: When the Aeromycology
immune status of the host is reduced, The Aeromycology is the study of air
fungi will induce or cause infection. borne fungi, its types and the seasonal
c. Toxigenic fungi: Toxins produced by variations of allergenic fungal spores in
fungi are responsible for the illness or the environment.
death of patients after ingestion of the There are certain fungal pathogens
contaminated food. which cause infections associated with
d. Allergenic fungi: Allergens are secreted workers in mycological laboratories.
by the fungi which cause allergic To avoid this safety procedures and
reaction in the human beings. equipments safety levels or bio safety
levels (BSL) are used. BSL - 1 is used for
9.1.1 Mycoses low - risk microorganisms and BSL - 4 is
Diseases caused by the medically used for highly risk pathogens.
important fungi are called Mycoses.
Mycoses are classified according to the Infobits
specific site of involvement.
a. Superficial Mycoses: The infection Medical Mycology in India
is limited to the outer most layers In India, the fungal infections are
of the skin and its appendages. known since the ancient civilization
Example: Malassezia and Piedra mentioned in Aryan documents
such as Atharva Veda Mycetoma is
infection
described as Padavamikam meaning
b. Cutaneous Mycoses: The infection ant hillfoot this was observed by
extends deeper into the epidermis John Gill in Madurai district of Tamil
and it also invades hair and nails. Nadu in 1842 which was designated as
Example: Dermatophytoses. ‘Madura foot’.
c. Sub cutaneous Mycoses: The infection
extends to dermis, subcutaneous tissue 9.1.2 Characteristics of Fungi
and muscles by any traumatic injury. Fungi are heterotrophic organisms that
Example: Mycetoma exist as saprophytes, commensal or
d. Systemic Mycoses: The infection parasites. They are found on decaying
originates from lungs and later spreads vegetative matter and also in soil.
systemically to other organs. Systemic Morphological features, cell structure,
mycoses along with the opportunistic reproduction, nutritional requirement and
fungal infection are known as deep thermal dimorphism in the pathogenic
mycoses. Example: Cryptococcosis fungi are described as follows:
e. Opportunistic Mycoses: The infection
occurs when the immune status of the i. Morphological Features
individuals is altered. It is common Fungi are eukaryotic with well defined
among immune compromised cell wall and internal membrane bound
and immune suppressed patients. organelles. The cell wall is composed of
Example: Candidiasis polysaccharides and chitin. A fungus
189

varies in size and shape. They are broadly specialized reproductive structures
divided into two main groups. called as conidia.
a. Yeasts: The yeasts are unicellular
organisms which reproduce by asexual HOTS
process known as budding or by fission.
The cell develops a protuberance Can you cultivate the molds at home?
that enlarges and separates from the
parental cell. The yeasts produce Depending on cell morphology
chains of elongated cells known as fungi are divided into four types,
Pseudohyphae. Some yeasts are they are Yeasts: These are unicellular
reproduced by sexual process Example: organisms that divide by budding
Cryptococcus neoformans. Germ tube is (Figure 9.1a & b). Example: Cryptococcus
special morphology found in Candida neoformans (Pathogenic), Saccharomyces
albicans. Some are commensal without cerevisiae (Non pathogenic).
any medical significance. Yeast – like fungi: These fungi reproduce
b. Molds: The molds grow by apical by budding but fails to separate and
extension, forming an interwoven mass hence elongation takes place forming
called as Mycelium, branching filaments pseudohyphae. Example: Candida
known as hyphae. Hyphae that grow species (Pathogenic). Molds: These
on the surface are called vegetative fungi produce spores which germinate
hyphae. They are responsible for the to form vegetative hyphae (Figure 9.2).
absorption of nutrients. The hyphae Example: Dermatophytes, Aspergillus,
that project above the surface are Penicillium, Mucor. Dimorphic
called aerial hyphae and they produce fungi: These Fungi exist in both
yeast at 37°C and filamentous form
at 25°C. This Phenomenon is known
as Fungal dimorphism (Figure 9.3).
Example: Histoplasma capsulatum,
Blastomyces dermatitidis.
Figure 9.1: (a) Morphology of yeast (b) Microscopic view of yeast

190

complex cytosol. It is composed of
g lycoprotein, lipids and ergosterol.
d. Cytosol: Cytosol comprises of
mitochondria, microtubules ribosome’s,
golgi apparatus, double membrane
endoplasmic reticulum and Nucleus.
The nuclei of the fungi are enclosed
by a membrane and contain most of
cellular DNA.
iii. Reproduction of fungi

Figure 9.2: Microscopic view of yeast Spores play a major role in reproduction.
Moulds
There may be asexual or sexual cell
divisions.
Phaeoid fungi: Most of true pathogenic
a. Asexual Reproduction: The asexual
fungi are dimorphic fungi which are
reproduction involves, budding or
composed of darkly coloured hyphal form
fission or mitosis. Fungi produce more
known as dematiaceous fungi. Some are
than one type of asexual spores. They
yeast like and also known as black yeasts.
are microspores (micro conidia) and
Vegetative Structures: Several macrospores (macro conidia). Spores
structures are formed by the vegetative that are present inside sporangium are
mycelia that have no reproductive value
but are important for the differentiation
Dimorphic fungi
of fungi eg. Chlamydospores and
25°C 37°C
Arthrospores. Chlamydospores are thick
Blastomyces
walled, resistant to adverse conditions and dermatitidis
are larger than other cells. Arthrospores
are rectangular spores which are thick
walled that are disposed on maturity.
Coccidiodes
ii. Cell structure immois
a. C apsule: Fungi produce an extra

cellular polysaccharide in the form of
capsule. Example: Cryptococcus.
Histoplasma
b. Cell wall: Fungi possess a multilayered capsulatum
rigid cell wall exterior to the plasma

lemma. The cell wall is made up of chitin,
a water insoluble, homo polymer of Paracocoididodes
N-acetyl glucosamine. Chitin synthase is brasilensis
responsible for the bio synthesis of chitin.

c. Plasma lemma: Cytoplasmic mem-
brane or plasma lemma encloses Figure 9.3: Dimorphic Fungi
191

Sporongiosphere Conidiospores or Conidio
Sporongium
Conidiospores
Sporongiosphere
Phiolides
Sporongiosphere Conidiospores
Cosnocylic
hyphae
Rhizoids
Figure 9.4: Asexual Spores of fungi
known as sporangiospores and those fungi is between 25°C and 37°C. The fungi
that are borne exogenously are called prefer acidic pH; do not require light for
conidiospores (Figure 9.4). Based on their growth. All fungi are heterotrophs
the arrangement of conidia they are requiring organic nutrients. They absorb
classified as Acropetal, Basipetal and their nutrient and do not ingest food.
Sympodial. Medically significant fungi are facultative
b. S exual Reproduction: The process of parasites, capable of causing disease or
sexual reproduction typically consists living on dead organic matter.
of plasmogamy (cytoplasmic fusion),
Karyogamy (union of two nuclei) 9.2 Superficial Cutaneous Mycoses
and meiosis (haploid formation). The superficial cutaneous fungal
Anamorphs and Telomorphs are the
infections involve the outer most layers
2 phases of sexual reproduction
of skin and its appendages like hair and
c. Mycelia Sterile: Mycelia sterile are fast nails. The causative agents colonize on
growing molds that do not produce epidermis or supra - follicular portions
spores or conidia. They are medically of hair and do not penetrate into deeper
significant fungi and are difficult to layers.
identify
The genus Malassezia is responsible
iv. Growth and nutrition for the superficial infection of the skin.
Fungi are ubiquitous in nature and grow Malassezia furfur is lipophilic yeast. It
readily in the presence of nitrogen and is a commensal of normal skin in the
carbohydrates. Medically significant fungi sebaceous glands of warm - blooded
are Mesophilic. The optimum temperature vertebrates. It may be pathogenic under
invitro for majority of the pathogenic certain conditions usually causing skin
192

conditions like Pityriasis versicolor, molds. They are divided into three main
Seborrheic dermatitis, Atopic anamorphic genera depending on their
dermatitis, Malassezia folliculitis morphological characteristics.
and systemic infection. Symptoms
i. Trichophyton [Cause infection in skin,
include macular, erythematous, hyper
hair and nails]
pigmented or hypo pigmented lesions
with fine scaling. ii. Microsporum [Cause infection skin
and hair]
Tinea nigra is responsible for the
superficial cutaneous infection of the iii. Epidermophyton [cause infection skin
skin. Hortaea werneckii is the phaeoid and nail]
(dematiaceous) fungi causes infection on The fungal species affecting humans
the palms and soles. It is also commonly are known as anthropophilic. Those
termed as Tinea nigra palmaris and Tinea inhabitating domestic and wild animals as
nigra plantaris. Symptoms includes brown well as birds are called zoophilic. Fungi
to black deeply pigmented non - scaly, species from soil are known as geophilic
macular lesions affecting skin of the palms dermatophytes.
and occasionally soles.
Piedra causes superficial infection of HOTS
hair shaft. The word Piedra is derived
from Spanish word Stone. There are two What are the sources of dermatophytes?
types of Piedra based on causative fungi
and characteristics of nodules. They are
Black piedra caused by Piedraia hortae 9.2.2 Pathogenesis and Pathology
and White piedra caused by Trichosporon The dermatophytes grow within
species. The symptoms include dead keratinized tissue and produce
development of firm, irregular nodules of keratinolytic proteases, which provide
fungal elements cemented to the hair. The means of entry into living cells. Fungal
piedra can be distinguished on the basis metabolic products cause erythema,
of shape, size and pigmentation of fungal vesicles and pustule on the site of infection.
cells of nodules which are found around Some dermatophytes species like soil
hair cortex. saprobes digest the keratinaceous debris
in soil and are capable of parasitizing
9.2.1 Dermatophytoses keratinous tissues of animals.
Dermatophytoses are the most common
cutaneous fungal infection seen in man 9.2.3 Clinical Features
and animals affecting skin, hair and nails. The clinical manifestations of
The fungi can invade the keratinized Dermatophytoses are also called Tinea or
tissues of skin and its appendages and they Ringworm depending on the anatomical
are collectively known as Dermatophytes site involved. Following are the common
or Tinea or ring worm infection. The clinical conditions produced by
dermatophytes are hyaline septate dermatophytes:
193

1. Tinea Capitis: This is an infection 6. Tinea Faciei: This is an infection
of the shaft of scalp hairs. It can be of skin of face except beard.
inflammatory (eg. Kerion, Favus) Erythematous annular plaques are
or non - inflammatory (Black dot, formed. It is one of the forms of Tinea
Seborrheic dermatitis). The infected incognito.
hairs appear dull and grey (Figure 9.5a). 7. Tinea Barbae: This is the infection
Breakage of hair at follicular orifice of the beard and moustache areas of
which creates patches of alopecia with the face. This is also called barber’s
black dots of broken hair. It is caused itch. It is caused by Trichophyton
by Trichophyton species. mentagrophytes, Trichophyton
2. Tinea Corporis: This is an infection rubrum and Microsporum canis.
on the glabrous (non - hairy) Erythematous patches on the face
skin of body. Erythematous scaly with scaling appear and these develop
lesions with sharply marginated folliculitis.
raised border appear on the infected 8. Tinea Pedis: This is an infection
areas (Figure 9.5b). It is caused by of the foot, toes and interdigital
Trichophyton rubrum. web spaces. This is seen among
3. Tinea Imbricata: It forms concentric the individuals wearing shoes for
rings of scaling on the glabrous skin, long hours and known as Athlete’s
leading to lichenification. It is caused foot (Figure 9.5c). Erythema and
by Trichophyton concentricum scaling associated with itching and
4. Tinea Gladiatorum: This infection burning sensation appear with
is common among wrestlers and thin fluid discharging from small
athletes. Lesions are seen on arms, vesicles. It is caused by Trichophyton
trunk or head and neck. It is caused mentagrophytes, Trichophyton
by Trichophyton tonsurans. rubrum and Epidermophyton
5. Tinea Incognito: It is steroid modified floccosum.
Tinea caused as a result of misuse of 9. Tinea Cruris: This is an infection of
corticosteroids in combination with the groin in men who use long term
topical antimycotic drugs. tight fitting garments. Erythematous
(a) Tinea Capitis (b) Tinea Corporis (c) Tinea Pedis

Figure 9.5: Clinical conditions of Dermatophytes
194

sharp margin lesions known as Jock a. Direct Examination
itch. It is caused by Trichophyton Samples are subjected to KOH (10%) wet
rubrum and Epidermophyton mount, the affected site were disinfected
floccosum. with alcohol before collecting the clinical
10. Tinea Manuum: This is an infection specimen.
of the skin of palmar aspect of b. Fungal culture
hands. It causes hyperkeratosis of
The samples are inoculated on Sabouraud
the palms and fingers. It is caused
dextrose agar (SDA) with antibiotics
by Trichophyton mentagrophytes,
and cycloheximide and are incubated at
Trichophyton rubrum and
25°C–35°C. The colony morphology can
Epidermophyton floccosum.
be identified.
11. Tinea Unguium: This is an infection
The three genera of dermatophytes
of the nail plates. The infection
are Trichophyton, Microsporum and
spreads on the entire nail plate
Epidermophyton (Table 9.1). Based on
infecting the nail bed. It results
morphology of the macro conidia, micro
in opaque, chalky or yellowish
conidia, their shape, position on the spore
thick ended nail. It is caused by
bearing hyphae such as spiral hypha,
Trichophyton mentagrophytes,
racquet hypha, nodular pectinate body,
Trichophyton rubrum and
they are identified.
Epidermophyton floccosum.
Figure 9.6 shows the microscopic view of ii. Special Techniques
major determatophytes 1. Wood’s Lamp Examination
Clinical samples are exposed to Wood’s
Infobits lamp. Wood’s glass consists of Barium
silicate containing 9% Nickel oxide. It
How do dermatophytes cause
transmits long wave ultra violet light
disease in humans?
with a peak of 365nm that shows a
Dermatophytosis is a common characteristic fluorescence produced by
contagious disease caused by the samples. The patterns of fluorescence
fungi known as dermatophytes. are bright green, golden yellow and
Dermatophytes belong to a group coral red. Microsporum species and
of organisms that are able to break Trichophyton species are differentiated
down the keratin in tissues such as the using this technique.
epidermis, hair, nails, feathers, horns
and hooves. 2. Hair brush sampling Technique
It involves brushing the scalp with a
sterile plastic hair brush, which is then
inoculated into an appropriate culture
i. Samples medium by plates, is incubated at
Skin scrapings, hair and nail samples were 25°C–35°C. The colony morphology can
collected be identified.
195

(a) T.rubrum (b) T.mentagrophytes (c) M.canis
(d) M.gypseum (e) E.flocossum
Figure 9.6: LPCB wet mount of major Dermatophytes
Table 9.1: Microscopic and macroscopic characteristics of Dermatophytes.

Macroscopic
S.No Dermatophytes Macro conidia Micro conidia
Morphology – SDA
1. Trichophyton Rare, Abundant
thin-walled,
smooth
2. Microsporum Numerous, Rare

thick-walled,
rough
3. Epidermophyton Numerous, Absent

smooth-walled
196

9.3.1 Mycetoma
Dermatophyte
infections, also Mycetoma is a slowly progressive,
known as tinea, are the chronic granulomatous infection of
most common fungal skin and subcutaneous tissues with
infections of the skin, hair, and nails. involvement of under lying fasciae and
The term “dermatophyte” refers to bones usually affecting the extremities.
fungal species that infect keratinized Mycetoma is commonly called Madura
tissue, and includes members of the foot or Maduramycosis (Figure 9.7).
Trichophyton, Microsporum, and They are classified into two categories,
Epidermophyton genera namely eumycetoma cased by fungi
and actinomycetoma caused by higher
bacteria of class actinomycets.
3. Hair perforation Test
It is used to differentiate T. mentagrophytes 9.3.2 Pathogenesis and Pathology
and T. rubrum. Wedge-shaped perforations The causative agent of Mycetoma is
in the hair shaft are observed in hair commonly present in saprobic soil
infected with T. mentagrophytes. source and is transmitted by accidental
4. Urease Test trauma by thorns or by injury into the
subcutaneous tissue. It is common
It is used to differentiate between
among farmers with minor trauma and
T. mentagrophytes and T. rubrum.
abrasions of the skin. Use of wicks for
T. mentagrophytes hydrolyzes urea and
removal of earwax is responsible for
becomes deep red, showing positive
Mycetoma of the ear.
result.
iii. Treatment HOTS
Whitfield’s ointment is used for all Tinea
infections. Oral griseofulvin is the drug Is mycetoma occupational disease?
of choice for nails and scalp infections.
Itraconazole and terbinafine may be given
as pulse therapy.
9.3 Subcutaneous Mycoses

The fungal infections are characterized
by development of lesions at the
site of infection by the traumatic
inoculation in the subcutaneous
tissues. Examples are Mycetoma,
Sporotrichosis, Chromoblastomycosis
and Rhinosporidiosis. Figure 9.7: Madura foot
197

9.3.3 Classification of Mycetoma KOH mount
The Mycetoma is classified on the Eumycotic grains show thick 2–6 µm
basis of the causative agent aerobic hyphae with large globose swollen
actinomycetes causes actinomycetoma cells with or without chlamydospores.
whereas hyaline and phaeoid fungi Actinomycotic grains show thin filaments
cause eumycetoma. of 0.5–1 µm with coccoid or bacillary
forms.
9.3.4 Clinical Features
Gram stain
The clinical entity depends upon the age of
Actinomycetoma grains show Gram-
the lesions and to size, shape and color of
positive branching filamentous bacteria
the grains. The painless localized swollen
with branches (Figure 9.8b).
lesions with purulent fluid lead to the
secondary bacterial infections. Important Ziehl - Neelson stain
features of Mycetoma are as follows: Nocardia species show red pink acid fast
i. Tumor like swelling filamentous bacteria.
ii. Multiple draining sinuses b. Culture
iii. Presence of grains or granules in sinuses. Crushed grains are washed several times
with normal saline without antibiotics
and inoculated on to Sabouraud dextrose
i. Samples agar, blood agar, Lowenstein -Jonson
The clinical sample in Mycetoma is media and brain-heart infusion agar. The
usually grains, pus exudates or biopsy plates are incubated at 25°C, 37°C and
were collected. 44°C for various organisms (Figure 9.8c).
a. Direct Examination ii. Treatment

Grams staining, modified Ziehl – Neelson 1. Ketoconazole 200 mg and Itraconazole
staining, LPCB and KOH wet mount are 100mg are given for 8–24 months to
used to visualize the organisms. treat eumycetoma.
The grains should be washed, crushed 2. Sulfonamides, tetracylines, streptomy-
and cultured on different media. Crushed cin, amoxicillin are administered to
grains are examined (Figure 9.8a). treat actinomycetoma.
(a) Biopsy-Black grains (b) Microscopic Morphology (c) Macroscopic Morphology

Figure 9.8: Laboratory Diagnosis of Mycetoma
198

9.4 Systemic Mycoses 9.4.3 Clinical Features
Systemic mycoses are caused by The disease is mostly asymptomatic. The
dimorphic fungi; these infections are development of symptom or symptomatic
acquired by inhalation of spores. These disease appears to depend on the intensity
primarily involve the respiratory system of exposure to conidia and cellular
and are self-limiting and asymptomatic. immune response of the host. The disease
If symptomatic, it spreads to other parts may be classified as follows.
of body through circulation. These 1. Acute pulmonary Histoplasmosis –
infections are caused by true fungal Fever, headache, chills, sweating, chest
pathogens. Systemic and opportunistic pain, cough and dyspnoea
infections together caused Deep
mycoses. 2. Chronic pulmonary Histoplasmosis –
Ulcerative lesions of the lips, mouth,
The organisms have a mycelial form
nose, larynx and intestines
when grown on fungal culture and have
yeast form in the tissue. The examples 3. Cutaneous, mucocutaneous
of systemic mycoses are Histoplasmosis, Histoplasmosis – Mucous lesions on
Blastomycosis. skin, abdomen wall and thorax.
4. Disseminated Histoplasmosis – Fever,
9.4.1 Histoplasmosis anoxia, anemia, leucopenia constant
Histoplasmosis is caused by dimorphic hepatosplenomegaly and multiple
fungus Histoplasma capsulatum. lymphadenopathies.
The fungi live inside the cells of the
reticuloendothelial system, where they 9.4.4 Laboratory Diagnosis
grow within macrophages and giant cells. i. Samples
This infection is also known as Darling’s Specimens collected are sputum, bone
disease. marrow and lymph nodes, cutaneous and
mucosal lesions and peripheral blood film.
9.4.2 Pathogenesis and Pathology
The infection with H. capsulatum a. Direct Examination
develops when conidia or mycelial Thick and thin smears should be prepared
fragments are in haled and converted from peripheral blood, bone marrow and
into yeasts in alveolar macrophages in stained with Calcofluor white, Giemsa or
the lungs. The oval yeast cells parasitize Wright stains.
macrophages, which are activated by The fungus is small, oval yeast like
T lymphocytes resulting in localized cells, 2–4 µm in diameter, within the
granulomatous inflammation. mononuclear or polymorpho nuclear cells
and occasionally in giant cells.
HOTS
b. Fungal culture
H. capsulatum is dimorphic fungi - justify The clinical samples is inoculated on
Sabouraud dextrose agar (SDA) and
199

Brain-heart infusion (BHI) agar with
antibiotics and actidione at 25°C and The fungus candida
37°C. On Sabourad dextrose agar the albicans is responsi-
colonies appear albino or brown. The ble for most vaginal
yeast infections. Your
albino type consists of white, fine aerial
vagina naturally contains a balanced
hyphae and brown type consists of flat
mix of yeast, including candida, and
colonies with light tan or dark brown in
bacteria. Certain bacteria (lactoba-
color in seven days. At 37°C the colonies
cillus) act to prevent an overgrowth
grow as granular to rough, mucoid and
of yeast. But that balance can be
cream-colored turning tan to brown in
disrupted.
14 days.
ii. Treatment Pathogenesis and Pathology

Amphotericin B is given for the treatment
Some of the virulence factors contributing
of disseminated and other severe forms of to pathogenicity are toxins, enzymes and
Histoplasmosis. adhesion. The organism adheres to the
epithelial and endothelial cells by proteinase
9.5 Opportunistic Mycoses production. Then the yeast cells of Candida
The opportunistic systemic mycoses encounter a particular host tissue and
are infections found in patients with colonization takes place at the local site or
underlying pre disposing conditions. they invade deeper into the host tissue and
It is produced by non pathogenic or induce various clinical symptoms.
contaminant fungi in a host, where the
immunological defense mechanisms are Clinical Features
weakened by endogenous causes like The Candida species are found as commensal
cancer, leukemia or exogenous causes on mucosal surfaces of the body. They
like immunosuppressive therapy and cause disease as and when conditions are
AIDS. The examples of opportunistic favourable. This yeast like fungi colonizes
mycoses are Candidiasis, Cryptococcosis, mucocutaneous surfaces, which can be
Aspergillosis and zygomycosis. portals of entry into deeper tissues when
the host defenses are compromised. They
9.5.1 Candidiasis may cause a simple lesion to event the life
Candidiasis is the commonest fungal threatening systemic infection.
disease found in humans affecting mucosa, The clinical manifestations of
skin, nails and internal organs of the Candidiasis are divided into two broad
body. It is caused by yeast like fungi called categories. They are:
Candida albicans. The infection may be
acute or chronic, superficial or deep and 1. Infectious Diseases
found mainly as secondary infection in a. Mucocutaneous Involvement
individuals with immune compromised i. Oral Candidiasis – Most common
condition. form Candida colonizes on the oral
200

2. Allergic Diseases
Allergic manifestation is caused due to the
metabolites of Candida. The cutaneous
allergies are urticaria and eczema, and
bronchial asthma.
Laboratory Diagnosis
i. Samples
Specimens collected are mucous membrane
Figure 9.9: Oral candidiasis from the mouth, vagina, skin and sputum
based on the site of involvement.
cavity (oral thrush) infection on the
buccal mucosa, gums, tongue, reddening a. Direct Examination
of the mucous membrane gives dry,
Gram staining LPCB, and KOH wet mount
smooth metallic taste and burning at the
are used to visualize the yeast cells.
local site (Figure 9.9).
Presence of yeast cells approximately
ii. Alimentary Candidiasis – Candida
4.8 µm with budding and pseudo
colonizes on the oesophagus causing
hyphae are observed. Other stains like
oesophagitis. It is mostly asymptomatic
periodic acid - Schiff stain and Gomori’s
or it may cause burning pain in the
methylamine silver stain are also used to
epigastrium or throat.
observe the fungal elements in tissue.
b. Cutaneous Dermatitis
b. Fungal culture
i. Diaper Dermatitis – Candida colonize
The clinical specimens can be cultured
on the cutaneous layer causes cutaneous
on Sabouraud dextrose agar (SDA) with
Candidiasis leads to maculopapules
antibiotics and incubated at 25°C and
vesicles with erythematous rash. This is
37°C (Figure 9.10). The colonies appear
common among infants and known as
in 3–4 days as cream coloured, smooth
Diaper rash.
and pasty.
ii. Intertrigo – This is an inflammatory
lesion of the skin folds due to candidal The some of the species of Candida
infection. are Candida albicans, Candida tropicalis,
Candida krusei and Candida glabrata.
c. Systemic Involvement
ii. Special Test
The Candida colonizes in various organs
and causes various manifestations Germ tube test
through the blood stream. Clinical The culture of Candida species is treated
features are found to be Urinary tract with sheep or normal human serum
Candidiasis, Candiduria, Endocarditis, and inoculated at 37°C for 2 to 4 hours.
Pulmonary Candidiasis, Arthritis, A drop of suspension is examined on the
Osteomyelitis, Meningitis, Candidemia slide. The germ tubes are seen as long
and Septicemia. tube–like projections extending from
201

Figure 9.10: Candida (a) Macroscopic Morphology (b) Microscopic Morphology
(c) Germ tube
the yeast cells. The demonstration of the neoformans. It is pathogenic to man and
germ tube is known as Reynolds – Braude animals. It causes opportunistic infection,
phenomenon. involving the lungs and disseminates to
Biochemical tests extra pulmonary sites through circulation
to different body organs particularly
Sugar fermentation and assimilation tests
to central nervous system causing
are used for the identification of Candidal
species. C.albicans ferments Glucose and Meningoencephalitis.
Maltose and assimilates Glucose, Maltose,
Sucrose, Lactose and Galactose. Infobits
Chlamydospores formation What does Cryptococcus cause?

Candida isolates are grown on corn meal, Meningitis can be caused by different
agar (CHN) or rice starch agar (RSA) germs, including bacteria, fungi,
and incubated at 25°C for 2–3 days. The and viruses. Two types of fungus can
formation of large, thick walled terminal cause cryptococcal meningitis (CM).
chlamydospores is demonstrated in They are called Cryptococcus
C.albicans and C. dubliniensis. neoformans (C. neoformans) and
Cryptococcus gattii (C. gattii). This
iii. Treatment disease is rare in healthy people.
1. 1% gentian violet is locally applied to
the affected areas.
Pathogenesis and Pathology
2. The azole creams like Clotrimazole,
Miconazole, Ketoconazole and Cryptococcal infection occurs
Econazole are also used. through inhalation of small forms or
basidiospores. The fungus may remain
9.5.2 Cryptococcosis dormant in the lungs until the immune
Cryptococcosis is an acute, sub acute system weakens and then can disseminate
or chronic fungal disease caused by to the central nervous system and other
encapsulated yeast called Cryptococcus body sites.
202

Figure 9.11: Cryptococcus (a) capsule staining (b) On SDA (c) On BSA
Clinical Features Laboratory Diagnosis

The clinical features of Cryptococcosis i. Samples
depend upon the anatomical sites. Specimens collected are mainly serum,
i. Pulmonary Cryptococcosis CSF and other body fluids.
The respiratory route is usually the portal a. Direct Examination

of entry for propagules in Pulmonary 10% Nigrosin or India ink staining, Gram
Cryptococcosis that subsequently staining and LPCB are used to visualize
disseminate to extra pulmonary sites. the yeast cell.
The symptoms are dry cough, dull chest
Biopsy material is stained with periodic
pain and milder or no fever with small
acid - Schiff and Gomoris’s methylamine
gelatinous granules all over the lungs.
silver stain to observe the fungal cells
ii. CNS Cryptococcosis in the tissue. Round budding yeast cells
This is an infection of brain and meninges with a distinct halo gelatinous capsule
leading to Meningoencephalitis. can be seen (Figure 9.11a). Gram positive
Nitrogenous source such as asparagines budding yeast cells are demonstrated by
and creatinine present in cerebrospinal Gram staining.
fluid enrich the yeast. The symptoms
b. Fungal Culture
are nausea, dizziness, impaired memory,
blurred vision and photophobia. The The clinical specimens can be cultured on
enlarged granulomatous cerebral lesions Sabouraud dextrose agar, Bird Seed agar
are called cryptococcoma. and incubated at 37°C. The colonies are
mucoid, cream to buff - colored in SDA
iii. Visceral Cryptococcosis (Figure 9.11b), whereas brown colored due
This infection usually spreads from a to conversion of the substrate into melanin
primary focus to invade the optic nerve by Phenoloxidase in BSA (Figure 9.11c).
and meninges. Visual loss in patients
ii. Treatment
is due to intra cranial pressure. There
are two distinct patterns of visual loss 1. Amphotericin B, Flucytosine is given
namely; rapid visual loss (within 12 hrs) together as induction and maintenance
and slow visual loss (within weeks to therapy.
months). 2. Fluconazole is also recommended.
203

3. is an example for
Saccharomyces dimorphic fungi
cerevisiae Fungemia:
a. Histoplasma b. Mucor
An Emerging Infectious
Disease. Saccharomyces c. Cryptococcus d. None of these
cerevisiae is well known 4. Phaeoid fungi is also called as
in the baking and brewing industry and
is also used as a probiotic in humans. a. Black yeast b. White yeast
However, it is a very uncommon cause of
c. Moulds d. None of these
infection in humans.
5. Tinea Barbae is also called as

Summary a. Athlete’s foot b. Onchomycosis
This chapter dealt with the general c. Barber’s itch d. None of these
classification of fungi in relationship
6. Tear shaped micro conidia is found in
with the host cells, classification of

mycoses and also about the vegetative
and reproductive structure, its growth a. T.mentagrophytes b. T.rubrum
and nutrition. Medically important fungi c. T.vercossum d. None of these
such as Dermatophytoses, Mycetoma, 7. is called as Madura foot
Histoplasmosis, Candidiasis and
a. Piedra b. Tinea pedis
Cryptococcosis its pathogenesis, clinical
features and laboratory diagnosis were c. Mycetoma d. None of these
discussed. 8. is known as Darling’s
disease
Evaluation a. Cryptococcosis b. Histoplasmosis
Multiple choice questions c. Candidiasis d. None of these
1. is the father of 9. Diaper rash is caused by
Medical Mycology a. Dermatophytes b. Histoplasmosis
a. Pasteur c. Candidiasis d. None of these
b. Raymond Jacques 10. Demonstration of is called
Sabouraud as Reynolds – Braude phenomenon
c. Robert koch a. Arthrospore b. Chlamydospore
d. Anton de Bary c. Germ tube d. None of these
2. is an example for
deep mycoses Answer the following
a. Systemic b. Opportunistic 1. Define Mycology.
c. Both d. None of these 2. What is host parasitic relationship?
204

3. What are toxigenic fungi? 15. Short note on classification of
4. Discuss about the types of mycoses? mycetoma.
5. Define Aeromycology? 16. Define deep mycoses
6. What is Pseudohyphae? 17. Elaborate on clinical feature of
histoplasmosis.
7. What are Dimorphic fungi?
18. What is Opportunistic mycosis.
8. Brief note on vegetative structure of
fungi. 19. Discuss on Lab diagnosis of
Candidiasis?
9. Account on reproduction of fungi.
20. Define Germtube.
10. Short note on taxonomy of fungi?
21. What is Cutaneous dermatitis?
11. What are the clinical features of
dermatophytes? 22. Note on Meningoencephalitis.
12. Define Ringworm infection. 23. What is Capsulated yeast?
13. Differentiate dermatophytic 24. Brief account on clinical feature of
fungi based on macroscopic and Cryptococcosis.
microscopic morphology 25. Differentiate between Candida and
14. What is maduramycosis. Cryptococcus.
205

Chapter
10 Medical Virology
Viruses are infectious

Learning Objectives agents with size ranging
After studying this chapter the from about 20 nm to
students will be able to, about 300 nm is diameter
and contain only one type
• Study the importance of viruses of nucleic acid (RNA
causing disease in humans and or DNA) as their genome. The nucleic
from Animals. Viral infection acid is encased in a protein shell, which
are very common so its our main may be surrounded by a lipid containing
concern to protect us from the viral membrane. The entire infection unit is
infection. termed as virion. Viruses are obligate
• Study the classification, structure intracellular parasite, they replicate only is
and cultivation of Viruses. living cells. The study of virus is called as
• Study about the virus its virology. Martinus Beijerinck is known as
pathogenesis, clinical feature and the Father of Virology.
its treatment and prophylaxis.
10.1 Evolutionary Origin of Viruses
• Study about the collection,
processing of the sample and its The origin of viruses is not known, but two
molecular diagnosis. theories of vital origin can be summarized
as follows;
i. Viruses may be derived from (DNA
Chapter Outline
or RNA) nucleic acid components
10.1 Evolutionary Origin of viruses of host cells to replicate and evolve
independently.
10.2 Cultivation of Viruses
ii. Viruses may be degenerate forms of
10.3 Herpes Viruses intracellular parasites.
10.4 Hepatitis Viruses
Morphology
10.5 Rabies Virus
Size
10.6 Human Immuno Deficiency Virus Viruses are smaller than bacteria, known
10.7 Arbo Virus as filterable viruses vary widely in size.
206

The largest among them is the Pox virus the host cell membrane when the progeny
measuring about 300nm. The smallest virus virus is released by budding. The envelope
is Parvo virus measuring about 20 nm. is lipoprotein in nature. The lipid is of
host cell origin while the protein is virus
Structure and Shape coded. Protein subunits may be seen as
The virion consists of nucleic acid projecting spikes on the surface of the
surrounded by a protein coat, the capsid. envelope and are known as Peplomers.
The capsid with the enclosed nucleic Overall shape of the virus particle
acids is known as the nucleo capsid. The varies; mostly animal viruses are roughly
capsid is composed of a large number of spherical. Some are irregular and
capsomers. The functions of the capsid pleomorphic. The rabies virus is bullet
are to protect the nucleic acid from the shaped, Ebola virus is filamentous and
deleterious agents and also to introduce pox viruses are brick shaped.
viral genome into host cells by adsorbing
readily to cell surfaces (Figure 10.1). Chemical Properties
Two kinds of symmetry encountered in Viral protein determines the antigenic
the virus are icosahedral (cubical) and specificity of the virus. Some viruses
helical. Virions may be enveloped or contain small amounts of carbohydrates.
non enveloped (naked). The envelope or Most Viruses do not possess any enzymes
outer covering of viruses is derived from but retro virus has a unique enzyme, such
Figure 10.1: Structure of Virus

207

as RNA dependent DNA polymerase or 2. Penetration
transcriptase which can transcribe RNA Bacteria possess rigid cell walls, only
into DNA. the viral nucleic acid is introduced
intracellularly by a complex mechanism.
Resistance Animal cells do not have rigid cell walls
Viruses are inactivated by sunlight, and the whole virus can enter and virus
UV rays and ionizing radiations. The particles may be engulfed by a mechanism
most active antiviral disinfectants resembling phagocytosis, a process
are oxidizing agents such as hydrogen known as ‘Viropexis’. In case of the
peroxide, potassium permanganate and enveloped viruses, the viral envelope may
hypochlorites. Organic iodine compounds fuse with the plasma membrane of the
are actively virucidal. Chlorination of host cell and release the nucleocapsid into
drinking water kills most viruses but its the cytoplasm.
efficacy is influenced by the presence
3. Uncoating
of organic matter. Some viruses such as
hepatitis virus, polio viruses are relatively Release of the viral nucleic acid from the
resistant to chlorination. capsid into the host cell with most viruses,
uncoating is affected by the action of
lysosomal enzymes of the host cell.
Antibiotics active
against bacteria are 4. Biosynthesis
completely ineffective Virus can synthesise viral nucleic acid,
against viruses. capsid protein and also the enzymes
necessary in the various stages of viral
Viral Multiplication synthesis, assembly and release. In
addition certain regulator proteins are
The genetic information necessary for also synthesized. Most DNA viruses
viral replication is contained in the v iral synthesise their nucleic acid in the
nucleic acid, and also depends on the syn- host cell nucleus. Most RNA viruses
thetic machinery of the host cell for repli- synthesise all their components in the
cation. The Viral multiplication cycle can cytoplasm.
be divided into six steps and they are as
Biosynthesis consists of the following
follows, 1. Adsorption or attachment,
steps:
2. Penetration, 3. Uncoating, 4. Bio
synthesis, 5. Maturation and 6. Release. i. Transcription of messenger RNA
(mRNA) from the viral nucleic acid.
1. Adsorption
ii. Translation of the mRNA into ‘early
Virions may come into contact with cells proteins’
by random collision but adsorption takes
iii. Replication of Viral nucleic acid.
place only if there is an affinity between
the virus and the host. The cell surface iv. Synthesis of late or structural proteins,
should contain specific receptor site for which are the components of daughter
the virus to attach on to it. virion capsids.
208

5. Maturation is from the stage of penetration till the
Assembly of daughter virions follows the appearance of mature daughter virions.
synthesis of viral nucleic acid and proteins. The virus cannot be demonstrated inside
Virions assembly may take place in the the host cell. The virus seems to disappear
host cell nucleus or cytoplasm. Herpes (Figure 10.2).
and adeno viruses are assembled in the
Viroids
nucleus, while picorna and pox viruses
are assembled in the cytoplasm. Viriods are small, single stranded
covalently closed circular RNA
6. Release molecules existing as highly base paired
In case of bacterial viruses, the release of rod like structure. The viroid depends
progeny virions takes place by the lysis of on the host for replication. These are
the infected bacterium. However, in the responsible for some of the transmissible
case of animal viruses, release usually plant diseases.
occurs without cell lysis. Eclipse phase
Nucleus Influenza vitus
Epithelial cell
1 Attachment 2 Penetration 3 Uncoating

Influenza virus becomes The cell engulfs the virus Viral contents are
attachedto a target by endocytosis. released.
epithelial cell.
6 Release 5 Assembly 4 Biosynthesis

New viral particles are made and New phage particles are viral RNA enters the nucleus,
released into the extracellular fluid assembled. where it is replicated by the
The cell. which is not killed in the viral RNA polymerase.
process. continues to make
new virus.
Figure 10.2: The Viral Life cycle

209

(pocks). Different viruses have different
HOTS
pock morphology. Example: varcola or
vaccinia
Which is more dangerous – bacteria or
Virus? b. Allantonic Cavity
Inoculation on the allantonic cavity
Prion provides a rich yield of influenza and
some paramyxo viruses.
Prions are small proteinaceous infectious
agents without genetic material. These are c. Amniotic Sac
responsible for a number of degenerative Inoculation into the amniotic sac is for
brain diseases (Example: Creutzfeldt) and the primary isolation of the influenza
hereditary dementia. virus.
10.2 Cultivation of Viruses d. Yolk Sac

Inoculation into the yolk sac is for
Viruses are obligate intracellular parasites;
the cultivation of some viruses like
they cannot be grown on any inanimate
Chlamydiae and Rickettsiae.
culture medium. Three methods are
employed for the cultivation of viruses – Allantonic inoculation is employed
inoculation into animals, embryonated for growing influenza virus for vaccine
eggs and tissue culture or cell culture. production (Figure 10.3).
i. Animal Inoculation iii. Tissue Culture

The earliest method for the cultivation First tissue culture in Virology was
of viruses causing human diseases was maintained by Steinhardt and colleagues
inoculation into human volunteers. (1913) for the vaccinia virus in fragments
Monkeys were used for the isolation of the of rabbit cornea. Bacterial contamination
polio virus by Landsteiner and Popper was the major limitation. Different types
(1909). The embryonated hen’s egg was of culture used are:
first used for cultivation of viruses by
Good pasture (1931). The embryonated a. Organ culture
egg offers several sites for the cultivation Small bits of organs can be maintained,
of viruses. Non human primates provide used for the isolation of some viruses.
the only method for virus cultivation. Example: Corona virus (respiratory
Mice are most widely employed animals pathogen) cultured on tracheal ring organ
in Virology. culture.
b. Explant culture
ii. Embryonated Eggs
Fragments of minced tissue are grown
a. Chorioallantonic Membrane (CAM) as ‘explants’. This is also known as tissue
Inoculation on the chorioallantonic culture. Example: Adeno virus cultured
membrane produces visible lesions on Adenoid tissue explants.
210

Herpes simplex virus
Chorioallantoic membrane inoculation Poxvirus
Rous sarcoma virus
Amniotic cavity
Shell Shell membrane

Air sac Influenza virus
Amniotic inoculation Mumps virus
Albumin
Allantoic Yolk sac inoculation Herpes simplex virus

cavity
Allantoic inoculation Influenza virus
Yolk sac Mumps virus
Chorioallantoic Newcastle disease virus
membrane Avian adenovirus
Figure 10.3: Cultivation of virus in Embryonated Egg
iv. Cell Culture c. Continuous cell lines

Tissues are dissociated into the component These are single type, derived from
cells by the action of enzymes (trypsin) or cancer cells that are capable of continuous
by mechanical process and are suspended serial cultivation. Example: Cells derived
in a growth medium (amino acids, from cancers, such as Hela, Hep-2 and KB
vitamins, salts, glucose) supplemented cell lines.
with fetal calf serum of antibiotics and
indicator (Phenol red). This media is Detection of virus Growth in Cell Cultures
dispensed in bottles, tubes or petridishes. Virus growth in cell cultures can be
The cells adhere to the glass surface and detected by the following methods.
on incubation divides to form a confluent
monolayer sheet of cells covering the 1. Cytopathic effect
surface within about a week. The cell Many viruses cause morphological changes
culture is classified into three types. in cultured cells in which they grow. These
changes are known as cytopathic effects
a Primary cell cultures
(CPE) and the viruses causing CPE are
In this culture, normal cells are taken from called as ‘cytopathogenic viruses’.
the body and cultured. They are capable of
only limited growth in culture. Example: 2. Metabolic inhibition
Monkey kidney, Human embryonic In normal cell cultures, the medium turns
kidney, Chick embryo cell culture. acidic due to cellular metabolism. This can
be made out by the colour of the indicator
b. Diploid cell strains
(Phenol red) incorporated in the medium.
These are cells of a single type that retain
the original diploid chromosome number 3. Haemadsorption
and serotype during serial sub cultivation When haemagglutinating viruses grow
for limited number of times. Example: in cell cultures, their presence can be
Human fibroblast. identified by the addition of guinea pig
211

erythrocytes. The erythrocytes will
adsorb on to the surface of viral cells
multiplying in the culture. This is known
as ‘haemadsorption’.
4. Interference
The growth of non cytopathogenic virus
in cell culture can be tested with the
cytopathogenic virus. The growth of the
first will inhibit infection by the second
virus by ‘interference’.
5. Transformation
Tumour forming viruses induce cell Figure 10.4: Structure of Herpes
Simplex Virus
transformation and loss of contact
inhibition, so that growth appears in a piled
Classification
up fashion producing ‘microtumours’.
The family Herpesviridae is divided into
6. Immuno fluorescence three subfamilies based on biological,
Cells from virus infected cultures are physical and genetic properties
stained by fluorescent conjugated (Table 10.1).
antiserum and examined under the UV
microscope for the presence of virus Table 10.1: Classification of Human
antigen. Herpes Viruses
Species
10.3 Herpes Viruses
Name Common name
The herpes virus family contains more
Human herpes virus Human Simplex
than a hundred species of enveloped DNA type1 virus type1
viruses that affect humans and animals.
Human herpes virus Human Simplex
Structure type 2 virus type2
Human herpes virus Varicella Zoster
The herpes virus capsid is icosahedral, type 3 virus
composed of 162 capsomers and enclosing Human herpes virus Epstein - Barr Virus
the core containing the linear double type 4
stranded DNA genome. The nucleocapsid Human herpes virus Cytomegalovirus
is surrounded by the lipid envelope derived type 5
from the host cell. The envelope carries Human herpes virus Human B cell
surface spikes (Figure 10.4). Teguments type 6 lymphotropic virus*
are present in between the envelope and Human herpes virus R K Virus *
capsid. The enveloped virion measures type 7
about 200nm and the naked virion about Human herpes virus -
type 8
100 nm in diameter.
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i. Alpha herpes viruses sources of infection are saliva, skin
They have relatively short replicative lesions or respiratory secretions. In type 2,
cycle (12–18 hours) and a variable host transmission occurs by close contact and
range. They cause latent infection in may be venereal in genital herpes.
sensory ganglia. Example: Herpes simplex The virus enters through defects in the
virus and varicella zoster virus. skin or mucous membranes and multiples
locally, with cell to cell spread. The herpes
ii. Beta herpes viruses
lesions are thin walled, umbilicated
They replicate slowly (more than vesicles, the roof of which breaks down,
24 hours) and have a narrow host range, leaving tiny superficial ulcers. They heal
grow well in fibroblasts. They cause latent without scarring.
infection of salivary gland and other
organs. Example: Cytomegalovirus. Clinical features
The clinical manifestations depend on the
iii. Gamma herpes viruses site of infection, age and immune status
They have a narrow host range and of the host and the antigenic type of the
replicate in lymphoblastoid cells. They virus. They are
are specific for either B or T lymphocytes •• Cutaneous infections
and causes latent infection in lymphoid
•• Mucosal infections
tissue Example: Epstein - Barr Virus. Eight
•• Ophthalmic infections
different types of herpes viruses are known
whose primary hosts are humans. They •• Nervous system infections
have been designated as ‘Human herpes •• Visceral infections
virus type 1–8. •• Genital infections
Laboratory diagnosis
1. Herpes Simplex
Microscopy
The herpes simplex virus (HSV) occurs Smears are prepared from the lesions,
naturally only in humans, but it can from the vesicles and stained with 1%
produce experimental infection in aqueous solution of toluidine blue ‘O’
laboratory animals. There are two types for 15 seconds. Multinucleated giant cells
of the herpes simplex virus. HSV type 1 with faceted nuclei with ground glass
(Human herpes virus type 1 or HHV type chromatin (Tzanck cells) was observed.
1) is isolated from lesions in and around
the mouth and is transmitted by direct Virus isolation
contact or droplet spread from carrier. Inoculation in mice and on chick embryo
HSV type 2 (Human herpes virus type 2 or CAM is insensitive. Primary human
HHV type 2) is responsible for the genital embryonic kidney, human amnion cells
herpes infections transmitted venereally. are susceptible, but human diploid
fibroblasts are preferred vesicle fluid,
Pathogenesis spinal fluid, saliva and swabs may be used.
Herpes simplex is one of the most Cytopathic changes may appear as early as
common viral infection humans, the 24–48 hrs.
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Serology a group of ubiquitous herpes viruses
Antibodies develop within a few days of of humans and animals. They are
infection and rise in titre of antibodies may characterized by enlargement of infected
be demonstrated by ELISA, neutralization cells and intranuclear inclusions. In 1926,
or complement fixation tests. cytomegalia presumed to be due to viral
infection was reported in the salivary
Chemotherapy glands of guinea pigs and children and
Indoxyuridine used topically in eye and the viral agent was called the ‘salivary
skin infection, acyclovir and vidarabine gland virus’.
are given for deep and systemic
infections. Infobits
2. Varicella Zoster CMV is the largest viruses in the

herpes virus family, being 150–200 nm
In 1889, Von Bokay had suggested that
in size.
varicella (Chicken pox) and herpes zoster
are different manifestations of the same
virus infection. The virus is therefore 4. Epstein – Barr Virus
called Varicella zoster virus (VZV). The
chicken pox follows primary infection in A number of different viruses apparently
a non immune individual, while herpes ‘Passenger Viruses’ were isolated from
zoster is a reaction of the latent virus cultured lymphoma cells. Epstein, Barr
when the immunity has fallen to infective and Achong in 1964 observed a new type
levels. of herpes virus named it has ‘EB Virus’
affecting cells of B lymphocyte. Only
VZV is similar to the herpes simplex
human and some sub human primate
virus in its morphology. It can be grown
B cells have receptors ( CD21 molecules)
in cultures of human fibroblasts human
for the virus.
amnion or HeLa cells. Chicken pox is one
of the mildest and most common of child The source of infection is usually the
hood infections. The disease may, occur saliva of infected persons who shed the
at any age. virus in orophryngeal secretions. Intimate
oral contact, as in kissing, appears to be the
Herpes gladiatorum predominant mode of transmission. This
is spread through accounts for infectious mononucleosis
skin-on-skin contact. being called as ‘The kissing disease’.
If you kiss someone
5. Human Herpes Virus Types 6,7,8
with a herpes cold sore on their lips,
you could become infected. A herpes virus, first isolated in 1986 from
the peripheral blood of patients with
lympho proliforative disease called as
3. Cytomegaloviruses human B lymph tropic virus, renamed
Cytomegaloviruses (CMV) formerly as HHV - 6. HHV- 7 was isolated in 1990
known as salivary gland viruses are from peripheral CD4 cells of a healthy
214

Type A Hepatitis (HAV)
Does HSV shorten
your lifespan? HAV is a 27nm non enveloped RNA virus
belonging to the picorna virus family. It
Becoming infected
with the herpes is designated as ‘entero virus 72’, HAV is
virus seriously complicates your social, recognised as new genus ‘Hepatovirus’. It
emotional and sexual life, but it is not can be grown in human and simian cell
otherwise a terribly dangerous condition cultures and is the only human hepatitis
to have. Having genital herpes virus can be cultivated in vitro.
does make it easier to get HIV (and thus HAV transmission is by the fecal oral
AIDS), but otherwise, the condition route. Infection is by ingestion. The virus
is not disabling, and does not reduce multiplies in the intestinal epithelium
lifespan. and reaches the liver by hematogenous
spread. Once jaundice develops, it is
rarely detectable in feces. The incubation
person appears to be widely distributed
period is 2- 6 weeks. The clinical disease
and transmitted through saliva.
consists of two stages the prodromal and
In 1994, DNA sequences presumed to the icteric stage. The onset may be acute
represent a new herpes virus from tissues
with fever, malaise, anorexia, nausea,
of Kaposi’s sarcoma from AIDS patients
vomiting and liver tenderness. These
named as HHV8. Later identified in
usually subside with the onset of jaundice.
Kaposi’s sarcoma in persons not infected
Recovery is slow, over a period of 4–6
with HIV and referred to as Kaposi’s
weeks. The disease is milder in children.
Sarcoma-associated herpes virus (KSHV).
Type A hepatitis caused by contaminated
food, water or milk. Over crowding and
10.4 Hepatitis Viruses
poor sanitation favour its spread.
The term viral hepatitis refers to a
primary infection of the liver, hepatitis Laboratory Diagnosis
viruses consists of types A, B, C, D, E and
G. Except for type B which is a DNA virus Diagnosis of type A hepatitis may be
all the others are RNA viruses. made by demonstration of the virus or
its antibody. Virus can be visualized by
Two types of viral hepatitis had been
Immunelectron Microscopy (IEM) in
recognised type one affects mainly
fecal extracts during the late incubation
children and young adults and transmitted
period.
by the fecal-oral route called as infective
or infectious hepatitits or type A IgM anti-HAV antibody appears
hepatitis. Second type transmitted mainly during the late incubation period
by receiving serum inoculation or blood disappears after 3-4 months. IgG appears
transfusion named as homologous serum peaks in 3-4 months and persists much
jaundice, serum hepatitis transfusion longer for life. ELISA kits for detection of
hepatitis or type B hepatitis. IgM and IgG antibodies are available.
215

A safe and effective formalin
inactivated, alum conjugaged vaccine
containing HAV grown in human diploid
cell culture. Course consists of two intra
muscular injections of the vaccine.
Protection begins 4 weeks after injection
and lasts for 10 to 20 years. No specific
antiviral drug is available.
Type B Hepatitis (HBV)

HBV is a 42nm DNA virus with an outer
envelope and an inner core 27nm in
Figure 10.5: Structure of Hepatitis B Virus
diameter. Enclosing the viral genome
and a DNA polymerase. It belongs to the The nucleocapsid encloses the viral
family Hepadna Viridae HBV is ‘Hepadna genome consisting of two linear strands
of DNA held in a circular configuration.
Virus type 1’. Australia antigen was
One of the strands incomplete (+ strand)
found to be associated with serum
DNA appears partially double stranded
hepatitis. It was the surface component
and partially single stranded. Associated
of HBV, so named as hepatitis B surface
with the + strand is a viral DNA
antigen (HBsAg).
polymerase (has both DNA dependent
3 types of particles are visualized, most DNA polymerase and RNA dependent
abundant form is a spherical particle, reverse transcriptase functions). This
22nm in diameter. The second type of polymerase can repair the gap in the
particle is filamentous or tabular with a plus strand and render the genome fully
diameter of 22nm both are antigenically double straned.
identical. Third type of particle for fewer Natural infection occurs only in
in number, is a double walled spherical humans. The virus is maintained in
structure 42 nm in diameter. This particle carriers whose blood contains circulating
is the complete hepatitis B virus, known virus for long periods carriers are of two
as Dane particle. categories, the highly infectious super
The envelope proteins expressed on carriers and the simple carriers. Former
the surface contains hepatitis B surface have high titre HBsAg along with HBsAg,
antigen (HBsAg). HBsAg consists of DNA polymerase and HBV in ciruculation.
two major polypeptides, one of which Simple carriers have low infectivity and
is glycosylated. The nucleocapsid or low titre HBsAg in blood.
core contains hepatitis B core antigen HBV is a blood borne virus and the
(HBcAg A) (Figure 10.5). Third antigen infection is transmitted by parenteral,
called the hepatitis B e antigen (HBeAg) sexual and perinatal models. The virus
is a soluble non particulate nucleocapsid may also be present in other body fluids
protein. and excreations such as saliva, breast
216

milk, semen, vaginal secretions, urine HBc appears in serum a week or two after
bile and feces of these semen and saliva the appearance of HBsAg. As anti HBc
are known to transmit the infection remains life long, it serves as a useful
very commonly. Transfusion of carrier indicator of prior infection with HBv.
blood once the most widely known mode HBeAg appears in blood concurrently
infection has largely been eliminated with HBsAg, indicating the high
by donor screening is strictly enforced. infectivity. Molecular methods such as
Infection by direct contact with open skin DNA: DNA hybridization and PCR at
lesions such as pyoderma, eczema, cuts present used for HBV DNA testing are
and scratches is very common among highly sensitive and quantitative.
young children in developing countries.
Certain groups and occupations carry Immunization
a high risk of infection. These include Both passive and active methods of
medical and paramedical staff of blood immunization are available. Active
banks, dialysis units, barbers, box immunization is more effective. The
workers. currently preferred vaccine is genetically
The incubation period is long about engineered by cloning the S gene for
1- 6 months. The onset is insidious and HBV in Baker’s yeast. A special vaccine
fever is not prominent. Extra hepatic containing all antigenic components
complications like arthralgia, urticaria of HBsAg (Pre-S1, Pre-S2 and s) has
and glomerulonephritis may occur. About been developed. No specific antiviral
90-95% of adults with acute hepatitis treatment is available for acute HBV
B infection recover within 1-2months infection.
of onset and eliminate the virus from
the body. They may be Asymptomatic 10.5 Rabies Virus
carriers or may progress to recurrent or
The Family Rhabdoviridae contains
chronic liver disease.
viruses that infects mammals, reptiles,
birds, fishes, insects and plants.
The disease in human being is called
Serology hydrophobia because the patient exhibits
Diagnosis of hepatitis B depends on the fear of water, being incapable of drinking
serological demonstration of the viral though subject to intolerable thirst.
markers. HBsAg is the first marker to Pasteur established that the rabies
appear in blood after infection, being virus was present in the brain of infected
detectable. It remains in circulation animals. By serial intracerebral passage
throughout the symptomatic course of in rabbits, he demonstrated fixed virus
the disease (2- 6months). Anti HBs is the that could be rendered immune by
protective antibody. a series of injections. Vaccine was
HBcAg is not demonstrable in prepared by drying pieces of spinal
circulation because it’s enclosed within card from rabbits infected with the
the HBsAg coat but its antibody, anti fixed virus.
217

Joseph Meister a nine year old boy, The rabies virus isolated from natural
severely bitten by a rabid dog and in grave human or animal infection is termed ‘the
risk of developing rabies, was given a street virus’. Rabies has been recognized
course of 13 inoculations of the infected from very ancient times as a disease
cord vaccine by Pasteur. The boy survived. transmitted to humans and animals by
This dramatic event was a mile stone in the bite of ‘mad dogs’. The name rabies
the development of medicine. comes from the Latin word rabidus,
meaning ‘mad’, derived from the Sanskrit
Morphology
root rabhas, for frenzy.
The rabies virus is bullet shaped, with
one end rounded or conical and the Pathogenesis
other planar or concave. The lipoprotein Human infection is usually caused by the
envelope, carries knob like spikes, bite of rabid dogs or other animals. The
composed of glycoprotein G responsible
virus present in the saliva of the animal
for pathogenesis, virulence and immunity
is deposited in the wound (Figure 10.7).
beneath the envelope is the matrix (M)
Rarely, infection can also occur following
protein layer which may be invaginated
non-bite exposures such as licks or
at the planar end. The membrane may
project outwards forming a bleb. The aerosols.
genome is unsegmented linear RNA The virus appears to multiply in the
(Figure 10.6). muscles, connective tissue or nerves at
Figure 10.6: Structure of Rabies Virus
218

Brain
6. Infection of brain
neurons with
neuronal
dysfunction
Eye
7. Centrifugal spread along
nerves to salivary glands,
skin, cornea, and
other organs Salivary glands
3. Virus binds to bicotinic

acetylcholine receptors at
neuromuscular junction at 5. Replication in motor
neuromuscular junction neurons of the spinal cord
and local dorsal root ganglia
and rapid ascent to brain
Dorsal root
ganglion
Sensory nerves
to skin
Skeletal 4. Virus travels within

muscle axons in peripheral
nerves via retrograde
tast axonal transport
2. Viral replication
in muscle Spinal cord
1. Virus inoclated
Figure 10.7: Mechanism of Rabies Infection
the site of deposition for 48-72 hours. It in persons bitten on the face or head and
penetrates the nerve endings and travels long in those bitten on the legs. This may
in the axoplasm towards the spinal cord be related to the distance the virus has to
and brain, at speed of about 3 mm per travel to reach the brain. The incubation
hour. The virus multiples and spreads period is generally shorter in children
centrifugally along the nerve trunks to than in adults.
various parts of the body including the The four stages of the disease are as
salivary glands. It multiplies in the salivary follows, prodrome, acute encephalitic
glands and is shed in the saliva. The virus phase, coma and death. The onset is market
reaches every tissue in the body and by symptoms such as fever, headache,
disseminated may be interrupted at any malaise, fatigue and anorexia. Anxiety,
stage by death. In humans the incubation agitation, irritability, nervousness,
period is usually from 1–3 months, insomnia or depression. The neurological
short as 7 days or as long as three year. phase begins with hyperactivity. Attempts
The incubation period is usually short to drink on such painful spasms of the
219

pharynx and larynx producing chocking Animal Rabies
or gagging that patients develop a dread The whole carcass of the animal suspended
of even the sight or sound of water to have died of rabies may be sent to the
(hydrophobia). laboratory. The brain may be removed
sent for biological test and microscopy
Animal Infection respectively. The portion of brain sent
In dogs, the incubation period is usually should include the hippocampus and
3–6 weeks but it may range from 10 days cerebellum as negri bodies are most
to a year. The initial signs are an alert, abundant. The following tests are done in
troubled air and restlessness, snapping the laboratory.
at imaginary objects, licking or gnawing 1. Demonstration of rabies virus antigen
at the site of the bite. by immuno fluorescence
After 2–3 days of this prodromal 2. Demonstration of inclusion
stage, the disease develops into either bodies - Negri bodies are seen as
the furious or dumb types of rabies. In intracytoplasmic, round or oval
furious rabies, dog runs biting without purplish pink with characteristic
provocation, the lower jaw droops and basophilic inner granules. Negribodies
saliva drools from the mouth. Paralysis vary in size from 3.27 Mm.
convulsions and death follow. In dumb
rabies, is the paralytic form, animals lies Infobits
huddled, unable to feed. About 60% of Local Treatment for rabies
rabid dogs shed the virus in saliva. Rabid
•• Prompte cauterization of the
dogs usually die in 3-5days.
wounds helps to destroy the virus.
Laboratory Diagnosis •• Antirabic serum may be applied
topically.
Human Rabies
•• Antitetanus measures and
The specimens tested are corneal smears
antibiotics to prevent sepsis.
and skin biopsy. Commonly used method
for diagnosis is the demonstration of rabies
virus antigens by immuno fluorescence. Antirabic Vaccines
Direct immunofluorescence is done
Antirabic vaccines fall into two main
using antirabies serum tagged with
categories neural and non-neural.
fluorescein isothiocyanate.
Negri bodies in the brain, are Neural Vaccines
demonstrated, Isolation of the virus by Suspension’s of nervous tissues of animals
intracerebral inoculation in mice can be infected with the fixed rabies virus.
attempted from the brain, CSF, saliva and Following are the modified forms.
urine. The mice are examined for signs of 1. Semple Vaccine: Vaccine developed by
illness, and their brains are examined at semple (1911). It is a 5% suspension of
dealth. sheep brain infected with fixed virus
220

and inactivated with phenol at 37°C
leaving noresidual live virus. Infobits
2. Beta propiolactone (BPL) Vaccine: Detecting HIV sooner

Beta propiolactone is used as the
Fourth generation test helps to detect
inactivating agent instead of Phenol.
HIV in blood earlier than previously
3. Infant Brain Vaccine: The recommended antibody test. It
enceptalitogenic factor in brain tissue identifies the viral protein, HIV-1 P24
is a basic protein associated with antigen, which appears in the blood
myelin. sooner than antibodies.
Vaccines were developed using infant Source: CDC
mouse, rat or rabbit brain. Infant brain
vaccine is impractical in India. Structure
Non-neural Vaccines HIV is a spherical enveloped Virus, about
Non-neural vaccines includes 90-120min size. The nucleo capsid has an
outer icosahedral shell and an inner cone
1. Egg Vaccines
shaped core, enclosing the ribonucleo
2. Tissue Culture Vaccines proteins. The genome is diploid, composed
3. Subunit Vaccine of two identical single stranded, positive
sense RNA copies. When the virus infects
Passive Immunisation a cell, the Viral RNA is transcribed by
Human rabies immune globulin (HRIG) the reverse transcriptase enzyme, first
into single stranded DNA and then to
is free from the danger of sensitization
double stranded DNA (provirus) which is
but should be ensured free from HIV and
integrated into the host cell chromosome.
hepatitis viruses.
The virus coded envelope proteins are the
projecting knob like spikes which binds
Vaccines for Animals
to the CD4 receptors on susceptible host
Antirabies immunization in animals is cells (Figure 10.8).
to be done as pre-exposure prophylaxis
concentrated cell culture vaccines –
inactivated virus gives good protection
after a single Intramuscular injection.
Injections are given at 12 weeks of age and
repeated at 1–3 years intervals.
10.6 Human Immuno Deficiency

Virus
HIV is known as Human Immuno
Deficiency Virus, the etiological agent of
AIDS, belongs to the lentivirus subgroup
of the family Retroviridae. Figure 10.8: Structure of HIV
221

Viral Genes and Antigens stranded DNA, which is integrated into
The genome of HIV contains the three the genome of the infected cell through
structural genes (gag, pol and env) as the action of the viral enzyme integrase,
well as other nonstructural and regulatory causing a latent infection. The primary
genes specific for the virus. These products pathogenic mechanism in HIV infection
of these genes, both structural and non is the damage caused to the CD4+T
structural act as antigens. lymphocyte. The T4 cells decrease is
numbers. Infected T4 cells do not release
Genes coding for structural proteins normal amounts of interleukin−2, gamma
1. The gag gene Determines the interferon and other lymphokines, this is
core and shell of the Virus. Precursor damping effect on cell mediated immune
protein, p55 and it is cleaved into three response.
proteins p15, p18 and p24. Major core
antigen p24 can be detected in serum Clinical Features
2. The env gene Determines the syn- AIDS is only the last stage in the wide
thesis of envelope glycoprotein gp160. spectrum in HIV infection.
Cleaved in to gp120 and gp41
1. Acute HIV infection
The pol gene Codes for the
3–6 weeks of infection, persons experience
polymerase reverse transcriptase and
low grade fever, malaise, headache,
other viral enzymes such as protease
lymphadenopathy, with rash. Antibodies
and endonucleases. It’s expressed as
a precursor protein, which is cleaved are usually negative at the onset of the
into protein p31, p51 and p66. illness but become positive during its
course called ‘Sero conversion illness’.
Pathogenesis 2. Asymptomatic or latent infection
Infection is transmitted when the All HIV infected persons, whether or not
Virus enters the blood or tissues of a they experience Sero conversion illness,
person and comes into contact with a pass through a phase of symptomless
suitable host cell, principally the CD4 infection which may last up to several
lymphocyte. The receptor for the virus years. The infection progresses in course
is the CD4 antigen and therefore the of time through various stages, CD4
virus may infect any cell bearing the CD4 lymphocytopenia, minor opportunistic
antigen on the surface. Specific binding infections, ARC AIDS-related complex,
of the virus to CD4 receptor is by the ultimately terminating to AIDS.
envelope glycoprotein gp120. Cell fusion
is brought about by transmembrane 3. Persistent generalized
gp41. After fusion virus with the host lymphadenopathy (PGL)
cell membrane, the HIV genome is It’s defined as the presence of enlarged
uncoated and internalized into the cell. lymph nodes at least 1cm, in diameter in
Viral reverse transcriptase mediate two or more non contiguous extrainguina,
transcription of its RNA into double sites persist for at least three months.
222

4. AIDS related complex (ARC) 6. Dementia
This group includes patients with Direct cytopathogenic damage in the CNS.
considerable immuno deficiency, It cross the blood-brain barrier and cause
suffering from various symptoms or encepthalopathy leading to dementia.
minor opportunistic infections. eg.
7. Pediatric AIDS
oral, Candidiasis, salmonellosis or
tuberculosis. Viral transmission may occur to the
fetus in pregnancy. Many of the infected
5. AIDS children may not survive for a year.
End-stage disease, poor immune defence Children may also acquire the infection
mechanism leading to the opportunistic from blood transfusion or blood products.
infection and malignancies.
a. Commonest symptoms
Lab diagnosis of HIV infection include
Drycough, dyspnea and fever.
tests for immuno deficiency in HIV
Pheumonia may be viral (cmv) or fungal
infection.
(Cryptococcus, Histoplasma).
A. Immunological tests
b. Gastrointestinal system
i. Total leukocyte and lymphocyte count
The mouth is often involved with
to demonstrate leucopenia and a
thrush, stomatitis, gingivitis, hairy
lymphocyte count usually below 2000/
leukoplakia. Dysphagia due to
mm3.
esophageal Candidiasis. Intestinal
pathogen in AIDS is cryptosporidium. ii. Platelet count will show
Other pathogens are Salmonellae, thrombocytopenia.
Mycobacteria, CMV or adeno viruses. iii. Raised IgG and IgA levels.
‘gay bowel syndrome’ is common among
B. Specific tests for HIV infection
the male homosexuals.
1. Antigen detection
c. Central nervous system Single massive infection, as by blood
The typical CNS opportunistic infections transfusion, the virus antigens may be
are toxoplasmosis and cryptococcosis. detectable in blood after about two weeks.
Lymphomas of the CNS are Common. The major core antigen p24 is the virus
marker to appear in blood.
d. Malignancies
Kaposi’s Sarcoma was the lesion seen 2. Polymerase Chain reaction
in male homosexuals. The tumours It the most senstitive and specific test.
commonly seen are lymphomas, both the Two forms of PCR have been used, DNA
Hodgkin and non Hodgkin types. PCR and RNA PCR.
e. Cutaneous 3. Antibody detection

Herpes lesions, Candidiasis, Dermatitis, Demonstration of antibodies is the
impetigo are common cutaneous lesions. simpest and widely employed technique.
223

2–8 weeks to months for antibodies to The virus enters the body through the
appear after infection, during part of bite of the insect vector. After multiplication
this period, the individual may be highly in the reticuloendothelial system, viremia
infectious. This sero negative infective of varying duration occurs, or the virus is
stage is known as the ‘window period’. transported to the target organs such as
Antibody can be detected by central nervous system in encephalitides,
1. ELISA the liver in yellow fever and the capillary
endothelium in hermorrhagic fever.
2. Western blot test.
Clinical syndromes are fever with or
Treatment without rash encephalitis, hemorrhagic
The treatment of AIDS include: fever with or without rash encephalitis,
hemorrhagic fever and systemic disease,
1. The treatment and prophylaxis of
yellow fever.
infections and tumours.
Diagnosis may be established by virus
2. General management
isolation or serology.
3. Immunorestrorative measure
Samples (Blood, CSF) are inoculated
4. Specific anti-HIV agents. intra cerebrally into sucking mice. The
Effective drugs are available, they are animal develop fatal encephalitis. Viruses
Zidovudine, Didanosine, Zalcitabine, may be isolated in tissue cultures or in eggs.
Lamivudine and Protease inhibitors like Isolates are identified by hemagglutination
Saquinavir, Ritonavir, Indinavir used as inhibition, complement fixation, gel
monotherapy or in various combination. precipitation, immunofluorescence.
10.7 Arbo Virus ELISA
Arbo Viruses (arthropod - borne viruses) Virus isolated from insect vectors and
are viruses of vertebrates biologically from reservoir animal.
transmitted by hematophagous insect
Toga Viruses
vectors. They multiply in blood sucking
insects and are transmitted by bite Toga viruses are spherical enveloped
to vertebrate hosts. Arbo viruses are viruses with a diameter of 50-70nm.
worldwide in distribution. Arbo viruses Single stranded RNA genome. The virus
have been named according to the replicates in the cyloplasm of the host
disease caused (yellow fever), the place cell and released by budding through host
of isolation of the virus (kyasanur forest cell membranes. The name Toga Virus is
disease) or the local name for the disease derived from ‘toga’ meaning the Roman
(chikungunya). They are classified into Mantle refers to the viral envelope.
Toga, Flavi, Bunya, Reo and Rhabdovirus The genus Alpha Virus was formerly
families. Arbo viruses have a very wide host classified as Group A arbo viruses which
range including many species of animals explains the name Alpha Virus. The genus
and birds. The most important arbo virus Alpha Virus contains 32 species of which
vectors are mosquitoes, followed by ticks. 13 infect humans. All are mosquito borne.
224

10.7.1 Chikungunya Virus viruses they are St. Louis encephalitis
The virus was first isolated from human Virus, Ilheus virus, west nile virus,
patients of Aedes aegypti mosquitoes murray valley encephalitis virus and
(Figure 10.9) from Tanzania in 1952. The Japanese encephalitis. Tick borne viruses
name Chikungunya is derived from the ore classified in to tick brone encephalitis
native word for the disease in which the viruses and tick borne hemorrhagic fevers.
patient lies ‘doubled up due to severe
10.7.2 Dengue
joint pains’. The virus first appeared in
India in 1963 in Calcutta, Madras and The name dengue is derived from the
Other areas. ‘Swahili ki denga pepo’, meaning a
The disease presents as a sudden sudden seizure by a demon. Dengue
onset of fever, Crippling joint pains, fever is similar to the illness caused by
lymphadenopathy and conjunctivitis. A chikungunya. Four types of dengue virus
maculopapular rash in common. The exist: DEN1, DEN2, DEN3 and DEN4.
fever is typically biphasic with a period
of remission after 1–6 days of fever. HOTS
The vector is Aedes aegypti. No animal
reservoir has been identified. Antibody to What is the best home remedy for
the virus has been demonstrated in horses, dengue fever?
cattle and other domestic animals.
Dengue presents after an incubation
Flavi viruses period of 3-14 days as fever of sudden
The family flaviviridae contains only one onset with headache, retrobulbarpain,
genus flavivirus. They are smaller than conjunctival injection, pain in the
alpha viruses, being 40nm in diameter. back and limbs (break bone fever),
There are over 60 arthropod borne flava lymphadenopathy and maculopapular
viruses classified as mosquito-borne and rash. The fever is typically biphasic
tick borne viruses. Examples of mosquito (saddle back) and lasts for 5–7 days.
borne group known as encephalitis Dengue may be more serious forms with
hemorrhagic manifestations (dengue
Hemorrhagic fever) or with shock (dengue
shock syndrome).
Dengue virus is transmitted from
person to person by Aedes aegypti
mosquitoes. The Incubation period
is 8–10days. All four types of dengue
virus are identified. Demonstration of
circulating IgM antibody provides early
diagnosis. IgM ELISA test offers reliable
diagnosis. Difference between Dengue
and Chikungunya is given in Table 10.2.
Figure 10.9: Aedes aegypti
225

Table 10.2: Difference between Dengue and Chikungunya
S.No Factors Chikungunya Dengue
1. Vector Aedes aegypti Aedes aegypti
2. Virus Toga viridae - alphavirus Flavi viridae - flavivirus
3. Incubation 3-7 days 4-7 weeks
Period
4. Symptoms Chikungunya begins as an Dengue is an acute febrile illness.
acute febrile illness. Febrile Phase: Lasts 2–7 days Fever with
Pain can be severe. Other Headache, retro-orbital pain, joint pain,
common symptoms muscle and or bone pain, rash, mild
include headache, muscle bleeding (nose or gums)
pain, joint swelling, and Critical Phase: Lasts 24–48 hours. Most
rash. patients improve but severe disease
Some patients have requiring hospitalization can occur.
persistence or relapse of Recovery Phase: Gradual reabsorption
rheumatologic symptoms of extravasated fluid from plasma
in the months following leakage over 48–72 hours. Dieresis,
acute illness. hemodynamic status stabilizes,patient
can temporarily become bradycardic.
5 Major Tremendous Joint pain Bleeding and breath discomfort
symptom
6 Person at Neonates exposed Some patients may develop life threatening
risk intrapartum, older consequences and require hospitalization.
adults, and persons with Infection with each dengue virus type
underlying medical confers lifetime immunity for that specific
conditions. virus type.
10.7.3 Zika Virus

Infobits
Zika virus is a mosquito-borne flavivirus
Which fruit is good to increase that was identified in Uganda in 1947 in
platelet count? monkeys. Zika spreads by daytime-active
Pomegranate is another great fruit Aedes mosquitoes, such as A. aegypti and
you can eat to increase platelets. A. albopictus. The infection is known as
As with all redfruits, the seeds of Zika fever or Zika virus disease. Zika
this delicious fruit are packed with iron, is related to the dengue, yellow fever,
an essential mineral for combating Japanese encephalitis, and West Nile
low platelet count. Pomegranate has viruses
been used since the ancient times for
Zika virus is enveloped and
its healthy and medicinal properties.
icosahedral and has a non segmented,
226

single-stranded, positive-sense (+) RNA Zika virus is primarily transmitted by
genome (Figure 10.10). A positive-sense the bite of an infected mosquito from
RNA genome can be directly translated the Aedes genus, mainly Aedes aegypti.
into viral proteins, the RNA genome The mosquitoes usually bite during the
encodes seven nonstructural proteins day, peaking during early morning and
and three structural proteins. One of the late afternoon or evening. This is the
structural proteins forms the envelope. The same mosquito that transmits dengue,
RNA genome forms a nucleocapsid along chikungunya and yellow fever. Zika virus
with copies of the 12-kDa capsid protein. is also transmitted from mother to fetus
during pregnancy, through sexual contact,
transfusion of blood and blood products,
and organ transplantation.
The incubation period of Zika virus
disease is estimated to be 3–14 days. The
majority of people infected with Zika virus
do not develop symptoms. Symptoms
are generally mild including fever, rash,
conjunctivitis, muscle and joint pain,
malaise, and headache, and usually last
for 2–7 days. Zika fever (also known as
Figure 10.10: Structure of Zika Virus Zika virus disease) is an illness caused by
Viral genome replication depends the Zika virus. Zika virus infection during
on the making of double-stranded RNA pregnancy is a cause of microcephaly
from the single-stranded, positive-sense and other congenital abnormalities in
RNA (ssRNA(+)) genome followed by the developing fetus and newborn. Zika
transcription and replication to provide infection in pregnancy also results in
viral mRNAs and new ssRNA(+) genomes. pregnancy complications such as fetal
Pathogenesis and Clinical features loss, stillbirth, and preterm birth.
Zika virus replicates in the mosquito’s mid Laboratory diagnosis
gut epithelial cells and then its salivary
Virus can be demonstrated from the
gland cells. After 5–10 days, the virus
blood or other body fluids, such as urine
can be found in the mosquito’s saliva.
or semen.
If the mosquito’s saliva is inoculated
into human skin, the virus can infect Zika virus grow well in a variety of
epidermal keratinocytes, skin fibroblasts mammalian and insect cell lines. Zika
in the skin and the Langerhans cells. The virus is identified by NAAT− Nucleic
pathogenesis of the virus is hypothesized acid Amplification test, Zika Antigen
to continue with a spread to lymph is detected by ELISA and PCR. Zika
nodes and the bloodstream. Antibody IgM is detected by MAC -
ELISA, IgG by ELISA and by PRNT-
plaque reduction neutralization test.
227

Prevention and Treatment b. cytopathogenic effects
Protection against mosquito bites during c. cytopathic effects
the day and early evening is a key measure d. None of these
to prevent Zika virus infection. It is
3. Cytomegalo viruses also called as
important to eliminate these mosquito

breeding sites, Health authorities may also
advise use of larvicides and insecticides a. Salivary gland virus
to reduce mosquito populations and b. Thymus gland virus
disease spread. There is no treatment c. Endocrine gland virus
available for Zika virus infection or its
d. None of these
associated diseases. No vaccine is yet
available for the prevention or treatment 4. is an example for
of Zika virus infection. Development of passenger viruses
a Zika vaccine remains an active area of a. HIV virus b. EB Virus
research. c. Rabies virus d. None of these
5. The process of elution is caused by
Summary enzyme
This chapter dealt with the history, a. Neuramidase b. Isomerase
morphology, chemical properties, viral
c. Polymerase d. None of these
replication, virus classification, cultivation
and detection of cytopathic effects of 6. Beta propiolactone (BPL. Vaccine is
virus. Most important viruses such as given for
Adeno, herpes, hepatitis, Influenza, rabies, a. HIV virus b. Influenza Virus
HIV and Arbo virus, its morphology, c. Rabies virus d. None of these
classification, pathogenesis and its 7. is an example for
laboratory diagnosis were discussed. mosquito-borne and tick borne
viruses
Evaluation a. Dengu virus b. Flavi virus
Multiple choice questions c. Chikungunya virus d. None of these
1. is an
example for smallest Answer the following
virus 1. What is Virology?
a. Pox virus 2. Define virion.
b. Parvo virus 3. Which is the largest virus?
c. Rabies virus 4. What is Nucleocapsid?
d. HIV virus 5. Brief note on Steps involved in viral
2. CPE stands for multiplication.
a. cytoplasmic effects 6. What is Viropexis?
7. Define Abortive infective.
228

8. What are Prions? 21. Note on Viral gene and antigen of
9. Write Short note on cultivation of HIV
Virus. 22. Write about the Clinical features of
10. What are Cytopathogenic Virus? HIV
11. Classification of Herpes virus. 23. Give the Lab diagnosis of HIV
12. Discuss on HSV-1 and HSV-2 24. Define Arbo Virus
13. Expand VZV 25. Give the Symptoms of hermorrhagic
fever.
14. What is EB Virus?
26. Short note on Chikungunya virus
15. Define Dane particle
27. Account on Mosquito borne virus
16. Genome structure of HBV
28. Write about the Structure of Zika
17. Give the Structure of Rabies Virus
Virus
18. What is Furious and dumb rabies?
29. What is Zika fever
19. Define Negri bodies
20. Discuss on Vaccine for rabies
229

Chapter
11 Immunology
foreign antigens, such as

Learning Objectives microbes (organisms such
After studying this chapter the as bacteria, fungi, and
parasites), viruses, cancer
cells, and toxins. The
• Understand the Antigen Antibody immune system works by
reactions recognising the difference between one’s
• Know the principle behind Western own body cells and alien cells, allowing
Blot techniques it to destroy anything that could be
potentially harmful. Immune deficiency
• Learn about Hypersensitivity diseases decrease the body’s ability to
• Gain knowledge about fight invaders, causing vulnerability to
Transplantation infections. In the previous year, we have
• Know Immunization/Vaccination elaborately discussed with the main
components and function of the immune
• Appreciate the Updated National
system. This chapter deals with the role of
Immunization Schedule chart. Immune system in both health and disease.
Chapter Outline 11.1 Antigen Antibody Reactions

The interaction between antigen and
11.1 Antigen Antibody Reactions antibody is called antigen-antibody
11.2 Western Blot Techniques reactions. It is abbreviated as Ag-Ab
reaction. This reaction is the basis of
11.3 Hypersensitivity
humoral immunity. The antigen and the
11.4 Transplantation antibody react to form immune complex.
11.5 Immunization/Vaccination Ag + Ab ------------ Ag − Ab complex
11.6 Updated National Immunization The reaction between antigen and
Schedule Chart antibody is highly specific. It is compared
to the lock and key system. The part of the
The immune system refers to a collection antigen that combines with the antibody
of cells and proteins that function to is called epitope or antigenic determinant.
protect the skin, respiratory passages, The part of antibody which combines with
intestinal tract and other areas from the antigen is called paratope or antigen
230

Figure 11.1: Methods in Immunofluorescence
determining site. Most of the antibodies the antibody labelled with the fluorescent
have two binding sites and IgM has 5–10 dye is added to the tissue. Anti-antibody
binding sites. specifically binds with already added or
linked unlabelled antibody (Figure 11.1).
Immunofluorescence
When antibodies are mixed with the Sandwich Method
fluorescent dyes such as fluorescein or
This is an immuno fluorescence method
rhodamine, they emit radiation. This
used to test the number of cells producing
phenomenon of emitting radiation by
antibodies for a specific antigen. In this
antibodies labelled with fluorescent
method, lymphocytes are fixed with
dye is called immuno fluorescence.
ethanol. These fixed cells are treated with
This reaction is well observed under
polysaccharide antigen of Pneumococcus.
fluorescent microscope. It is used to locate
This antigen combines with those
and identify antigens in tissues.
lymphocytes which have the capacity to
Types of Immunofluorescence produce antibody against pneumococcal
• Direct method antigen. Now fluorescent antibody is added.
• Indirect method Antigen is sandwiched between antibodies.
• Sandwich method
ELISA (Enzyme Linked Immuno
Direct Method Sorbent Assay)
In this method, the antibody labelled with ELISA (Enzyme-Linked Immuno Sorbent
fluorescent dye is directly applied on the Assay) is a plate-based assay technique
tissue section. The labelled antibody binds designed for detecting and quantifying
with specific antigen. This can be observed substances such as peptides, proteins,
under the fluorescent microscope. antibodies and hormones. It is also known
as Enzyme Immuno Assay (EIA).
Indirect Method In 1971, after the descriptions of Peter
In this method, unlabelled antibodies Perlmann and Eva Engvall at Stockholm
are directly applied on the tissue sections University in Sweden, ELISA has become
which bind with the specific antigens. Then the system of choice when assaying
231

soluble antigens and antibodies. All assays The enzyme system consists of:
for antibody production depend upon the 1. An enzyme: Horse Radish
measurement of interaction of elicited Peroxidase(HRP), alkaline phosphatase
antibody with antigen. which is labelled or linked, to a specific
antibody.
Principle
2. A specific substrate:
The principle of ELISA is very simple. •• O-Phenyl-diamine-
The test is generally conducted in micro dihydrochloride for peroxidase
titre plates. (Figure 11.2 Micro titre plate). •• P Nitrophenyl Phosphate- for
If the antigen is to be detected the Alkaline Phosphatase
antibody is fixed in the micro titre plate Substrate is added after the antigen-
and vice versa. Test sample is added in antibody reaction. The enzyme hydrolyses
the microtitre plate, if there is presence the substrate to give a yellow colour
of Ag or Ab in the test sample, there will compound in case of alkaline phosphatase
be Ag-Ab reactions (with immobilized Ab (Figure 11.3). The intensity of the colour is
or Ag). Later enzyme labelled antibody is proportional to the amount of antibody or
added in the reaction mixture, which will antigen present in the test sample, which
combine with either test antigen or Fc can be quantified using ELISA reader
portion of test antibody. (Figure 11.4 ELISA reader)
Figure 11.2: Micro titre plate Figure 11.4: ELISA Reader
Figure 11.3: Basic steps in ELISA

232

Figure 11.5: Types of ELISA
Types added finally which is hydrolysed by the
enzyme which develops a colour.
There are four kinds of ELISA assay tests.
They are: Direct ELISA, Indirect ELISA, iii. Sandwich ELISA
Sandwich ELISA and Competitive ELISA Sandwich ELISA is used to detect antigen.
(Figure 11.5). A known antibody is coated on the micro
i. Direct ELISA titre plate. A test antigen is added to each
well and allowed to react with the bound
An antigen is immobilized in the well of an
antibody.
ELISA plate. The antigen is then detected
by an antibody directly conjugated to If the patient’s serum contains antigen
an enzyme such as HRP. Direct ELISA specific to the antibody, the antigen will
detection is much faster than other ELISA bind to the antibody. Specifically bound
techniques as fewer steps are required. antigen and antibody will remain in the
The assay is also less prone to error since wells even after washing. The second
fewer reagents and steps are needed, i.e. antibody is added and allowed to react
no potentially cross-reacting secondary with bound antigen. Substrate is added to
antibody needed. Finally, the direct ELISA measure colour reaction.
technique is typically used when the
iv. Competitive ELISA
immune response to an antigen needs to be
analyzed. It is used for the detection of antigens.
Antibody is first incubated with a
ii. Indirect ELISA sample-containing antigen. The antigen
Indirect ELISA is used to detect antibody. and antibody complex is added to the
A known antigen is coated on the micro antigen coated microtitre well. If more
titre plate. If the patient’s serum contains antigen present in the sample, the less
antibody specific to the antigen, the free antibody will be available to bind to
antibody will bind to the antigen. After the antigen coated well. Addition of an
incubation the wells are washed and the enzyme conjugated secondary antibody
enzyme labelled with anti Human Gamma specific to the primary antibody can
Globulin (HGG) is added to the well. Anti- be used to determine the amount of
HGG can react with antigen antibody primary antibody bound to the well. It is a
complex. The substrate for the enzyme is quantitative test for the antigen detection.
233

Application Steps
An ELISA test may be used to diagnose: Step I: Extraction of Protein
HIV, Lyme disease, pernicious anaemia, The most common protein sample
Rocky Mountain spotted fever, rotavirus, used for Western blotting is cell lysate.
squamous cell carcinoma, syphilis, The protein from the cell is generally
toxoplasmosis, varicella-zoster virus, extracted by mechanical means or by
which causes chickenpox and Zika virus. adding chemicals which can lyse the cell.
The extraction step is termed as tissue
11.2 Western Blot techniques preparation. Protease inhibitor is used to
Macromolecules immobilized or fixed prevent the denaturing of proteins. Using
on nitrocellulose membrane i.e., blotted spectroscopy the concentration of the
can be subjected to a variety of analytical protein sample is analysed and diluted in
loading buffer containing glycerol. This
techniques more easily. Southern blotting
will help the sample to sink in the well.
was the first blotting technique developed
Bromothymol blue is used as tracking dye
which made the analysis and recording
and is used to monitor the movement of
of DNA easy. Later the technique was
the sample.
extended for analysis of RNA and proteins
and they have acquired the jargon
Step II: Gel electrophoresis
terms Northern and Western Blotting
The protein sample is loaded in well of
respectively. Western blotting is also
SDS-PAGE Sodium dodecyl sulfate-
known as immunoblotting because it uses
poly-acryl amide gel electrophoresis.
antibodies to detect the protein. Western
The proteins are separated on the basis
blotting is a quantitative test to determine
of electric charge, isoelectric point,
the amount of protein in sample.
molecular weight, or combination of all
these. Proteins are negatively charged, so
Principle
they move toward positive (anode) pole
Western blotting technique is used for the as electric current is applied. Smaller
identification of a particular protein from proteins move faster than the larger
the mixture of proteins. In this method, proteins.
the proteins are first extracted from the
sample. Extracted proteins are subjected Step III: Blotting
to Poly Acryl amide Gel Electrophoresis Blotting refers to the transfer of the
(PAGE). Transfer of proteins from poly protein from the gel to the nitrocellulose
acryl amide to the nitrocellulose paper is paper by capillary action. Electro blotting
achieved by applying electric field. When is done nowadays to speed up the
radio labelled specific antibody is added process. In electro-blotting nitrocellulose
on such membrane it binds to the specific membrane is sandwich between gel and
complementary protein. Finally the cassette of filter paper and then electric
proteins on the membrane can be detected current is passed through the gel causing
by staining or through ELISA technique. transfer of protein to the membrane.
234

Step IV: Blocking Step VI: Treatment with suitable
The nitrocellulose membrane is non- substrate
specifically saturated or masked by using Finally, the reaction mixture is incubated
casein or Bovine serum albumin (BSA) with specific substrate. The enzyme
before adding the primary antibody. This convert the substrate to give visible
blocking step is very important in western coloured product, so band of colour can be
blotting as antibodies are also proteins and visualized in the membrane (Figure 11.6).
they are likely to bind to the nitrocellulose
paper. Application
Step V: Treatment with primary and 1. The size and concentration of protein
secondary antibody in given sample is determined by
western blotting.
The primary antibody is specific to desired
protein so it forms Ag-Ab complex. The 2. It is used in the detection of antibody
secondary antibody is enzyme labelled against virus or bacteria in serum and
and is against primary antibody (anti- helps in the disease diagnosis.
antibody) so it can bind with Ag-Ab 3. Western blotting technique is the
complex. Alkaline phosphatase or confirmatory test for HIV. It detects
Horseradish peroxidase (HRP) is labelled anti HIV antibody in patient’s serum.
with secondary antibody. 4. Useful to detect defective proteins.
Figure 11.6: Western blot technique

235

11.3 Hypersensitivity Type I: Immediate (Atopic or
anaphylactic) Hypersensitivity
Hypersensitivity is defined as the
exaggerated immunological response This type of hypersensitivity is an allergic
leading to severe symptoms and even death reaction provoked by the re-exposure to
in a sensitized individual when exposed for a specific antigen. The antigen can make
the second time. It is commonly termed as its entry through ingestion, inhalation,
allergy. The substances causing allergic/ injection or direct contact. The reaction
hypersensitivity is known as allergens. may involve skin, eyes, nasopharynx and
Example:. Drugs, food stuffs, infectious gastrointestinal tract. The reaction is
microorganisms, blood transfusion and mediated by IgE antibodies (Figure 11.7).
contact chemicals. IgE has very high affinity for its receptor
on mast cells and basophils. Cross linking
Classification of Hypersensitivity of IgE receptor is important in mast
(Coombs and Gell Classification) cell trigerring. Mast cell degranulation
is preceded by increased Ca++ influx.
Type I: Immediate (Atopic or anaphylac-
Basophils and mast cells release
tic) Hypersensitivity
pharmacologically active substances such
Type II: Antibody–dependent Hypersen- as histamines and tryptase. This causes
sitivity inflammatory response. The response is
Type III: Immune complex mediated immediate (within seconds to minutes).
Hypersensitivity Hence, it is termed as immediate
Type IV: Cell mediated or delayed Hyper- hypersensitivity. The reaction is either
sensitivity local or systemic.
Type I Anaphylactic Hypersensitivity (Acute)
Allergen Allergen
IgE
IgE receptor
IgE
APC
production
B cell
Mast cell
Presentation TCR basophil
to T helper
cells Degranulation
IL-4
IL-5 histamines, leukotrienes
TH2 prostaglandins, PAF
IL-13
Vasodilation
Wheal and flare
• Edema
• Redness
• Itching
Figure 11.7: Type I hypersensitivity

236

Hay Fever
Allergic rhinitis is commonly known
as hay fever. Allergic rhinitis develops
when the body’s immune system becomes
sensitized and overreacts to something in
the environment like pollen grains, strong
odour of perfumes, dust etc. that typically
causes no problem in most people. When
a sensitive person inhales an allergen the
body’s immune system may react with the
symptoms such as sneezing, cough and Figure 11.8: Type II hypersensitivity
puffy swollen eyelids.
Type II Hypersensitivity: Antibody Type III Hypersensitivity: Immune

dependent hypersensitivity complex mediated hypersensitivity
In this type of hypersensitivity reactions When a huge amount of antigen enters
the antibodies produced by the immune into the body, the body produces higher
response binds to antigens on the patient’s concentrations of antibodies. These
own cell surfaces. It is also known as antigens and antibodies combine together
cytotoxic hypersensitivity and may affect to form insoluble complex called immune
variety of organs or tissues. Ig G and Ig complex. These complexes are not
M antibodies bind to these antigens and completely removed by macrophages.
form complexes. This inturn activates These get attached to minute capillaries
the classical complement pathway and of tissues and organs such as kidneys,
eliminates the cells presenting the foreign lung and skin (Figure 11.9). These
antigen. The reaction takes hours to day antigen-antibody complexes activate the
(Figure 11.8). classical complement pathway leading to
vasodilation. The complement proteins
Drug induced haemolytic anaemia
and antigen-antibody complexes attract
Certain drugs such as penicillin, leucocytes to the area. The leukocytes
cephalosporin and streptomycin can discharge their killing agents and promote
absorb non-specifically to protein on massive inflammation. This can lead to
surface of RBC forming complex similar tissue death and haemorrhage.
to hapten-carrier complex. In some
patients these complex induce formation Arthus reaction
of antibodies, which binds to drugs on It was first observed by Arthus. It is a local
RBC and induce complement mediated immune complex reaction occurring in
lysis of RBC and thus produce progressive the skin. Horse serum and egg albumin
anaemia. This drug induced haemolytic are the antigens that induce the arthus
anaemia is an example of Type II reaction. It is characterized by erythema,
hypersensitivity reaction. induration, oedema, haemorrhage and
237

Experimentally induced Type III hypersensitivity
Locally injeceted antigen Activation of complement releases Local inflammation, movement of

in immune individual Local immune-complex inflammatory mediators C5a, C3a fluid and protein into tissue and
with IgG antibody formation and C4a. C5a also induces blood vessel occlusion
mast-cell degranulation
1-2 hours
Immune complex deposition in the skin and resultant

pathology is known as the Arthus reaction
Figure 11.9: Type III hypersensitivity
necrosis. This reaction occurs when days to develop. Type IV hypersensitivity

antibody is found in excess. It appears in is involved in the pathogenesis of many
2–8 hours after injection and persists for autoimmune and infectious diseases such
about 12–24 hours (Table 11.1). as tuberculosis and leprosy. T lymphocytes,
monocytes and macrophages are involved
Type IV hypersensitivity: Cell Mediated
in the reaction. Cytotoxic T Cells cause
Delayed Hypersensitivity
direct damage whereas the T helper cells
It is often called as delayed hypersensitivity secrete cytokines and activate monocytes
reaction as the reaction takes two to three
Table 11.1: Difference between Immediate Hypersensitivity and Delayed Hypersensitivity

Sn. No. Immediate Hypersensitivity Delayed Hypersensitivity
1. It appears and disappears rapidly It appears slowly and last longer.
2. It is induced by antigens or haptens by Induced by infection, injection
any route of antigen intra dermally or with
adjuvants of by skin contact.
3. The reaction is antibody mediated The reaction is T-cell mediated
B-cell response response.
4. Passive transfer is possible with serum Cannot be transferred with serum but
can be transferred by lymphocytes
5. Desensitization is easy, but does not Desensitization is difficult but long
last long lasting.
238

A. Delayed-type hypersensitivity
Cytokines Inflammation
CD4+
T cell
CD8+
T cell
APC presenting
tissue antigen Tissue injury
Normal
tissue
B. T cell-mediated cytolysis
CD8+
CTLs
Cell killing and

tissue injury
Figure 11.10: Type IV delayed hypersensitivity
and macrophages and cause the bulk If the graft is placed into its normal
damage (Figure 11.10). anatomic location, the procedure is called
orthotopic transplantation. If the graft
Tuberculin reaction (Mantoux Reaction)
is placed in a different site it is called
When a small dose of tuberculin is injected heterotopic transplantation.
intra dermally in an individual already
Transplantation is the only form of
having tubercle bacilli, the reaction
treatment for most end-stage organ failure.
occurs. It is due to the interaction of
In clinical practice, transplantation is used
sensitized T cell and tubercle bacterium.
to overcome a functional and anatomic
The reaction is manifested on the skin
deficit in the recipient. Transplantation
very late only after 48–72 hours.
of kidneys, hearts, livers, lungs, pancreas
11.4 Transplantation and bone marrow are widely done today.
Transfer of living cells, tissues or organs Methods of Transplantation

from one part of the body to another or
•• Auto grafting: The transfer of self
from one individual to another is known
tissue from one body site to another in
as transplantation.
the same individual
A tissue or organ that is removed from
•• Allografting: The transfer of organs or
one site and placed to another site usually
tissues from human to human
in a same or different individual is called
graft. The individual who provides the •• Xenografting: The transfer of
graft is called donor and the individual who tissue from one species to another
receives the graft is called host or recipient. (Figure 11.11).
239

Allograft Rejection
Types of allograft rejection
•• Acute rejection–Quick graft rejection.
It is due to stimulation of thymocytes
and B lymphocytes
•• Hyperacute rejection–It is a very quick
rejection. It is due to pre-existing
humoral antibodies in the serum of
the host as a result of presensitization
with previous grafts.
•• Insidious rejection–It is a secret
rejection due to deposition of immune
complex on the tissues like glomerulus
membrane that can be demonstrated
Figure 11.11: Types of Grafting in kidney by immune fluorescence.
Graft Acceptance Mechanism of Allograft Rejection
When transplantation is made between Immunological contact

genetically identical individuals the When tissue is implanted the graft as can
graft survives and lives as healthy as pass into local lymph nodes of the host.
it is in the original places. When the The graft antigens then make contact with
graft tissue remains alive, it is said to be the lymphocytes of the host. Production of
accepted and the process is called graft sensitized T cells and cytotoxic antibodies
acceptance. are produced in the host. This brings
about graft rejection.
Graft Rejection
First set rejection
When transplantation is made between When the graft is made between genetically
genetically distinct individual the graft different individuals, the graft gets blood
tissue dies and decays. When the graft supply from the host and it appears to
tissue dies, the graft is said to be rejected be normal for the first 3 days. But on the
and the process is called graft rejection. It 5th day, sensitized T cells, macrophages
is of two types. They are and a few plasma cells invade the graft.
i. Host Verses Graft Reaction Inflammation starts in the graft. This leads
ii. Graft Verses Host Rejection. to necrosis. It is similar to the primary
immune response to an antigen.
Host Verses Graft Reaction (HVG) Second set rejection
The graft tissue antigens induce an When a graft is implanted in an individual
immune response in the host. This type who has already rejected a graft is second
of immune response is called host versus set rejection. This is similar to the
graft reaction. secondary immune response of our body.
240

Cell mediated cytotoxic reaction cell. Activated T cell further activates the
The 1st set of rejection of allograft is B cells. The stimulated B cell reacts with
brought about mainly by CMI response. the self antigen and causes the damage.
In this process the cells involved in the How to prevent graft rejection?
cytotoxic mediated immunity involves.
Before transplantation the following
On stimulation of these cells interferon
things should be done to avoid graft
causes the lysis of the graft.
rejection.
Antibody mediated cytotoxic reaction •• Perform blood grouping and Rh
The 2nd set rejection of graft is brought grouping
about mainly by HMI response. This is •• HLA typing should be done
one of the hyperacute rejection brought •• Immuno suppressive drugs should be
about by the antibodies. Complement, administered
macrophages, mast cells, platelets, B cells •• Suitable graft should be used
bring about this reaction.
11.5 Immunization/Vaccination
Graft versus Host Rejection (GVH)
Father of Immunization is Edward Jenner.
Sometimes the graft tissue elicits an
He produced the vaccine for small pox
immune response against the host
from cow pox virus.Vaccine is a substance
antigens. This immune response is called
that is introduced into the body to prevent
graft versus host reaction. It occurs when:
the disease produced by certain pathogens.
•• Graft remains inside the host and the Vaccines consist of dead pathogens or live
host should not reject the graft. but attenuated (artificially weakened)
•• The graft should have immune organisms.
competent T cells. Immunization programmes and the
•• The transplantation antigens of the development of new vaccines play an
host should be different from that of important role in protecting individuals
the graft. against illness. Vaccination works by
safely exposing individuals to a specific
A transplanted heart pathogenic microbe, artificially increasing
usually beats slightly their immunity to it.
faster than normal
because the heart Vaccines are made from
nerves are cut during surgery. •• Live micro-organisms that have been
‘treated’ so that they are weakened
Mechanism of the graft (attenuated) and are unable to cause
disease.
The graft lymphocytes aggregate in the host
lymphoid organs and are stimulated by the •• Dead micro-organisms.
lymphocytes of the host. The stimulated •• Some part or product of the micro-
lymphocytes produce lymphokines. organism that can produce an immune
Lymphocytes in turn activate the host T response.
241

Principles of Vaccination Vaccine Types
•• The primary goal in vaccination is •• Live attenuated vaccines: These
to provide protective immunity by vaccines contain modified strains of
inducing a memory response to an a pathogen that have been weakened
infectious microorganism using a but are able to multiply within the
non-toxic antigen preparation. It is body and remain antigenic enough
important to produce immunity of the to induce a strong immune response.
appropriate kind: antibody / or cellular Example: Oral Polio vaccine
immunity.
•• Heterologous vaccine: These are
•• Antibodies produced as a result of
a group of live attenuated vaccines
immunization are effective primarily
produced from the strains that are
against extracellular organisms and
pathogenic in animals and not in
their products e.g., toxins. Passively
humans. It is a vaccine that confers
administered antibodies have the same
effect as induced antibodies. protective immunity against a
pathogen that shares cross-reacting
•• Cell-mediated immunity (T cells,
antigens with the microorganisms in
macrophages) induced by vaccination
the vaccine. Example: Cow pox virus
is important particularly in preventing
that protects against small pox in
intracellular bacterial and viral
humans.
infections and fungal infections.
•• The ultimate goal of any immunization •• Killed inactivated vaccines: These
program is the eradication of the groups of vaccine are produced either
disease. by killing or inactivating the bacteria
or virus by chemical treatment or
•• This requires that the infection is
heat. Example:; Polio virus
limited only to humans, with no
animal or environmental reservoir, •• Sub unit vaccine: Uses the antigenic
and the absence of any subclinical or determinant / epitope (the very
carrier state in humans. specific part of the microbe) is used
•• Achieving elimination requires a high to prepare the vaccine.
level of herd immunity to prevent •• DNA Vaccines: When the genes for
person to person spread. microbe’s antigens are introduced into
•• This requires considerable the body some cells will take up the
infrastructure support to ensure that DNA. The DNA then instructs those
all at-risk populations are targeted for cells to make the antigen molecules.
immunization. The cells secrete the antigens adn
•• This has been achieved for small pox, display them on their surfaces. The
although we are close to the elimination body’s own cells become vaccine
of polio. generating factories.
242

Routes of Administration globulins from a person who is already
•• Deep – most vaccines immunized to a non- immune person.
subcutaneous or •• Passive immunization is the
intramuscular administration of preformed
route antibodies either intravenously or
•• Oral route – Oral BCG intramuscularly.
vaccine •• It is used to provide rapid protection
•• Intradermal route – BCG vaccine in certain infections such as diphtheria
•• Scarification – Small pox or tetanus or in the event of accidental
vaccine exposure to certain pathogens such as
•• Intranasal route – Live attenuated hepatitis B.
influenza virus •• It is also used to provide protection in
Types of Immunization immune compromised individuals.
Immunization is of two types: Passive natural immunization -

acquired from the mother before and after
1. Passive Immunization
birth. Before birth, immunity is transferred
2. Active Immunization
from mother to the fetus in the form of
1. Passive Immunization maternal antibodies through placenta. After
•• Passive immunization is produced birth, the antibodies (Ig A) are transferred
without challenging the immune through breast milk (Table 11.2).
system of the body. It is done by Passive artificial immunization -
administration of serum or gamma developed by injecting previously prepared
Table 11.2: Passive Immunization

Infection Source of Antiserum Indications
Tetanus Immune human; horse Post exposure (plus vaccine)
Diptheria Horse Post-exposure
Gas gangrene Horse Post-exposure
Botulism Horse Post-exposure
Varicella-Zoster Immune human Post-exposure in immunodefi-
ciency
Rabies Immune human Post exposure (plus vaccine)
Hepatitis B Immune human Post-exposure prophylaxis
Hepatitis A Pooled human Ig Prophylaxis
Measles Immune human Prophylaxis
Snakebite Horse Post-bite
Some autoimmune Pooled human ig Acute thrombocytopenia and neutro-
disease penia
243

antibodies using serum from humans or most immunizations since nearly all
animals. This type of immunity is useful infectious agents gain entrance through
for providing immediate protection against the mucosal surfaces.
acute infections like tetanus, measles etc. Active natural immunization involves
2. Active Immunization activation of immune system in the body
to produce antibodies. It is achieved in
Active immunization is the
both clinical and subclinical infections.
administration of vaccines containing
microbial products with or without Active artificial immunization is
adjuvants in order to obtain long term achieved by the administration of vaccines
immunological protection against the or toxoids.
offending microbe. Antigen preparations
At present the normal route of Most vaccines consist of attenuated
vaccination in most instances is either organisms, killed organisms, inactivated
intramuscularly or subcutaneously. toxins, or sub cellular fragments and
Oral immunization is the method more recently genes for antigens in viral
of choice for polio and Salmonella ‘vectors’, and DNA itself. Thus, vaccines
typhi vaccines. However, there is an must be capable of targeting the immune
increasing awareness that this route system appropriately i.e. cellular/or
of immunization may be the best for humoral mechanisms (Table 11.3).
Table 11.3: Antigen Preparations Used in Vaccines
Examples
Type of antigen
Viruses Bacteria
Normal heterologous organism Vaccinia (Cowpox)
Measles BCG
Mumps Typhoid (New)
Rubella
Living attenuated organism
Polio (Sabin)
Yellow fever
Varicella-Zoster
Rabies Pertussis
Whole killed oranism Poli (Salk) Typhoid
Influenza Cholera
Diphtheria
Inactivated toxin (toxoid) Tetanus
Cholera (New)
Meningococcus
Pneumococcus
Capsular polysaccharide
Haemophilus
Typhoid (New)
Surface antigen Hepatitis B
244

Adjuvants having adjuvant activity, but at present
Nonliving vaccines, especially those only aluminium or calcium salts are
consisting of small molecules require generally used in humans.
the inclusion of agents to enhance their Adjuvants should enable antigens to be
effectiveness. slowly released, preserve antigen integrity,
These adjuvants include microbial, target antigen presenting cells and induce
synthetic and endogenous preparations cytotoxic lymphocytes.
Table 11.4: National immunization schedule

Sn. No. Vaccine Due age Max age Route
1. BCG At birth till one year age Intra dermal
2. Hepatitis B-Birth dose At birth within 24 hours Intra muscular
3. OPV-O At birth within the first Oral
15 days
4. OPV 1, 2 & 3 At 6 weeks, till 5 years of Oral
10 weeks & 14 age
weeks
5. Pentavalent 1, 2 & 3 At 6 weeks, 1 year of age Intra muscular
(Diphtheria + Pertuss is 10 weeks & 14
+ Tetanus + Hepatitis B weeks
+ Hib)
6. Inactivated polio At 6 & 14 weeks 1 year of age Intra muscular
vaccine
7. Rotavirus (where At 6 weeks, 1 year of age Oral
applicable) 10 weeks & 14
weeks
8. Pneumococcal At 6 weeks & 1 year of age Intra muscular
conjugate vaccine 14 weeks. At
(where applicable) 9 completed
months –
booster
9. Measles/Rubella 1st At 9 completed 5 years of age Subcutaneous
dose months – 12
months
10. DPT Booster-1 16–24 months 7 years of age Intra muscular
11. Measles/Rubella 2nd 16–29 months 5 years of age Subcutaneous
dose
12. OPV Booster 16–24 months 5 years Oral
13. DPT Booster – 2 5–6 years 7 years of age Intra muscular
14. TT 10 years & 16 16 years Intra muscular
years
245

11.6 Updated National the mixture of proteins. The most common
Immunization Schedule Chart protein sample used for Western blotting is
cell lysate. Blotting refers to the transfer of
Immunization/vaccination produce a
the protein from the gel to the nitrocellulose
response in the body that is similar to
paper by capillary action. The substances
the body’s response in the body that is
causing allergic/hypersensitivity is known
similar to the body’s response to a natural
as allergens. Allergic rhinitis develops
infection (Table 11.4). Immunization or
when the body’s immune system becomes
vaccines can therefore protect the body
sensitized and overreacts to something in
from a disease before the disease has a
the environment like pollen grains, strong
chance to cause illness. Immunization
odour of perfumes, dust etc. Certain drugs
has helped to reduce the impact of
such as penicillin, cephalosporin and
communicable disease on health and
streptomycin can absorb non-specifically
well being. Some diseases have been well
to protein on surface of RBC forming
controlled and other has been eliminated
complex similar to hapten-carrier complex.
from some parts of the world because of
vaccination. Stopping vaccination may Transfer of living cells, tissues or organs
lead to epidemic. from one part of the body to another or
from one individual to another is known as
Summary transplantation. The graft tissue antigens
induce an immune response in the host.
The reaction between antigen and antibody
This type of immune response is called
is highly specific. It is compared to the lock
host versus graft reaction. The ultimate
and key system. ELISA (Enzyme-Linked
goal of any immunization program is the
Immuno Sorbent Assay) is a plate-based
eradication of the disease. Active natural
assay technique designed for detecting and
immunization involves activation of
quantifying substances such as peptides,
immune system in the body to produce
proteins, antibodies and hormones.
antibodies. It is achieved in both clinical
There are four kinds of ELISA assay tests.
and subclinical infections Immunization
They are: Direct ELISA, Indirect ELISA,
has helped to reduce the impact of
Sandwich ELISA and Competitive ELISA.
communicable disease on health and well
Western blotting technique is used for the
being.
identification of particular protein from
246

Evaluation 7. produce antibodies.
Multiple choice questions a. T cells
1. Antibody reacts with b. B cells
to give agglutination. c. Ts cells
a. Particulate antigen d. Plasma cells
b. Hapten and antigen 8. Sabin is vaccine.
c. Antibody and a. Injection
soluble antigen b. Recombination
d. Carrier and antibody c. Oral
2. Anaphylaxis refers to d. Subunit
a. Immediate hypersensitivity 9. is an injectable
polio vaccine.
b. Hyposensitivity
a. Salk
c. Delayed hypersensitivity
b. TAB
d. Auto sensitivity
c. Sabin
3. Atopy occurs due to
d. BCG
a. House dust b. Egg
10. Immunisation done in a community
c. Pollen d. all the above is called immunity.
4. In type II reaction, a. Combined
is involved.
b. General
a. IgG antibody c. Local
b. IgG and IgM antibodies d. Herd
c. IgM antibody
d. IgE antibody Answer the following
5. acts as an ACP. 1. What do you understand by the term

antigen presentation?
a. Macrophage
2. Define: Pathogenicity.
b. RBC
c. T cells
a. MMR - Subunit vaccine
d. Mucosal cells
b. Salk - Triple vaccine
6. Phagocytosis is enhanced by
c. HBV - Recombinant vaccine
process.
d. Sabin - Killed vaccine
a. Pinocytosis
e. Influenzae - Live vaccine
b. Opsonisation
4. What is meant by attenuation?
c. Endocytosis
5. Describe toxin with examples.
d. None
247

6. Match: 7. Write a note on Lymphocytes.
a. Mast cell - Myelomaprotein 8. Define the following:
b. Primary - IgG
a. Immunity
immune
c. S econdary - IgM b. Innate immunity
immune c. Acquired Immunity
response d. Active immunity
d. S ecretory - IgA e. Passive immunity
antibody
e. Plasma cell - IgE
tumor
248

Chapter
12 Microbial Genetics
12.4 Translation
Learning Objectives
12.5 Types of Mutation
After studying this chapter the 12.6 Formation of Mutants
12.7 Transfer of Genetic Material
• Define gene, genome, genetic code,
12.8 Recombinant DNA Technology
genotype, phenotype, mutagen, wildtype
• Describe transcription and translation 12.9 Vectors – Types and
Characteristics
• Classify mutations and its types and
Understand how mutants are formed 12.10 Restriction Enzymes
• Know the mode of action of physical 12.11 Techniques in Genetic
and chemical mutagens Engineering
• Identify the purpose of and outline the 12.1 Concept of Gene
procedure for Ames test
The fundamental unit
• Compare the gene transfer mechanisms
of information in living
• Know the types of cloning vectors used systems is the gene.
in genetic engineering Genome is the set of all
• Describe how plasmids and genes and genetic signals
bacteriophages are used to transfer of a cell. The information
foreign DNA contained in genes is converted to molecules
• Explain the role of restriction enzymes that determine the metabolism, structure
in recombinant DNA technology and form of microorganisms. Gene is
• Know the types of restriction enzymes expressed through a sequence of events.
• Understand agarose gel electrophoresis A gene can be defined biochemically as a
and PCR techniques segment of DNA (or, in a few cases, RNA)
that encodes the information required to
• Explain RAPD and RFLP
produce a functional biological product.
The final product is usually a protein. Not
Chapter Outline all genes are involved in protein synthesis;
some code instead for rRNA and tRNA.
12.1 Concept of Gene
The central dogma of molecular biology,
12.2 Transcription
comprises the three major processes
12.3 Genetic Code (Figure 12.1). The first is replication, the
249

Nucleus
DNA
information
Replication
DNA replicates
DNA
information
Transcription
RNA synthesis
(+) Sense RNA

m RNA
RNA
information
Cytoplasm
Translation
Protein synthesis
Ribosomes
Protein
Protein
Figure 12.1: Central dogma of molecular biology
copying of parental DNA to form daughter a template. The DNA strand copied into
DNA molecules with identical nucleotide RNA molecule is called CODING OR
sequences. The information contained in SENSE STRAND.
the base sequence of DNA is copied into The synthesis of RNA consists of five
protein molecule through an RNA molecule. discrete stage (Figure 12.2):
The second is transcription, production of 1. Promoter recognition: RNA polymerase
mRNA from DNA. It is the process by which binds to DNA within a specific base
the segment corresponding to a particular sequence (20–200 bases long) called a
gene is selected and an RNA molecule is promoter. The sequence TATAAT (or a
synthesized. The third is translation, The nearly identical sequence) often called
production of an amino acid sequence from a pribnow box or – 10 region is found
an RNA base sequence. The genetic message as part of all prokaryotic promoters.
encoded in messenger RNA (mRNA) is The RNA polymerase of the
translated on the ribosomes into a polypeptide bacterium E.coli consists of five protein
with a particular sequence of amino acids. subunits. Four of the subunits comprise
The order of amino acid in a polypeptide the core enzyme (catalyzes the joining
chain is determined by DNA base sequence. of the nucleoside triphosphates to the
RNA) and fifth subunit, the σ subunit
12.2 Transcription (required for promoter binding).
An important feature of RNA synthesis is 2. Local unwinding of DNA occurs
that even though the DNA molecule being and RNA polymerase forms an open
copied is double stranded, in any particular promoter complex.
region of DNA only one strand serves as
250

Transcription
Unit Termination
Promoter (Gene to be tansaited) Site
CODING STRAND once RNA polymerase has polymerized
′
5
DNA ′
3
50–60 nucleotides. In bacteria most mRNA
DNA Template
molecules are degraded within a few
Initiation minutes after synthesis. This degradation
(Binding of RNA Polymerase)
enables cells to dispense with molecules
5′ 3′
that are no longer needed.
3′
RNA Polymerase
5′
In prokaryotes mRNA molecules
ELONGATION
commonly contain information for the
(sigma factor
dissociates from
amino acid sequences of several different
5′
polymerase)
3′ polypeptide chains. In this case, such a
3′ molecule is called polycistronic mRNA.
5′ Cistron is a term used to mean a base
TERMINATION
sequence encoding a single polypeptide
chain. The genes contained in polycistronic
5′
mRNA molecule (Figure 12.3) often encode
3′ 5′
the different portions of a metabolic
pathway. For example, in E. coli the ten
enzymes needed to synthesize histidine are
encoded in one mRNA molecule.
5′ 3′
NEWLY 3’ PROMOTER OPERATOR CISTRON 1 CISTRON 2 CISTRON 3 5’

SYNTHESIZED RNA
(RNA TRANSCRIPT)
MULTIPLE TRANSLATION START SITE
Figure 12.2: Major events in transcription POLYCISTRONIC

mRNA
5’ 3’
3. The first nucleoside triphosphate is placed

at polymerization start site (near to the
Protein 1 Protein 2 Protein 3
SINGLE TRANSLATION START SITE

initial binding site) and synthesis begins. EUKARYOTIC
5’ 3’
MRNA
4. RNA polymerase then moves along the MONOCISTRONIC
DNA, adding ribonucleotides, to the

growing RNA chain. Protein 1
5. RNA polymerase reaches chain

Figure 12.3: Polycistronic mRNA
termination sequence and both the
newly synthesized RNA and the In prokaryotes the immediate product
polymerase are released. Two kinds of of transcription (called the primary
termination events are known those transcript) is mRNA, in contrast in
that are self – terminating (dependent eukaryotes the primary transcript must
on the base sequence only) and be converted to mRNA. This conversion
those that require the presence of the called RNA processing consists of two types
termination protein Rho. of events- modification of termini and
Initiation of a second round of excision of untranslated sequences (non-
transcription need not await completion of coding sequence or introns) embedded
the first, for the promoter becomes available
251

within coding sequences (exons). Introns bases can form only 42 = 16 pairs, and
excision and the joining of exons to there are 20 amino acid. Triplets of bases
form an mRNA molecule is called RNA would suffice because, these can form 43
splicing. The introns are present in almost = 64 triplets. In fact, the genetic code is
all eukaryotic transcripts but are rare in a triplet code, and all 64 possible codons
the free – living unicellular eukaryotes carry information of some sort. Several
such as yeast. Some bacterial genes do different codons designate the same amino
contain introns. acid. Furthermore, in translating mRNA
molecules the codons do not overlap but
Synthesis of rRNA and tRNA are used sequentially. The same genetic
Ribosomal RNA and tRNA are also code is used by almost all biological
transcribed from genes. The production systems and hence is said to be universal
of these molecules is not as direct as (exceptions are mitochondria and a few
synthesis of bacterial mRNA. The main unusual microorganisms). The codons are
difference is that these RNA molecules are by convention written with the 5′ end at
excised from large primary transcripts. the left. The complete code is shown in
Highly specific RNA excise rRNA and Table 12.1.
tRNA from these large transcripts, and
other enzymes produce the modified Features of the Code:
bases in tRNA. •• Sixtyone codons correspond to amino
acids. Four codons are signals. These
12.3 Genetic Code are the three stop codons – UAA, UAG,
A tRNA molecule “reads” the base UGA – and the one start codons, AUG.
sequence of mRNA. The language read by The start codons (initiation codon) also
the tRNA molecules is called the genetic specifies the amino acid methionine.
code, which is a set of relations between In rare cases, certain other codon (E.g.
sequences of three adjacent bases on an GUG) initiate translation. No normal
mRNA molecule and particular amino tRNA molecule has an anticodon
acids. An RNA base sequence (a set of (a sequence of three bases on tRNA that
3 bases) corresponding to a particular can base – pair with a codon sequence
amino acid is called a codon. The genetic in the mRNA) complementary to
code is the set of all codons. Only four any of the stop codons UAG, UAA or
bases in DNA serve to specify 20 amino UGA, which is why these codons are
acids in proteins, so some combination of stop signals.
bases is needed for each amino acid. Before •• The code is highly redundant i.e. more
the genetic code was elucidated, it was than one codons code for an amino
reasoned that if all codons were assumed acid. Only tryptophan and methionine
to have the same number of bases, then are specified by one codon. The
each codon would have to contain at least synonymous codons usually differ
three bases. Codons consisting of pairs of only in third base (except for serine,
bases would be insufficient because four leucine and arginine).
252

The 20 amino acids and their
abbreviations
Amino acid 3-letter
abbreviation
Alanine Ala
Arginine Arg
Asparagine Asn
Aspatic acid Asp
Aspartic acid & Asx
Asparagine
Cysteine Cys
Glutamine Gln
Glutamic acid Glu
Glutaine or Glx
Glutamic acid
Glycine Gly
Histidine His
Isoleucine IIe
Leucine Leu
Lysine Lys
Methionine Met
Table 12.1: Genetic code
Not all the base sequences in an mRNA

HOTS is translated into amino acid sequences
of polypeptides. Initiation of polypeptide
1. How many of the 64 codons can be synthesis may begin hundreds of
made from the three nucleotides nucleotides from the 5′ – P terminus of
A, U, and C? the RNA. The section of untranslated
RNA before the region encoding the
2. If codons were four bases long,
first polypeptide chain is called a leader,
how many codons would exist in a
which in some cases contains regulatory
genetic code?
sequences that influence the rate of protein
synthesis. The major events in translation
12.4 Translation are (Figure 12.4).
RNA is translated 1. An mRNA binds to the surface of
from the 5′ end of the a protein synthesizing particle, the
molecule toward the 3′ Ribosome.
end. Polypeptides are 2. The tRNA – amino acid complexes
synthesized from the (made by the aminoacyl tRNA
amino terminus toward synthetases) bind sequentially, one
the carboxyl terminus, by adding amino by one, to the mRNA molecule that is
acids one by one to the carboxyl end. attached to the ribosome.
253

Large
Amino acid
subunit
Initiator tRNA tRNA
U A C
A U C G A UG A U A G C U C G A C
A C A U A A
Small
subunit Anticodon
2 Large and small ribosomal U A C

subunits join to form a functional
A U C G A UG A U A G C U C G A
ribosome. A C A U A
Amino acid mRNA

Codons
Initiator tRNA
3 Anticodon of Incoming tRNA pairs
Anticodon with next mRNA codon beside
mRNA initiator tRNA.
U A C
A U C G A U G A U A G C U C G A
A C A U A
mRNA Small
binding subunit
Start
site
codon
1 Initiator tRNA A U C
attaches to a A U C A U G U AG A G C U C G
start codon. A C A U A A
New
peptide
bond
4 Amino acid on initator tRNA
forms a peptide bond with the
amino acid beside it.
A U C
A U G U A G C U G C UG
A U C G G A A U C
C
A U A U C G G A U U A GG C U C G A
A C UA G
Stop mRNA
codon movement
6 Protein synthesis stops when 5 Intiator tRNA leaves the ribosome;

the ribosome reaches the stop ribosome shifts by one codon;
codon on mRNA. tRNA binds to newly exposed
codon; steps 3 repeat.
5
Key: Growing
mRNA protein Complete protein
A = Adenine
G = Guanine
C = Cytosine
U = Uracil
summary of movement of ribosome along mRNA
Figure 12.4: Major events in Translation
3. Peptide bonds are made between •• When an amino acid has become
successively aligned amino acids. attached to a tRNA molecule, the tRNA
4. Finally the chemical bond between the is said to be acylated or charged
tRNA and its attached amino acids is •• An important feature of initiation
broken and the completed protein is of polypeptide synthesis in both
removed. prokaryotes and eukaryotes is the use
•• The 3′ terminal of the tRNA molecule of a specific initiating tRNA molecule.
(Figure 12.5) is covalently linked to In prokaryotes this tRNA molecule is
the amino acid corresponding to the acylated with the modified amino acid
particular mRNA codon N – formyl methionine (fMet). This
254

Amino acid
3′ Redundancy and the Wobble Hypothesis
5′ The identity of the third base of a codon
appears to be unimportant. (The first base
in a codon is at the 5′ end and the third base
is at the 3′ end). Wobble refers to the less
T loop
D loop stringent requirement for base pairing at the
third position of the codon than at the first
two positions. That is the first two bases must
Anticodon loop
follow Watson and Crick base pairing rule
(A with U, or G with C), but the third base
Anticodon CUC pair can be of a different type (for example,
GU C C A G G A G CC A U A G G with U). The Wobble hypothesis explains
3′
5′
mRNA the pattern of redundancy in the code in
Codon
that certain anticodons. (For example, those
Figure 12.5: tRNA containing U and G in the first position of
the anticodons) can pair with several codons
tRNA is often designated tRNAfMet. during translation (Figure 12.6).
Both tRNAfMet and tRNAMet recognize
the codon AUG, but only tRNAfMet 12.5 Types of Mutations
is used for initiation. All prokaryotic The base sequence of DNA determines
proteins while being synthesized the amino acid sequence of a protein. The
have fMet at the amino terminus. chemical and physical properties of each
However, this amino acid is frequently protein are determined by its amino acid
deformylated or removed later. sequence, so a single amino acid change
•• The usual form of translation unit is a is capable of altering the activity of, or
polyribosome or polysome wherein an even completely inactivating, a protein.
mRNA is covered with ribosomes. Genotype refers to the genetic composition
Leu Wobble Hypothesis Leu 3
5
3 3
5 5
Identical 1 2 3
leucine 5 mRNA 3
tRNAs
if these bases are in
if these bases are in
first , or wobble,position
third , or wobble,position
of codon of mRNA
of codon of mRNA
C A G U C A G U U
G U C A then the condon may G U C A C then the condon may

be recognized by a
be recognized by a
I I U G tRNA having these I U G A tRNA having these
bases in third position
bases in first position
I of anticodon U of anticodon
G A G G A G
. . . . . .
mRNA . . . Normal pairing mRNA . . . wobble pairing
. . . . . .
C C C C C U
5... ...3 5... ...3
Figure 12.6: Wobble hypothesis

255

is altered.The alteration can be a single base
Infobits pair substitution, insertion or deletion.
In 1970 Howard Temin and David Mutations can be divided into two
Baltimore independently discovered general categories:
the enzyme reverse transcriptase that 1. Base–pair substitution mutation
retroviruses use to produce DNA copies involves a change in the DNA such that
of their RNA genome. This enzyme can one base pair is replaced by another.
be used to construct DNA copy, called •• A mutation from one purine –
complementary DNA (cDNA), of any pyrimidine base pair to the other
RNA as shown in figure below . Thus purine –pyrimidine base pair is a
genes or major portions of the gene can transition mutation (Figure 12.7a).
be synthesized from mRNA. E.g. AT to GC, CG to TA.
cDNA from mRNA
•• A mutation from a purine -
RNA Poly(A) tail
5 3 pyrimdine base pair to a pyrimidine
AAAAAAAA
TTT T – purine base pair is a transversion
Viral reverse Oligo (dT) mutation (Figure 12.7b). E.g. AT to
transcriptase primer
RNA TA, CG to GC.
5
AAAAAAAA 2. Base pair insertion or deletions
T T T T 5
3
cDNA involves the addition or deletion of
Hairpin loop one base pair. If one or more base pairs
NaOH degrades mRNA
are added to or deleted from a protein
coding gene, the reading frame of an
T T T T 5 mRNA can change downstream of the
3
mutation. An addition or deletion of
DNA polymerase I one base pair, for example, shifts the
cDNA mRNA’s downstream reading frame
5 by one base, so that incorrect amino
acids are added to the polypeptide
S1 nuclease
chain after the mutation site. This
(single strand-specific) type of mutation, called a frame shift
mutation (Figure 12.8) usually results
3 5
in a nonfunctional protein.
5 3
Double-stranded cDNA
Frame shift mutations:
•• May generate new stop codons,
resulting in a shortened protein.
of an organism. Phenotype is an observable
property of organism. The functional •• May result in a read through of the
normal stop codon, resulting in longer
form of a gene is called Wildtype because
than normal proteins
presumably this is the form found in nature.
• • Or may result in a complete alteration
Mutation is the process by which the of the amino acid sequence of a
sequence of base pairs in a DNA molecule protein.
256

Sequence of part of a normal gene Sequence of mutated gene
(a) Transition mutation (AT to GC in this example)
5′ TCTCAAAAATTTAGG 3′ 5′ TCTCAAGAATTTACG 3′
3′ AGAGTTTTTAAATGC 5′ 3′ AGAGTTCTTAAATGC 5′
(b) Transversion mutation (CG to GC in this example)
5′ TCTCAAAAATTTAGG 3′ 5′ TCTGAAAAATTTACG 3′
3′ AGAGTTTTTAAATGC 5′ 3′ AGACTTTTTAAATGC 5′
Figure 12.7:
(a) Transition mutations (b) transversion mutations
Wild-type
DNA template strand 3 T A C T T C A A A C C G A T T 5
5 A T G A A G T T T G G C T A A 3
mRNA 5 A U G A A G U U U G G C U A A 3
Protein Met Lys Phe Gly
Stop
Amino end Carboxyl end
Extra A A Missing
3 T A C A T C C A A A C C G A T T 5 3 T A C T T C A A C C G A T T 5T
5 A T G T A A G T T T G G C T A A 3 5 A T G A A G T T G G C T A A 3
Extra U U Missing
5 A U G U A A G U U U G G C U A A 3 5 A U G A A G U U G G C U A A 3
Met Met Lys Leu Ala
Stop
Frameshift causing immediate nonsense (1 base-pair insertion) Frameshift causing extensive missense (1 base-pair delection)
Figure 12.8: Frameshift mutations
Point mutations are single base the proteins function. Consequently, the
changes, that do not affect the reading phenotype does not change.
frame, that is, the mutation only makes
a single change in a single codon, and 3. A silent mutation (Figure 12.9c) is also
everything else is undisturbed. a subset of missense mutations that
Mutations can also be defined according occurs when a base – pair change in a
to their effects on amino acid sequences gene alters a codon in the mRNA such
in proteins. They are that the same amino acid is inserted
1. A missense mutation (Figure 12.9a) is in the protein. In this case, the protein
a gene mutation in which a base – pair obviously has a wild type function.
change in the DNA changes a codon 4. A nonsense mutation (Figure 12.9d) is
in an mRNA so that a different amino a gene mutation in which a base – pair
acid is inserted into the polypeptide. change in the DNA, changes a codon
2. A neutral mutation (Figure 12.9b) is a in an mRNA to a stop (nonsense)
subset of missense mutations in which codon (UAG, UAA or UGA).
the new codon codes for a different amino Nonsense mutation cause premature
acid that is chemically equivalent to the chain termination so instead of
original and therefore does not affect complete polypeptides, shorter than
257

NORMAL DNA Template
DNA
A A A A T A C G T G C A Strand
U U U U A U G C A C G U
NORMAL mRNA
polypeptide
NORMAL Phe Tyr Ala Arg
Mutated template Mutated template

DNA Strand DNA Strand
A A A A T A C C T G C A A A G A T A C C T G C A
U U U U A U G C A C G U U U C U A U G C A C G U
Mutated mRNA Mutated mRNA
Phe Tyr Gly Arg Slightly different amino Phe Tyr Gly Arg NO change in amino acid
acid sequence sequence of polypeptide
A Missense mutationa C Silent mutation

Mutated template
Mutated template
DNA Strand
DNA Strand A A A A T T C C T G C A
A A A A T A C C T G C A U U U U A A G C A C G U
U U U U A U G C A C G U
Mutated mRNA
Mutated mRNA
Slightly different amino Phe STOP Polypeptide synthesis ceases

Phe Tyr Gly Arg CODON
acid sequence
B Neutral mutation D Nonsense mutation
Figure 12.9:
(a) Missense (b) neutral c) silent (d) nonsense mutation respectively
normal polypeptide fragments (often mutagenesis. In nature and in the laboratory,

nonfunctional) are formed. mutations sometimes arise spontaneously,
Forward mutations change the without any help from the experimenter.
genotype from wild type to mutant and This is called spontaneous mutagenesis. The
reverse mutations (or reversions or two mechanisms that are most important
back mutations) change the genotype for spontaneous mutagenesis are
from mutant to wild type or to partially 1. Errors occurring during replication
wild type. An organism which has and
reverted is Revertant. The effects 2. Spontaneous alteration of bases.
of mutation may be diminished or
Mutations can also be induced
abolished by a suppressor mutation.
experimentally by application of
Suppressor mutation is a mutation at a
mutagens. Mutagens are agents that cause
different site from that of the original
mutations.
mutation. A suppressor mutation masks
or compensates for the effects of the
Mutagens and their Mode of Action
initial mutation, but it does not reverse
the original mutation. Physical Mutagens
UV radiation: UV light causes mutations
12.6 Formation of Mutants because the purine and pyrimidine
bases in DNA absorb light strongly in
The term mutant refers to an organism in
the ultraviolet range (254 to 260 nm).
which either the base sequence of DNA or
At this wavelength, UV light induces
the phenotype has been changed. A mutant
point mutations primarily by causing
is an organism whose genotype differs
photochemical changes in the DNA. One
from that found in nature. The process of
of the effects of UV radiation on DNA
formation of mutant organism is called
258

is the formation of abnormal chemical substances. These mutagens can be
bonds between adjacent pyrimidine grouped into different classes on the basis
molecules in the same strand, or between of their mechanism of action. They are
pyrimidines on the opposite strands, of i. Base analogs are bases that are similar
the double helix. This bonding is induced to the bases normally found in DNA.
mostly between adjacent thymines, Like normal bases, base analogs exist in
forming what are called thymine dimmers normal and rare tautomeric states. In
(Figure 12.10), usually designated TT. each of the two states, the base analog
This unusual pairing produces a bulge pairs with a different normal base in
in the DNA strand and disrupts the DNA. Because base analogs are similar
normal pairing of T’s (thymines) with to the normal nitrogen bases, they may
corresponding A’s(adenines) on the be incorporated into DNA in place of
opposite strand. If UV induced genetic normal bases. E.g. 5 – bromouracil
damage is not repaired, mutations or cell (5-BU). If 5-BU is incorporated in its
death may result. more common normal state, it pairs
5
TT with adenine. If it changes into its rare
state during replication, it pairs with
3
AA guanine instead. In the next round of
replication, the 5-BU – G base pair is
DNA is damaged 1. Dimer forms
resolved into a C – G base pair instead
of the T – A base pair. By this process a
T T lesion
transition mutation is produced, from
TA to CG (Figure 12.11).
A A
PRE Not all base analogs
blue light
2. Dimer repaired are mutagens. For
example, AZT
T T
(Azidot hymidine),
one of the approved drugs given to
A A patients with AIDS, is an analog of
thymidine, but it is not a mutagen,
3. Normal pairing because it does not cause base pair
restored
changes.
TT
ii. Base Modifying Agents are chemical

AA that act as mutagens by modifying
the chemical structure and properties
Figure 12.10: UV induced DNA damage
of bases. The three types of mutagens
Chemical Mutagens that work in this way are
1. A deaminating agent e.g.: Nitrous
Chemical mutagens include both naturally
acid removes amino groups (- NH2)
occurring chemicals and synthetic from the bases guanine, cytosine, and
259

Add 5 BU
5BU shifts
to rate states
5BU shifts back

DNA to normal state
replication
DNA
replication
DNA
replication
Add 5 BU
5BU shifts
to rate states
DNA
replication
DNA
replication
DNA
replication
Figure 12.11: Mutagenic effects of 5-BU
adenine. When cytosine is treated only be CG to TA transitions

with nitrous acid it removes amino (Figure 12.12b).
group from cytosine which changes to 3. Alkylating agents like methymethane
uracil resulting in transition mutation sulfonate (MMS) introduces alkyl groups
(Figure 12.12a).
onto the bases at a number of location.
2. Hydroxylamine (NH 2 OH) is a For example after treatment with MMS,
hydroxylating mutagen that react some guanines are methylated at 6 –
specifically with cytosine, modifying oxygen to produce O6 – methyl guanine.
it by adding a hydroxyl group (OH)
The methylated guanine pairs with
so that it can pair solely with adenine
thymine rather than cytosine giving G C
instead of with guanine. Mutations
to AT transitions (Figure 12.12c).
induced by hydroxylamine can
Modified base Predicted

Original base Mutagen
Pairing partner transition
Nitrous acid (H2NO)
Cytosine Uracil Adenine CG TA
Hydroxylamine
Hydroxyl amino
Cytosine (NH2OH) CG TA
cystosine Adenine
Methylmethane
sulfonate(MMS)
Guanine O-Methylguanine Thymine CG TA
(alkylating agent)
Figure 12.12: Action of three base modifying agents.

260

iii. Intercalating agents agents can cause either additions or
Acridine, proflavin, ethidium bromide are deletions.
examples few examples of intercalating
The Ames Test: A Screen for Potential
agents. These insert (intercalate)
Carcinogens
themselves between adjacent bases in
Everyday we are exposed to a wide variety
one or both strands of the DNA double
of chemicals in our environment, such as
helix. Due to this an extra base (chosen at
drugs, cosmetics, food additives, pesticides,
random, G in the Figure 12.13a) must be
and industrial compounds. Many of these
inserted into the new DNA strand opposite
chemicals can have mutagenic effects,
the intercalating agent. Intercalating
(a) Mutation by addition
Molecule of
intercalating agent
Template DNA strand 5′ 3′

AT C A G T T A C T
New DNA strand T A G T C G A AT G A
3′ 5′
A randomly chosen
base is inserted opposite
intercalating agent; here
the base is G
Subsequent replication
of new strand
5′ 3′
AT C A G C T T A C T
T A G T C G A AT G A
3′ 5′
(b) Mutation by deletion Results: frameshift mutation
due to insertion of
one base pair(CG)
5′ 3′
Template DNA strand
AT C A G T T A C T
New DNA strand T A G T C AT G A

3′ 5′
Intercalating agent
Replication of new strand after

intercalating agent lost
5′ 3′
AT C A G T A C T
T A G T C AT G A
3′ 5′
Figure 12.13: Mutations due to intercalating agents

261

Infobits
Isolation and detection of Mutants

Once mutations are induced, then, they must be detected if they are to be studied.
Selection and screening procedures historically have helped geneticists isolate
mutants of interest from a heterogenous mixture in a mutagenized population.
When isolating mutants of a particular organism, one must know the normal
or wild type characteristics so as to recognize an altered phenotype. A suitable
detection system for the mutant phenotype under study also is needed. Detection
systems in bacteria and other haploid organisms are straightforward because
any new allele should be seen immediately, even if it is recessive mutation. The
detection of mutants can be direct and complex. For example, the replica plating
technique is used to detect auxotrophic mutants (mutants which are deficient in
synthesizing a particular biochemical compound). Replica plating technique
distinguishes between mutant and wild type strain based on their ability to grow
in the absence of a particular biosynthetic end product Figure below. A lysine
auxotroph, for instance, will grow on lysine supplemented media but not on a
medium lacking an adequate supply of lysine because it cannot synthesize this
amino acid.
Mutagen 1 Inoculate bacteria

onto complete
medium containing
Iysine x
Bacteria
4 Stamp replica plates

Incubation with velvet.
Bacterial 2 Bacterial colonies
suspension grow. A few may
be. lysine
auxotrophs.
Most are
wild type.
x x
Complete medium Medium lacking Iysine
containing Iysine
5 Identify auxotroph
3 Stamp sterile velvet as colony growing
Incubation
onto plate, picking on complete
up cells from each medium but not
colony. on lacking medium.
Sterile velvet
surface
All colonies grow Iysine auxotroph

cannot grow
6 Inoculate auxotroph
colony into complete
medium.
262

including genetic diseases and cancer. is also set up which does not contain the
Some banned chemical warfare agents chemical. After incubation the control
(e.g. mustard gas) also are mutagens. plates may have few colonies resulting
A number of chemicals (subclass of from spontaneous reversion of the his-
mutagens) induce mutations that result strain. Compared to the control plates if
in tumorous or cancerous growth. These there are increased number of colonies on
chemical agents are called chemical test plate, it indicates that the chemical
carcinogens. Directly testing the chemicals has reverted the mutant strain back to
for their ability to cause tumors in wild type. This chemical is likely to be a
animals is time consuming and expensive. carcinogen. Figure 12.14 shows steps in
However, the fact that most chemical Ames test.
carcinogens are mutagens led Bruce Ames Mix together the salmonella strain, rat liver extract, and suspected
mutagen. The suspected mutagen is omitted from the control sample.
Provides a
mixture of
to develop a simple, inexpensive, indirect Control

enzymes that
may activate a
mutagen
assay for mutagens. In general Ames test is

Rat liver
an indicator of whether the chemical is a
Suspected Rat liver
mutagen extract extract
mutagen. The Ames test assays the ability Salmonella

strain
Salmonella
strain
of chemicals to revert mutant strains of

(requires (requires
histidine) histidine)
the bacterium Salmonella typhimurium Plate the mixtures onto petri

plates that lack histidine.
to wild type. The mutant strain of

S.typhimurium is auxotrophic to histidine The control plate
indicates that
(his-), that is it requires histidine for its Incubate overnight to there is a low
allow bacterial growth. level of
spontaneous
growth and cannot grow in the absence of

mutation
histidine. The mutant strain is grown in A large number of colonies
a histidine deficient medium containing

suggests that the suspected
mutagen causes mutation.
the chemical to be tested. A control plate Figure 12.14: Steps in Ames test
Infobits
DNA Repair
Both prokaryotes and eukaryotes have a number of repair systems that deal with
different kinds of DNA damage. All the systems use enzymes to make correction.
Without this repair systems lesions would accumulate and be lethal to the cell or
organism. Not all lesions are repaired, and mutations do appear, but at low frequencies.
At high doses of mutagens, repair systems are unable to correct all of the damage,
and cell death may result. We can group repair systems into different categories on
the basis of the way they operate. Some systems correct damaged areas by reversing
the damage. This type of repair is called direct correction or direct reversal. Other
systems excise the damaged areas and then repair the gap by new DNA synthesis.
Some of the DNA repair systems are
•• Mismatch repair by DNA polymerase proofreading
•• Repair of UV induced pyrimidine dimers- Photo reactivation or Light repair
•• Base excision repair
•• Nucleotide excision repair
263

to another. The exchange of genes between
With recombinant two cells of the same generation is referred
DNA technology it is to as horizontal gene transfer. Mechanisms
possible to mutate a
like transformation, transduction and
gene at specific posi-
conjugation takes place naturally and may
tions in the test tube
bring about genetic variation and genetic
by SITE SPECIFIC MUTAGENESIS and
recombination. These gene transfer
then introduce the mutated gene back
mechanisms are also employed in genetic
into the cell and investigate the pheno-
typic changes produced by the mutation engineering to introduce desired gene into
in vivo. Such techniques enable geneti- the cells. Introducing a foreign gene or
cists to study, for example, genes with un- recombinant DNA into the cells is one of
known function and specific sequences the techniques used in genetic engineering.
involved in regulating a gene’s expression. The success of cloning depends on the
efficiency of gene transfer process. The
most commonly employed gene transfer
12.7 Transfer of Genetic Material methods are transformation, conjugation,
Normally, genes and the characteristics transduction, electroporation, lipofection
they code for are passed down from and direct transfer of DNA. The choice
parent to progeny. This is called vertical of the method depends on the type of
gene transfer. Bacteria and some lower host cell (bacteria, fungi, plant, animal).
eukaryotes are unique in that they can pass Figure 12.15 shows methods of DNA
DNA from one cell of the same generation transfer.
Methods of DNA Transfer
Natural Methods Artificial Methods
1. Conjugation Physical Methods Chemical Methods

2. Bacterial transformation
3. Transposition 1. DNA transfer by calcium
4. Phage transduction phosphate method
1. Microinjection
5. Retroviral transduction 2. Transfer of DNA by use of
2. Protoplasts fusion
6. Agrobacterium mediated polyethylene glycol
3. B olistics transformation
transfer 3. Use of DEAE – Dextran for
DNA transfer
4. Liposome mediated transfer
Electrical Methods
1. Electroporation
2. Electrofusion
Figure 12.15: Methods of DNA transfer
Note: The term Transfection is used for the transfer of DNA into eukaryotic cells by
various physical or chemical means.
264

12.7.1 Transformation a pulse of heat shock. Electroporation is
Transformation is genetic alteration of another method of promoting competence.
a cell resulting from the direct uptake, Using this method, the cells are briefly
incorporation and expression of exogenous shocked with an electric field of 10–20 kV/
genetic material (exogenous DNA) from cm which is thought to create holes in the
its surroundings. Transformation occurs cell membrane through which the plasmid
naturally in some species of bacteria, DNA may enter. After the electric shock,
but it can also take place by artificial the holes are rapidly closed by the cell’s
means in other cells. For transformation membrane-repair mechanisms.
to happen, bacteria must be in a state of
competence. Competence refers to the
state of being able to take up exogenous
DNA from the environment. There are
two forms of competence: natural and
artificial. Transformation works best with
DNA from closely-related species. The
naturally-competent bacteria carry sets of
genes that provide the protein machinery Figure 12.16: Mechanism of transformation
to bring DNA across the cell membrane(s). 1. Binding of DNA; 2. Degradation of one
There are some differences in the strand; 3. Entry of ssDNA; 4. Integration
mechanisms of DNA uptake by gram into host chromosome.
positive and gram negative cells. However,
they share some common features that 12.7.2 Conjugation
involve related proteins. The DNA first The initial evidence for bacterial
binds to the surface of the competent cells conjugation, came from an experiment
on a DNA receptor, and passes through performed by Joshua Lederberg and
the cytoplasmic membrane via DNA Edward L Tatum in 1946. Later in 1950,
translocase. Only single stranded DNA Bernard Davis gave evidence that physical
may pass through, one strand is therefore contact of the cells was necessary for
degraded by nucleases in the process, and conjugation. During conjugation, two live
the translocated single-stranded DNA bacteria (a donor and a recipient) come
may then be integrated into the bacterial together, join by cytoplasmic bridges (e.g.
chromosomes. Figure 12.16 shows pilus) and transfer single stranded DNA
mechanism of transformation. (from donor to recipient).
Artificial competence can be induced Inside the recipient cell, the new DNA
in laboratory procedures that involve may integrate with the chromosome (rather
making the cell passively permeable to rare) or may remain free (as is the case with
DNA. Typically, the cells are incubated in a plasmids). Conjugation can occur among
solution containing divalent cations; most the cells from different genera of bacteria,
commonly, calcium chloride solution under while transformation takes place among
cold condition, which is then exposed to the cells of a bacterial genus.
265

A plasmid called the fertility or F factor a very high efficiency of chromosomal
plays a major role in conjugation. The gene transfer in comparison with F+
F factor is about 100 kilobases long and cells. In F+ cells the independent F factor
bears genes responsible for cell attachment rarely transfer chromosomal genes hence
and plasmid transfer between specific the recombination frequency is low.
bacterial strains during conjugation. F Figure 12.17b shows formation of HFR
factor is made up of cell. When an HFR cell is mated with
a. tra region (tra operon / transfer genes): F- cell the F- recipient does not become
genes coding the F pilus and DNA F+ unless the whole chromosome is
transfer, transferred as explained in Figure 12.17c.
b. Insertion sequence: genes assisting The connection usually breaks before
plasmid integration into host cell this process is finished. Thus, complete F
chromosome. factor usually is not transferred, and the
Thus, the F factor is an episome - a recipient remains F-.
genetic material that can exist outside the Because the F plasmid is an episome,
bacterial chromosome or be integrated it can leave (deintegrate) the bacterial
into it. chromosome. Sometimes during this
During F+ × F – mating or conjugation process, the plasmid makes an error
(Figure 12.17a) the F factor replicates by in excision and picks up a portion of
the rolling circle mechanism and a copy the chromosomal material to form
moves to the recipient. The channel for an F′ plasmid. Figure 12.17d shows
DNA transfer could be either the hollow formation of F′. During F′XF- conjugation
F pilus or a special conjugation bridge (Figure 12.17e) the recipient becomes F′
formed upon contact. The entering strand and is a partially diploid since it has two
is copied to produce double – stranded set of the genes carried by the plasmid.
DNA. The natural phenomenon of
F factor can integrate into the bacterial conjugation is now exploited for
chromosome at several different locations gene transfer and Recombinant DNA
by recombination between homologous technology. In general, the plasmids lack
insertion sequences present on both the conjugative functions and therefore, they
plasmid and host chromosomes. The are not as such capable of transferring
integration of F factor into bacterial DNA to the recipient cells. However, some
chromosome results in formation of plasmids with conjugative properties can
HFR (High Frequency Recombination) be prepared and used.
cell. When integrated, the Fplasmid’s tra
operon is still functional; the plasmid 12.7.3 Transduction
can direct the synthesis of pili, carry out Transduction is the transfer of bacterial
rolling circle replication, and transfer genes from one bacteria to other
genetic material to an F- recipient cell. An by viruses. Example: Bacteriophage
HFR cell is so called because it exhibits (Bacterial viruses). To understand the
266

a
F plasmid Chromosome b
Conjugation pilus Bacterial
chromosome
+
F cell Hfr cell
Donor cell attaches to a recipient

cell with its pilus. The pilus draws the
cells together.
F+ cell F– cell
The cells contact one another. F factor
The F factor is integrated

into the chromosome.
Crossing over takes place between
F factor and chromosome
One strand of plasmid DNA
transfers to the recipient.
The recipient synthesizes a

complementary strand to become
+
an F cell; the donor synthesizes
F+ cell F– cell
a complementary strand,
restoring its complete plasmid.
d Hfr Cell F’ Cell

Integrated
F plasmid
F’ plasmid
c
Chromosomal DNA
Hfr F– Hfr F–
Chromosome Chromosome
Conjugation Merozygot
X X e tube
F’ F– F’ F– F’ F’
Hfr F– Hfr F– A
X
Host
DNA Primer F’ F
Secondary F’
Figure 12.17: Mechanism of conjugation (a) F+ × F– (b) HFR cell Formation (c) HFR × F–
(d) F′ formation (e) F′ × F–
267

role of bacteriophage in gene transfer, Bacterial viruses that reproduce using
the lifecycle of bacteriophage is described a lytic cycle often are called virulent
below briefly. bacteriophages (e.g. T phages) because
After infecting the host cell, a they destroy the host cell. The genome
bacteriophage (phage for short) often takes of many DNA phages such as the lambda
control and forces the host to make many phage, after adsorption and penetration
copies of the virus. Eventually the host do not take control of its host and does
bacterium bursts or lyses and releases new not destroy the host. Instead the viral
phages. This reproductive cycle is called a genome remains within the host cell and
lytic cycle because it ends in lysis of the host. is reproduced along with the bacterial
chromosome. The infected bacteria may
The lytic cycle (Figure 12.18) has four
multiply for long periods while appearing
phases.
perfectly normal. Each of these infected
1. Attachment - Virus particle attaches to bacteria can produce phages and lyses under
a specific receptor site on the bacterial appropriate environmental conditions.
surface. This relationship between phage and its
2. Penetration - the genetic material, host is called lysogeny (Figure 12.19).
which is often double stranded DNA,
then enters the cell. Infection (absorption
and penetration)
3. Biosynthesis - After adsorption and

penetration, the virus chromosome forces Phage DNA cyclizes
Cell division
the bacterium to make viral components-

viral nucleic acids and proteins. Integration of phage DNA
Cell division Lysogenic
to form prophage
clone
4. Assembly - Phages are assembled from

the virus components. Phage nucleic acid
is packed within the virus’s protein coat. Figure 12.19: Lysogeny
5. Release - mature viruses are released Bacteria that can produce phage particles
under some conditions are said to be
by cell lysis.
lysogens or lysogenic bacteria. Phages
which are able to establish lysogeny are
called temperate phages.
Infection (absorption
and penetration)
Reinfection The latent form of virus genome
that remains within the host without
Phage DNA cyclizes destroying the host is called the prophage.
The prophage usually is integrated into
Cell lysis
the bacterial genome. Sometimes phage
Phage DNA replicates (rolling circle)
reproduction is triggered in a lysogenized
culture by exposure to UV radiation or other
Phage heads, tails, and
DNA assemble into
factors. The lysosomes are then destroyed
progery phage
and new phages released – This phenomenon
Figure 12.18: Lytic cycle is called induction (Figure 12.20).
268

Infection (absorption
and penetration)
Reinfection
Cell division
Phage DNA cyclizes
Cell lysis Integration of phage DNA

to form prophage Cell division Lysogenic
Phage DNA replicates (rolling circle) clone
Phage heads, tails, and

DNA assemble into
progery phage
Figure 12.20: Induction of lysogen
Sometimes, bacterial genes are chromosome can be transferred. Once the

incorporated into a phage capsid because DNA has been injected it may integrate
of errors made during the virus life cycle. into the recipient cell’s chromosome to
The virus containing these genes then preserve the transferred genes. About
infects them into another bacterium, 70 to 90% of the transferred DNA is not
resulting in the transfer of genes from one integrated but is often able to survive and
bacterium to the other. Transduction may express itself. However, if the transferred
be the most common mechanism for gene DNA is degraded gene transfer is
exchange and recombination in bacteria. unsuccessful.
There are two very different kinds of Specialized Transduction
transduction. (Figure 12.21b) is also called restricted
1. Generalized transduction transduction in which only specific
2. Specialized transduction portions of the bacterial genome is carried
by the phage. When a prophage is induced
Generalized transduction
to leave the host chromosome, exicision
(Figure 12.21a) occurs during the lytic
is sometimes carried out improperly. The
cycle of virulent and temperate phages.
resulting phage genome contains portions
During the assembly stage, when the viral
of the bacterial chromosome next to
chromosomes are packaged into protein
capsids, random fragments of the partially the integration site. When this phage
degraded bacterial chromosome also may infects another bacterium, it transfers the
be packaged by mistake. The resulting bacterial genes from the donor bacterium
virus particles often injects the DNA along with phage DNA. Here only the
into another bacterial cell but does not bacterial genes that are close to the site
initiate a lytic cycle. Thus in generalized of prophage are transferred. So, this
transduction any part of the bacterial transduction is called specialized.
269

Generalized transduction
Phage infects
bacterial cell
Stable gene transfer into

recipient cell chromosome
Destruction of
bacterial DNA
Lysis of bacterial cell,

phage release, & infection
of new cell
Virus capsid synthesis

a. and assembly
Specialized transduction
Crossover and stable gene

transfer into recipient cell
Phage DNA integrates chromosome
into chromosome
Prophage de-integrates
and picks up piece of Lysis of bacterial cell,
bacterial chromosome phage release, infection
of new bacterial cell
Replication of viral DNA and Virus capsid synthesis

b. destruction of bacterial DNA and assembly
Figure 12.21: (a) Generalized Transduction (b) Specialized Transduction
270

parts of genes and normally is accompanied
Infobits by a phenotypic change.
The chromosomes of bacteria, viruses, There are many diverse and complex
and eukaryotic cells contain pieces of techniques involved in gene manipulation.
DNA that move around the genome. Such However, the basic principles of
movement is called transposition. DNA recombinant DNA technology are
segments that carry the genes required reasonably simple, and broadly involve
for this process and consequently move the following stages (Figure 12.22).
about chromosomes are transposable
elements or transposons. Transposons
are also called jumping genes because
they can jump from one DNA to another,
resulting in mutation of the cell. They
were first discovered in 1951 by Barbara
McClintock whose significant discovery
was ignored by scientific community for
many years. She was awarded the Nobel
Prize in 1983.
12.8 Recombinant DNA Technology

One of the practical applications of
microbial genetics and the technology
arising from it is the recombinant DNA
technology. The deliberate modification
of an organism’s genetic information by
directly changing its nucleic acid genome
is called genetic engineering and is
accomplished by a collection of methods
known as recombinant DNA technology.
Recombinant DNA technology opens up
totally new areas of research and applied
biology. Thus, it is an essential part of
biotechnology, which is now experiencing
a stage of exceptionally rapid growth
and development. In general sense, Figure 12.22: Basic principles of
recombination is the process in which recombinant DNA technology
one or more nucleic acids molecules are
1. Isolation of DNA from the source
rearranged or combined to produce a
(Donor)
new nucleotide sequence. Usually genetic
material from two parents is combined to 2. Generation of DNA fragments and
produce a recombinant chromosome with selection of the desired piece of DNA
a new, different genotype. Recombination 3. Insertion of the selected DNA into a
results in a new arrangement of genes or cloning vector (Example: a plasmid) to
271

create a recombinant DNA or chimeric 12.9 Vectors, Types and
DNA. Characteristics
4. Introduction of the recombinant
Vectors are the DNA molecules, which carry
vectors into host cells (Example:
a foreign DNA fragment to be cloned. They
bacteria)
are cloning vehicles, examples of which
5. Multiplication and selection of clones
containing the recombinant molecules are Plasmids, Bacteriophages, cosmids,
6. Expression of the gene to produce the phagemids and artificial chromosomes.
desired product. The vector types differ in the molecular
properties they have and in the maximum
Cloning in the molecular biology sense size of DNA that can be cloned into each.
(as opposed to cloning whole organisms)
Characteristics of an ideal vector.
is the making of many copies of a segment
1. Should be small in size
of DNA, such as a gene. Cloning makes
it possible to generate large amounts 2. Should contain one or more restriction site
of pure DNA, such as genes, which can 3. Should be self replicating
then be manipulated in various ways, 4. Should containing an origin of
including mapping, sequencing, mutating replication sequence (ori)
and transforming cells. An overview of 5. Should possess genetic markers (to
cloning strategies in recombinant DNA detect the presence of vectors in
technology is shown in Figure 12.23. recipient cells)
GENERATION OF DNA FRAGMENTS
(Restrictions endonuclease digestion,
Plasmid Cloning Vectors
cDNA synthesis, restriction PCR, Bacterial plasmids are extra chromosomal
chemical synthesis)
elements that replicate autonomously in
cells. Their DNA is circular and double
INSERTION INTO A CLONING VECTOR
stranded and carries sequences required
(Ligation of blunt ends or cohesive ends, for plasmid replication (ori sequence) and
homopolymer tailing, linker molecules) for the plasmid’s other functions. (Note:
A few bacteria contain linear plasmids.
Example: Streptomyces species, Borellia
INTRODUCTION INTO HOST CELLS burgdorferi). The size of plasmids varies
(Transformation, transfection, transduction) from 1to 500 kb. Plasmids were the first
cloning vectors. DNA fragments of about
570kb are efficiently cloned in plasmid
SELECTION OR SCREENING cloning vectors. Plasmids are the easiest
(Hybridization, PCR, immonochemical methods, to work with. They are easy to isolate and
protein-protein interactions, purify, and they can be reintroduced into
functional complementation)
a bacteria by transformation. Naturally
occurring plasmid vectors rarely possess
Figure 12.23: An overview of cloning
strategies in recombinant DNA all the characteristics of an ideal vector.
technology Hence plasmid cloning vectors are
272

derivatives of natural plasmids and are Figure 12.24a and 12.24b shows genetic
“engineered” to have features useful for maps of plasmid cloning vectors PUC19
cloning DNA. and PBR322 respectively.
Examples of plasmid cloning vectors Plasmid cloning vector PUC 19 has 2,686
: pBR 322 (plasmid discovered by –bp and has following features:
Bolivar and Rodriguez 322) and pUC 19
1. It has a high copy number; so many
(plasmid from University of California).
copies of a cloned piece of DNA can be
Herbert Boyer and Stanley Cohen in
generated readily.
1973 showed it was possible to transplant
DNA segments from a frog into a strain of 2. It has amp R (ampicillin resistant)
Escherichia coli using pSC101, a genetically selective marker
modified plasmid, as the vector. The 3. It has a number of unique restriction
work laid the foundation for the birth of sites clustered in one region, called
Genetech, the first company dedicated to a multiple cloning site (MCS) or
commercialization of recombinant DNA. polylinker
EcoRI
BamHI
Pstl Sall
Ampicillin Tetracycline
resistance resistance
(AmpR) (TetR)
pBR322
(4361 bp)
Origin of
replication
(ori) Pvull
Figure 12.24: (a) PUC19 (b) PBR322
273

DNA 3’ DNA insertion in
Plasmid pUC19 fragments disrupted lacZ gene
5’
3’ 5’
5’ 3’
lacZ+ gene
(part)
ampR ampR ampR
Plasmid confers resistance Restriction cut in polylinker Plasmid confers ampicillin

to ampicillin and can make resistance, but cannot make
functional b-galactosidase functional b-galactosidase
Figure 12.25: Insertion of a piece of DNA into the plasmid cloning vector pUC19 to
produce a recombinant DNA molecule
4. The MCS is inserted into part of the Phage λ (Figure 12.26) consists of a
E.coli β – galactosidase (lac Z+) gene. head and a tail (both proteins). The DNA,
Figure 12.25 illustrates how a piece of located in the head, is a linear molecule
DNA can be inserted into a plasmid of about 50 kb. At each end of the DNA,
cloning vector such as pUC19 there are single stranded extensions called
Bacteriophage as Cloning Vectors Infobits

They are viruses that replicate within the
bacteria. A phage can be employed as vector Terms associated with plasmids:
since a foreign DNA can be spliced into 1. Low copy number plasmids are
phage DNA, without causing harm to phage plasmids that occur low in number
genes. The phage will reproduce (replicate in each cell.
the foreign DNA) when it infects bacterial 2. High copy number are plasmids that
cell. Both single and double stranded occur high in number in each cell.
phage vectors have been employed in
3. Conjugative plasmids carry a set
recombinant DNA technology. Derivatives
of transfer genes (tra genes) that
of phage can carry fragments up to about
facilities bacterial conjugation.
45 kb in length. PI bacteriophage can carry
fragments up to 95 kb 4. Non – conjugative plasmidsare
plasmids that do not possess transfer
genes.
5. Stringent plasmids are plasmids
that are present in a limited number
(1–2 per cell).
6. Relaxed plasmids are plasmids that
occur in large number in each cell.
7. F plasmids possess genes for their
own transfer from one cell to another
8. R plasmids carry genes resistance to
antibiotics.
Figure 12.26: Structure of phage λ
274

cohesive (cos) ends. On attachment with The main advantage of using phage
tail to E.coli, phage injects its DNA into vectors is that foreign DNA can be packed
the cell. Inside E.coli the phage linear DNA into the phage (invitro packaging),
cyclizes and gets ligated through cos ends the latter in turn can be injected into
to form a circular DNA .The phage DNA the host cell very effectively (Note: no
has two fates – lytic cycle and lysogenic transformation is required). Figure 12.28
cycle (Figure 12.27). shows how a λ phage is used for cloning.
Only about 50% of phage λ DNA is Cosmids: Cosmids are the vectors
necessary for its multiplication and other possessing the characteristics of both
functions. Thus, as much as 50% (i.e. up to plasmid and bacteriophage. Cosmids
20 kb) of the phage DNA can be replaced can be constructed by adding a fragment
by a donor DNA for use in cloning of phage λ DNA including cos site, to
experiments. However, several restriction plasmids. Once inside the host cell,
sites are present on phage which is not by cosmids behave just like plasmids and
itself a suitable vector. The λ based phage replicate. The advantage with cosmids is
vectors are modifications of the natural that they carry larger fragments of foreign
phage with much reduced number of DNA (35–45 kb) compared to plasmids.
restriction sites.
Y CLE Lysogenic
LYTIC C cycle
cos
cation
Repli
Circular DNA E. coli chromosome
Cleavage Excision
integration
Phage packaging
Phage
particles
Host lysis Replication
Figure 12.27: Life cycle of phage λ

275

EcoRI EcoRI EcoRI
cos
cos
EcoRI
EcoRI
Left arm EcoRI
Add insert ( ( & ligate

Recombinant DNA
Recombinant DNA
+
l Packaging system
Infectious phages
Infect E.coli
Figure 12.28: Cloning using a λ phage
Phagemids: Phagemids are the regions, and therefore the recombinant

combination of plasmid and phage and DNA can be maintained like a yeast
can function as either plasmid or phage. chromosome.
Since they posses functional origins of 2. (BACs) Bacterial Artificial
replication of both plasmid and phage chromosomes: BACS can accept DNA
λ they can be propagated (as plasmid or inserts around 300 kb.
phage) in appropriate E.coli.
A major part of the sequencing of human
Artificial chromosome Vectors: genome has been accomplished by using
Artificial chromosomes are cloning a library of BAC recombinant. BACS are
vectors that can accommodate very large vectors containing the origin replication
pieces of DNA, producing recombinant of a natural plasmid called the F factor, a
DNA molecules resembling small MCS, a selectable marker and often some
chromosomes. other features.
1. Yeast artificial chromosome YAC is a Plasmid shuttle Vectors: The plasmid
synthetic DNA that can accept large vectors that are specifically designed to
fragments of foreign DNA between 0.2 replicate in two or more different host
to 2.0 Mb (particularly human DNA). organisms(say in E.coli and yeast) are
In addition to origin of replication referred to as shuttle vectors. The origins
sequence and selectable markers they of replication for two hosts are combined
possess centromeric and telomeric in one plasmid.
276

Expression vectors: An expression vector 12.10 Restriction Enzymes
is a cloning vector containing the regulatory
In 1960s Swiss microbiologist Werner
sequences (promoter sequence) necessary
Arber and American microbiologists
to allow the transcription and translation of
Hamilthon Othanel Smith and Daniel
a cloned gene or genes. Expression vectors
Nathans discovered restriction enzymes.
are essentially derivatives of the plasmid
The discovery, for which the three men
cloning vectors used in the host. They are
shared the 1978 Nobel Prize for Physiology
used to produce the protein encoded by
or Medicine. Restriction enzymes or
a cloned gene in the transformed host.
restriction endonucleases are one of the
For example, the biotechnology industry
most important groups of enzymes for
produces pharmaceutically active proteins
the manipulation of DNA. It is one of the
with the use of expression vectors and the
important molecular tools used by a genetic
appropriate host.
engineer. These are the bacterial enzymes
that recognize a specific base sequence in a
A genomic library DNA molecule (from any source) and make
is a collection of two cuts one in each strand generating 3′
clones that contains – OH and 5′ – P termini. They were first
at least one copy of discovered in E.coli. E.coli produces the
every DNA sequence in an organism’s restriction enzyme to cut the viral DNA
genome. Like libraries with books, and protect itself. The host E.coli DNA is
genomic libraries are a great source protected by its own restriction enzyme since
of information; in this case, the its methylated. Since these enzymes restrict
information is about the genome. the viral replication the word restriction is
Specific sequences in cDNA libraries added to these enzymes. Hind II was the
and genomic libraries can be identified first discovered restriction endonuclease.
via a number of approaches, including The site where the DNA is cut by a
the use of specific antibodies, cDNA restriction enzyme is called recognition
probes and oligonucleotide probes sequence. Restriction endonucleases can
Human artificial chromosome specifically recognize DNA with a particular
(HAC)-based vectors offer a sequence of 4-8 nucleotides and cleave. Each
promising system for delivery and recognition sequence has two fold rotational
expression of full-length human genes symmetry i.e. the same nucleotide sequence
of any size into human cells, and a tool occurs on both strands of DNA which
for determining human chromosome run in opposite direction. Such sequences
function. It does not have the problem are referred to as palindromes, since they
of limited cloning capacity of other read similar in both directions (forwards
vectors, and it also avoids possible and backwards). Majority of restriction
insertional mutagenesis caused by endonucleases (particularly type II) cut
integration into host chromosomes by DNA at defined sites within recognition
viral vector. sequence. Type II restriction enzymes make
277

Figure 12.29: Two types of cuts made by restriction enzymes
two single – stand breaks) one break in each Application of Recombinant DNA
strand. There are two distinct arrangements Technology
of these breaks 1. both breaks at the center of a. Production of medically useful proteins
symmetry (generating flush or blunt ends) such as somatostain, insulin, human growth
or 2. breaks that are symmetrically placed hormone and interferon. It decreases the
around the line of symmetry generating dependency on human tissues and solves
cohesive ends. Figure 12.29 shows two problem of limited production.
types of cuts made by restriction enzymes.
b. Development of synthetic vaccines
The arrow indicates the cleavage site.
for instance, vaccines for malaria and
The dashed line is the center of symmetry of
rabies a recombinant hepatitis vaccine
the sequence (Table 12.2). is already commercially available.
No of Enzyme
Type Cleavage site Examples Bacterial source
and sub units
I One with 3 sub units for 1000 bp from EcoK1 Cfr A1 Escherichia coli
recognition cleavage and recognition site Citrobacter fruendii
methylation
II Two different enzymes Same as recognition Eco R1 Alu I Escherichia coli
to cleave or modify the or close to Arthrobacter luteus
recognition sequence recognition site
III One with 2 subunits 24- 26 bp from Hinf III Pst II Haemophilus influenzae
recognition site Providencia stuarti
Table 12.2: Types and features of restriction enzyme

278

c. Gene therapy the positive electrode while the positively
d. Diagnosis of infection diseases. charged molecules migrate towards the
e. To manufacture industrially important negative electrode. Gel electrophoresis is a
products like enzymes using bacteria, routinely used analytical technique for the
fungi and cultured mammalian cells. separation and purification of specific DNA
fragments. The gel is composed of either
12.11 Techniques in Genetic polyacrylamide or agarose. Polyacrylamide
Engineering gel electrophoresis (PAGE) is used for the
separation of smaller DNA fragments while
There are several techniques
agarose electrophoresis is convenient for
used in recombinant
the separation of DNA fragments ranging in
DNA technology or gene
size from 100 base pairs to 20 kilobase pairs.
manipulation. The most
frequently used methods are Gel electrophoresis can also be used for the
agarose gel electrophoresis, separation of RNA molecules. A diagramatic
isolation and purification of view of the agarose gel electrophoresis unit
nucleic acids, nucleic acid blotting techniques, is shown in Figure 12.30a.
DNA sequencing, chemical synthesis of
Steps
DNA, gene transfer methods, polymerase
chain reaction, construction of gene library, 1. Gel is set with wells on one end.
radiolabeling of nucleic acids etc, few of them 2. The gel is placed in an electrophoresis
are discussed here. apparatus and covered with buffer
solution.
12.11.1 Agarose Gel Electrophoresis
3. The DNA samples along with tracer
Electrophoresis refers to the movement of dye are placed in the wells of gel.
charged molecules in an electric field. The
negatively charged molecules move towards
Figure 12.30: (a) agarose gel electrophoresis unit (b) DNA electrophoresis gel
279

4. Power supply is switched on and gel is 12.11.2 Polymerase Chain Reaction
run till the tracer dye reaches the end (PCR)
of the gel. The PCR technique has already proven
As the DNA is negatively charged, DNA exceptionally valuable in many areas
fragments move through the gel towards of molecular biology, medicine, and
the positive electrode. The rate of biotechnology. PCR technique has
migration of DNA is dependent on the
great practical importance and impact
size and shape. In general, smaller linear
fragments move faster than the larger on biotechnology. Between 1983 and
ones. Hence, gel electrophoresis can be 1985 American biochemist Kary Mullis
conveniently used for the separation of developed PCR technique that made it
a mixture of DNA fragments, based on possible to synthesize large quantities
their size. The bands of the DNA can be of a DNA fragment without cloning it.
detected by soaking the gel in ethidium Mullis received the 1993 Nobel Prize for
bromide solution (Ethidium bromide Chemistry for his invention. PCR is a cell
can also be added to molten agarose
free amplification technique.
prior to setting the gel). When activated
by ultraviolet radiation, DNA base pairs
in association with ethidium bromide, Infobits
emit orange fluorescence. And in this
Cloned genes and other DNA
way the DNA fragments separated in
sequences often are analyzed to
agarose electrophoresis can be identified
determine the arrangement and
(Figure 12.30b).
specific locations of restriction sites.
PAGE is composed of chains of acryl The analytical process involves
amide monomers crosslinked with methylene cleavage of the DNA with restriction
bisacryalmide units. The pore size of the gel enzymes, followed by separation of the
is dependent on the total concentration of resulting DNA fragments by agarose gel
monomers and the cross links. PAGE is used electrophoresis. The sizes of the DNA
for the separation of single stranded DNA fragments are calculated, enabling
molecules that differ in length by just one restriction maps to be constructed.
nucleotide. Agarose gels cannot be used for The many DNA fragments produced
this purpose. This is because polyacrylamide by cleaving genomic DNA show a wide
gels have smaller pore sizes than agarose range of sizes, resulting in a continuous
gels and allow precise separation of DNA smear of DNA fragments in the gel.
molecule from 10–1500 bp. In this case, specific gene fragments
can be visualized only by transferring
HOTS them to membrane filter by southern
blotting, hybridizing a specific labelled
1. Explain how gel electrophoresis probe with the DNA fragments,
can be used to determine the size and detecting the hybrids. A similar
of a PCR product. procedure, Northern blotting is used
to analyze the sizes and quantities of
RNAs isolated from cell.
280

Figure 12.31 outlines how PCR polymerases employed in PCR technique
technique works. To amplify (make large are able to function at high temperatures.
quantities) a particular DNA sequence by 4. Four deoxyribonucleoside triphos-
PCR a reaction mixture (often 100ml or phates (dNTPs)- dCTP, dATP, dCTTP,
less in volume) containing the following dTTP
are required.
5’
DNA
3’ Nucleotides:
Taq polymerase
lacks proof reading
dATP
one piece Targeted dCTP
START sequence dGTP
of DNA
5’
dTTP
exonuclease (3′–5′)
3’
activity which might
primer has base sequence
homology to DNA
5’ 3’ 1 Heat briefly
3’5’
TA
AT PRIMER
to separate DNA
strands contribute to errors in the products
DNA STRAND C G
G
GC of PCR. Some other thermostable
5’
DNA polymerases with proof
T 3’
ETC 2 Cool to allow
primers to hydrogen-
Primers: bond
Cycle 1
yields
reading activity have been identified.
Example: Tma DNA polymerase from
2
Primers molecules
Thermotoga maritana.
3 DNA polymerase
adds nucleotides to
the 3’ end of each
primer
two pieces
of DNA
Steps in PCR
heat, cool polymerize
1. Denaturation: The target DNA
Cycle 2 containing the sequence to be amplified
yields
is heat denatured to separate its
four pieces
of DNA 4
molecules
complementary strands at temperature

94 °C–95 °C.
heat, cool polymerize
eight Cycle 3
pieces
of DNA
yields
8
2. Annealing: The temperature is
lowered to 37 °C–55 °C so that the
molecules
primers can hydrogen bond or anneal

Figure 12.31: Steps in PCR to the DNA on both sides of the target
1. Target DNA sequence. Because the primes are
2. Two primers–These are synthetic present in excess the targeted DNA
oligonucleotides, usually about strands normally anneal to the primers
20 nucleotides long. These are rather than to each other.
fragments with sequences identical to 3. Extension: Heat resistant DNA
those flanking the targeted sequence. polymerase extends the primers and
3. Thermostable DNA polymerase– Two synthesizes copies of the target DNA
popular enzymes employed in the PCR sequence using the deoxyribonucleoside
technique are Taq polymerase from triphosphate’s at 70 °C–75 °C.
the thermophilic bacterium Thermus
The three – step cycle (Figure 12.32)
aquaticus and the vent polymerase
is repeated to obtain copies of target
from Theromococcus litoralis. These
DNA in large numbers. At the end of one
281

cylcle, the targeted sequences on both used in PCR procedures. Cellular RNAs
strands have been copied. When the three and RNA viruses may be studied even
– step cycle is repeated, the four strands when the RNA is present in very small
from the first cycle are copied to produce amounts (as few as 100 copies can be
eight fragments. The third cycle yields transcribed and amplified). Quantitative
16 products. Theoretically, 20 cycles will PCR is quite valuable in virology and gene
produce about one million copies of the impression studies. PCR is modified as
target DNA sequence. Each cycle of PCR per the specific demands of the situation.
takes about 3 – 5 minutes. Thus there are many variations in the
original PCR Examples nested PCR,
Denaturation
inverse PCR, reverse transcription PCR,
time quantitative PCR, RAPD, RFLP,
AFLP.
Annealing Elongation
Figure 12.32: Three steps PCR cycle

The PCR technique has now been
automated and is carried out by a specially
designed machine (Figure 12.33) PCR
machines are now fully automated and ON/OFF 1 2 3
microprocessor controlled. They can process START/STOP

4 5 6
7 8 9
up to 96 samples at a time. PCR machines ENTER
0
can carry out 25 cycles and amplify DNA

105 times in as little as 57 minutes.
The PCR has many applications in
research and in commercial arena, including
generating specific DNA segments for Figure 12.33: PCR machine
cloning or sequencing, amplifying DNA
to detect specific genetic defects, and
amplifying DNA for fingerprinting in
crime scene investigation.
PCR technology is improving
continually. Various forms of PCR are
available. RNA too can be efficiently
282

called as polymorphisms can be mapped
HOTS
and identified. The DNA markers are
highly useful for genetic mapping of
Both PCR and Cloning allow for the genomes. RFLPS (Restriction Fragment
production of many copies of a DNA
Length Polymorphisms), VNTRs (mini
sequence. What are the advantages
satellites or Variable Number Tandem
of using PCR instead of cloning to
Repeats), STRs (Microsatellites or
amplify a DNA template?
Simple Random Repeats), SNPs (Single
What advantages are there to
Nucleotide Polymorphisms) are types of
using a DNA polymerase for PCR
DNA sequences (stretch of DNA) which
that has proofreading activity?
can be used as markers. These markers
are used in disease diagnosis and DNA
12.11.3 Molecular Markers – RFLP, fingerprinting.
RAPD Restriction fragment length
A molecular marker is a DNA sequence polymorphism (RFLP) was the very
in the genome which can be located and first technology employed for the
identified. As a result of genetic alterations detection of polymorphism, based on
the DNA sequence differences. RFLP is
(mutations, insertions, deletions), the base
mainly based on the altered restriction
composition at a particular location of
enzyme sites, as a result of mutations
the genome may be different in different
and recombinations of genomic DNA.
cells. These differences, collectively
An outline of the RFLP analysis is given
Hind III
Isolation of genomic DNA
Digestion by
200 bp 800 bp
restriction Source A
Hind III EcoR1
endonucleases
DNA pieces
200 bp 450 bp 350 bp
Source B
Digestion by
restiction enzymes
Gel electrophoresis
Separated by ager gel
electrophoresis
with enzymes with enzymes
Hind III EcoR1
A B A B
800 bp 1000 bp
DNA pieces transferred 350 bp
200 bp
to membrane filter 650 bp
Incubated with
monomorphic Polymorphic
cloned and pattern pattern
labeled probes
Figure 12.34: (a) An outline of RFLP analysis as a molecular marker (b) A schematic
RFLP bands detected
representation of restriction fragment length polymorphism (RFLP) analysis as a
molecular marker
283

in Figure 34 a. The procedure basically Isolation of
genomic DNA
involves the isolation of genomic DNA,
its digestion by restriction enzymes, Denatured DNA
separation by electrophoresis, and finally
hybridization by incubating with cloned Annealing of DNA
and labeled probes (Figure 12.34 b). template with primers
Based on the presence of restriction sites,

Complementary
DNA fragments of different lengths can be strand synthesis
generated by using different restriction

enzymes. In the Figure 12.34 b. two DNA Amplified products separated
by gel electrophoresis
molecules from two sources (A and B) and identified
are shown. In source A, a mutation has Figure 12.35: An outline of RAPD
analysis as a molecular marker
Infobits
DNA Fingerprinting Or DNA Profiling:

DNA fingerprinting is the present day genetic detective in the practice of modern
medical forensics. The underlying principles of DNA fingerprinting are briefly
described.
The structure of each person’s genome is unique. The only exception being
monozygotic identical twins (twins developed from a single fertilized ovum). The
unique nature of genome structure provides a good opportunity for the specific
identification of an individual. The DNA fingerprint is an analysis of the nitrogenous
base sequence in the DNA of an individual.
The original DNA fingerprinting technique was developed by Alec Jaffreys in
1985. Although the DNA fingerprinting is commonly used, a more general term DNA
profiling is preferred. This is due to the fact that a wide range of tests can be carried
out by DNA sequencing with improved technology.
Applications of DNA fingerprinting:
The amount of DNA required for DNA fingerprint is remarkably small. The minute
quantities of DNA from blood strains, body fluids, hair fiber or skin fragments
are enough. Polymerase chain reaction is used to amplify this DNA for use in
fingerprinting. DNA profiling has wide range of applications – most of them related
to medical forensics. Some important ones are listed below.
•• Identification of criminals, rapists, thieves etc.
•• Settlement of paternity disputes.
•• Use in immigration test cases and disputes.
In general, the fingerprinting technique is carried out by collecting the DNA from
a suspect (or a person in a paternity of immigration dispute) and matching it with that
of a reference sample (from the victim of a crime, or a close relative in a civil case).
284

occurred leading to the loss of restriction
site that can be digested by EcoRI. Infobits
The result is that when the DNA In 1970s American molecular

molecules are digested by the enzyme biologists Allan M. Maxam and
Hind III, there is no difference in the Walter Gilbert and English biochemist
DNA fragments separated. However, Frederick Sanger developed some of the
with the enzyme EcoRI, source A DNA first techniques for DNA sequencing.
molecules is not digested while source B Gilbert and Sanger shared the 1980
DNA molecule is digested. This results in Nobel Prize for Chemistry for their
a polymorphic pattern of separation. work. Dideoxy procedure is one of the
Random amplified polymorphic procedure used to sequence DNA.
DNA (RAPD) is a molecular marker
based on PCR amplification. An outline
of RAPD is depicted in Figure 12.35.
Summary
The DNA isolated from the genome is The fundamental unit of information in
denatured. The template molecules are living systems is the gene. Genome is the
annealed with primers, and amplified set of all genes and genetic signals of a
by PCR. Single short oligonucleotide cell. Gene is expressed through a sequence
of events. The central dogma of molecular
primers (usually a 10-base primer)
biology, comprises the three major
can be arbitrarily selected and used for
processes replication, transcription and
the amplification of DNA segments of translation. The genetic message encoded
the genome (which may be distributed in messenger RNA (mRNA) is translated
throughout the genome). The amplified on the ribosomes into a polypeptide with
products are separated on electrophoresis a particular sequence of amino acids.
and identified. An RNA base sequence (a set of 3 bases)
Based on the nucleotide alterations corresponding to a particular amino acid
in the genome, the polymorphisms is called a codon. The genetic code is the
of amplified DNA sequences differ set of all codons. The genetic code is a
which can be identified as bands on gel triplet code, and all 64 possible codons
electrophoresis. Genomic DNA from two carry information of some sort. The code
different sources often results in different is highly redundant. Polypeptides are
amplification patterns i.e. RAPDs. synthesized from the amino terminus
toward the carboxyl terminus, by adding
amino acids one by one to the carboxyl
end. An important feature of intiation of
polypeptide synthesis in both prokaryotes
and eukaryotes is the use of a specific
initiating tRNA molecule.
Mutation is the process by which the
sequence of base pairs in a DNA molecule
is altered. Mutations can be divided into
285

base pair substitution mutation and base exogenous DNA from the environment.
pair insertion or deletions. During conjugation, two live bacteria
Frame shift mutation usually results (a donor and a recipient) come together,
in a nonfunctional protein. An addition join by cytoplasmic bridges (e.g. pilus) and
or deletion of one base pair, for example, transfer single stranded DNA (from donor
shifts the mRNA’s downstream reading to recipient). Transduction is the transfer of
frame by one base, so that incorrect amino bacterial genes from one bacteria to other
acids are added to the polypeptide chain by viruses, e.g. Bacteriophage (Bacterial
after the mutation site. Point mutations viruses). Recombination is the process in
are single base changes, that do not affect which one or more nucleic acids molecules
the reading frame, that is, the mutation are rearranged or combined to produce a
only makes a single change in a single new nucleotide sequence. Cloning in the
codon, molecular biology sense (as opposed to
cloning whole organisms) is the making of
Mutations can also be defined
many copies of a segment of DNA, such as a
according to their effects on amino acid
gene. Cloning makes it possible to generate
sequences in proteins. They are missense
large amounts of pure DNA, such as genes,
mutation, silent mutation, nonsense
which can then be manipulated in various
mutation. Forward mutations change
ways
the genotype from wild type to mutant
and reverse mutations (or reversions or Vectors are the DNA molecules, which
back mutations) change the genotype carry a foreign DNA fragment to be cloned.
from mutant to wild type or to partially They are cloning vehicles, examples of which
wild type. are Plasmids, Bacteriophages, cosmids,
phagemids and artificial chromosomes.
The term mutant refers to an organism
Bacterial plasmids are extra chromosomal
in which either the base sequence of DNA
elements that replicate autonomously in
or the phenotype has been changed. The
cells. They are viruses that replicate within
process of formation of mutant organism is
the bacteria. Cosmids are the vectors
called mutagenesis. Ames test is an indicator
possessing the characteristics of both
of whether the chemical is a mutagen. The
plasmid and bacteriophage. Phagemids
Ames test assays the ability of chemicals
are the combination of plasmid and
to revert mutant strains of the bacterium
phage, and can function as either plasmid
Salmonella typhimurium to wild type. The
or phage. The plasmid vectors that are
most commonly employed gene transfer
specifically designed to replicate in two
methods are transformation, conjugation,
or more different host organisms (say in
transduction, electroporation, lipofection
E. coli and yeast) are referred to as shuttle
and direct transfer of DNA. Transformation
vectors. An expression vector is a cloning
is genetic alteration of a cell resulting from the
vector containing the regulatory sequences
direct uptake, incorporation and expression
(promoter sequence) necessary to allow the
of exogenous genetic material (exogenous
transcription and translation of a cloned
DNA) from its surroundings. Competence
gene or genes. Restriction enzymes are the
refers to the state of being able to take up
286

bacterial enzymes that recognize a specific a. Recognition of a codon
base sequence in a DNA molecule (from b. Recognition of an anticodon
any source) and make two cuts one in each
c.
Ability to distinguish one amino
strand generating 3′ – OH and 5′ – P termini.
acid from another
There are several techniques used in
d. Recognition of DNA molecule
recombinant DNA technology or gene
manipulation. The most frequently used 2. Which chain termination codon
methods are agarose gel electrophoresis, could be formed by a single base
isolation and purification of nucleic change from UCG, UGG and UAU?
acids, nucleic acid blotting techniques, a. UAA b. UAG
DNA sequencing, chemical synthesis of c. UGA d. AUG
DNA, gene transfer methods, polymerase 3. Which of the following base-pair
chain reaction, construction of gene changes are transitions?
library, radiolabeling of nucleic acids etc.
a. AT → TA b. AT → GC
Gel electrophoresis is a routinely used
analytical technique for the separation and c. Both a and b d. GC → AT
purification of specific DNA fragments. 4. UV light usually causes mutations by
PCR is a cell free amplification technique. a mechanism involving
The three – step cycle is repeated to obtain a. One-strand breakage in DNA
copies of target DNA in large numbers. b. Deletion of DNA segments
The DNA markers are highly useful c. Induction of thymine dimers and
for genetic mapping of genomes. their persistence
RFLPS (Restriction Fragment Length
d. Inversion of DNA segments
Polymorphisms), VNTRs (mini satellites
or Variable Number Tandem Repeats), 5. The form of genetic information used
STRs (Microsatellites or Simple Random directly in protein synthesis is
Repeats), SNPs (Single Nucleotide a. DNA b. mRNA
Polymorphisms) are types of DNA c. rRNA d. tRNA
sequences (stretch of DNA) which can be 6.
used as markers. These markers are used in display one anticodon each
disease diagnosis and DNA fingerprinting.
a. eukaryotic mRNAs
Evaluation b. transfer RNAs
c. ribosomal RNAs
d. mRNAs
1. Which of the fol-
lowing properties 7. contains exons and
is essential for the introns.
function of a tRNA a. Eukaryotic mRNAs b. rRNA
molecule? c. tRNAs d. primers
8. The symbol lac+ refer to
287

a. genotype b. phenotype b. Conjugation
c. both a & b d. none c. Transformation
9. sequence terminates d. Lysis
protein synthesis 16. The F plasmid makes a cell
a. UAA b. UAG a. Donor b. Recipient
c. UGA d. All the above c. Resistant d. None
10. The principal start codon corresponds 17. Which of the following is more efficient
to which amino acid? in transferring chromosomal DNA
a. Valine a. F+cell b. F-cell
b. arginine c. Hfr cell d. R+cell
c. Methionine 18. Which of the following statement is true
d. Isoleucine a. Protein is the only gene product
11. Number of nucleoprotein subunit in a
b.
A functional gene product is
prokaryotic ribosome
protein or might also be one of
a. 2 b. 4 several classes of RNA molecules
c. 5 d. 6 c.
Carbohydrate is the only gene
12. A deletion occurs that eliminates a product
single amino acid in a protein. How d. L
ipids are the only gene product
many base pairs were deleted?
19. DNA is transcribed into
a. 1 b. 2
a. mRNA b. tRNA
c. 3 d. 4
c. sRNA d. hnRNA
13. During conjugation plasmids undergo
20. Which of the following is found as
a. Theta replication
part of all prokaryotic promoters
b. rolling circle replication
a. Pribnow box
c. sigma replication
b. Shine dalgarno sequence
d. gamma replication
c. AUG sequence
14. If a plasmid is mobilizable but
d. UAG sequence
nonconjugative, what function does it
lack?
Answer the following
a. Antibiotic resistance
1. What is the direction of synthesis of
b. Fertility
RNA?
c. Colicinogenic
2. Define coding strand.
d. Restriction sequences
3. What parts of a mRNA molecule not
15. The uptake of naked DNA from the
translated? Ans. Leader & Introns
surrounding is known as
4. How many codons could be contained
a. Transduction
in a four-letter code? Ans 44=256
288

5. What is the principal start codon 13. Explain the process by which an
and to what amino acid does it infected bacterium releases progeny
correspond? phage.
6. Restriction endonucleases are 14. Define coding strand.
naturally found in bacteria. What 15. Distinguish a missense and a nonsense
purpose do they serve? mutation.
7. There are many varieties of cloning 16. By what mechanism does
vectors that are used to propagate 5-bromouracil induce mutations?
cloned DNA. One type of cloning
17. Define the term conjugative.
vector used in E.coli is a plasmid
vector. What features does a plasmid 18. How does an Hfr cell differ from F+ cell?
vector have that makes it useful for 19. How are F′ plasmids produced?
constructing and cloning recombinant 20. Define a lysogen.
DNA molecules?
21. Restriction enzymes generate two
8. What is shuttle vector and why is it types of termini. What are they?
used?
22. Explain cosmids and the advantages
9. What information and materials are resulting from the use of a cosmid?
needed to amplify a segment of DNA
23. Explain the use of bacteriophage in
using PCR?
cloning DNA fragment.
10. In most PCR reactions, a DNA
24. What are expression vectors?
polymerase that can withstand short
periods of very high (near boiling) 25. Diagramatically describe the plasmid
temperatures is used. why? cloning vector PUC19.
11. The sequence of nucleotides in an 26. How is the natural phenomenon of

mRNA is 5′-AUG-ACCCAUU- conjugation used to transfer foreign
CAUUGGUCUCGUUAG-3′. Assum- gene?
ing that ribosomes could translate 27. List the stages involved in
this mRNA, how many amino acids Recombinant DNA technology.
long would you expect the resulting 28. Discuss RAPD and RFLP.
polypeptide chain to be?
12. The N-terminus of a protein has the
sequence Met-His-Arg-Lys-Val-His-
Cys-Gly. A molecular Biologist wants
to synthesize a DNA chain that can
encode this portion of the protein.
How many DNA sequences can
encode this polypeptide?
289

MICROBIOLOGY
PRACTICAL
290
XII_Micro_Biology_Practical_Manual.indd 290 27-02-2019 15:13:24

Std XII Microbiology Practical Manual
Practicals Page. No.

Gram’s staining of curd/idly batter/yeast
Identification of the fungus(aspergillus/mucor/Rhizopus) by
wet mount using LPCB.
Blood grouping
Blood staining
Test for catalase
Widal test (slide test)
Demonstration of rhizobium from root nodules and its isolation
Spotters
II A) Specimen
Root nodules of leguminous plant
Tikka leaf spot of groundnut plant
Mushroom
Sand fly
Ascaris
II B) Slide
Cyst of Entamoeba histolytica
Penicillium species
Microfilariae
Egg of Ascaris lumbricoides
Heterocysts of Nostoc
Acid fast bacilli
II C) Spotter
Antibiotic sensitivity plate set up by Kirby Bauer technique
Sugar fermentation tube showing acid and gas
Agarose gel electrophoresis apparatus.
Spoiled food
291

Higher Secondary – First Year Practical Examination
Microbiology
Marking Scheme
Allotment of Marks
Internal Assessment 05 marks
External Assesment 15 marks
Total 20 marks
Internal Assessment (Practicals) Marks Break Up
1. Record Note Book 03 marks
2. Skill of performing Experiments 02 marks
Total 05 marks
External Assessment Mark Break Up
1. Major Practical 09 marks
2. Spotters 06 marks
Total 15 marks
I. Major Practical (Any one out of 5 questions) 9×1 = 9 marks
• Aim 01 mark
• Principle 02 marks
• Procedure 03 marks
• Diagram 01 marks
• Observation 01 marks
• Results 01 marks
Total 09 marks
II. Spotters (Any three – one from each category) 2×3 = 6 marks
• Identification ½ marks
• Two salient points 1 mark
• Diagram ½ mark
Total 02 marks × 3 spotters = 6marks
292

Key for Practical Examination
I. Major Practical (Any one) 9×1 = 9 marks

1. Determine the gram nature of bateria present in the given sample (curd/idly
batter/yeast)
2. Identify whether the given fungus is Aspergillus or Mucor or Rhizopus based
on its microscopic characteristics.
3. Determine the blood group of the given blood sample.
4. Carry out blood staining using field’s stain and observe the erythrocytes and
leucocytes.
5. Identify whether the given culture is catalase positive.
II. Spotters
A. Specimen 2 marks
B. Slide 2 marks
C. Spotter 2 marks
293

1. Gram’s staining of curd/idly batter/yeast
Aim: To determine the gram nature of bacteria present in the given sample(curd/idly
batter/yeast) by Gram’s staining technique.
Theory and Principle:

Based on gram staining reaction bacteria can be divided into two large groups called gram
positive and gram negative. Gram positive bacteria have thicker peptidoglycan compared
to the gram negative bacteria. The lipid content of the cell wall of gram negative bacteria is
higher than that of gram positive bacteria. Both gram positive and gram negative bacteria
take up the primary stain crystal violet and stain red. When decolorized the porosity and
permeability of the gram negative bacteria increases due to which the crystal violet iodine
complex is given out by the gram negative bacteria. Further the gram negative bacteria
takes up the counterstain safranin and stains red. Hence when bacteria are observed under
microscope after gram staining the gram positive cells appear violet and gram negative
cells appear red.
Requirements:
■■ Clean grease free slide
■■ Nichrome loop
■■ Given culture
■■ Crystal violet
■■ Grams iodine
■■ Decolorizer(Acetone Alcohol)
■■ Safranin
■■ D/W
Procedure:
1. Take a loopful of the given culture and place on the slide.
2. Prepare a smear and heat fix it.
3. Cover the smear with Crystal Violet for one minute.
4. Wash gently
5. Add Grams iodine for one minute
6. Decolorise with acetone alcohol
7. Wash the slide immediately
8. Cover the smear with safranin for a minute
9. Wash and Air dry.
10. Observe the slide under high power and oil immersion objectives.
11. Record your observations.
294

Diagram: (Any one Diagram)

Observation Table: ( any one shape and stain)

Colour of Colour of
Sr. no Morphology Arrangement Cytoplasm Background Inference
1. Rod (bacilli) Singles, chains Violet colourless Gram positive
2. Oval yeast cells Singles, budded Violet colourless Gram positive
Results: Gram staining of the given culture revealed gram positive violet colored rod-
shaped bacteria in chains.
2. Identification of the fungus (Aspergillus/Mucor/Rhizopus) by wet mount

using LPCB.
Aim: To identify whether the given fungus is Aspergillus or Mucor or Rhizopus based
on microscopic characteristics by wet mount method using lactophenol cotton blue stain.
Theory and Principle :

Filamentous fungi are reliably identified by their characteristics microscopic morphology
such as shape, size and arrangement of spores and hyphae. Fungi are eukaryotic and range
from unicellular yeast to multicellular molds. They reproduce by producing spores.
Common fungi are Aspergillus, Mucor and Rhizopus. They are filamentous and
collectively form mycelium. The morphology of the hyphae and spores can be identified
using a simple wet mount technique using lactophenol cotton blue stain.
The organism suspended in the stain are killed due to the presence of phenol. Lactic
acid preserves fungal structures and cotton blue stains the fungal cell wall.
Fungi Characteristics of Hyphae Spores borne in
Aspergillus sp. Septate Conidiophore bear conidia
Mucor and Rhizopus sp. Aseptate Sporangiosphore bear sporangium
containing sporangiospore.
295

Requirements :
■ Clean grease free slide
■ Coverslip
■ Forcep
■ Teasing needle
■ Distilled water
■ Lactophenol Cotton Blue
Procedure:
1. Take a clean slide.
2. Place a drop of water on the slide.
3. With the help of forceps transfer the fungal mycelium.
4. Tease it with needle to separate the filaments (hyphae).
5. Add a drop of lactophenolcotton blue.
6. Gently place a coverslip avoiding air bubble formation.
7. Observe under low power and high power objective lens.
8. Read the observations and interpret.
Diagram:
296

OBSERVATION;
Filamentous hyphae bearing sporangia were observed.
Results:
Wet mount using lactophenol cotton blue was carried to identify the fungus sample.
Hyphae with sporangium bearing sporangiospores were observed. It is likely to be of
mucor species.
3. Blood Grouping
Aim: To determine the blood group of the blood sample by the slide agglutination test.

Blood grouping is an essential requirement before blood is transfused from one person to
another.It is also useful in settling paternity disputes and medicolegal problems.
Red blood cells contain blood group antigens. Antibodies to the blood group antigens
are present in the blood plasma.The antigens are generally determined and are responsible
for blood types. When RBCs of a person are mixed with corresponding antiserum,
agglutination occurs due to antigen-antibody reactions.
Materials Required
■■ Blood sample ( anticoagulated)
■■ Sterile cotton
■■ Sterile lancet
■■ Clean dry grease free slides or white tile
■■ Toothpicks
■■ Marker pen
■■ Commercially available Anti A sera, Anti B sera and Anti D sera
Procedure
1. Prick the finger under aseptic conditions
2. Place a drop of blood on the slide on each side marked as A, B and D.
3. Add a drop of antiserum A , B and D on A, B and D side respectively.
4. Mix with toothpick using separate toothpicks for each mixture.
5. Wait for 2 mins and observe for clumping reaction if any confirm it by observing
under microscope.
6. Interpret the results and report.
297

Interpretation
If agglutination on A side the blood group is A
If agglutination seen on B side the blood group is B
If Agglutination on both A and B side the blood group is AB
If No agglutination on A and B side the blood group is O
If agglutination is seen on D side the blood group is Rh(D) positive
If No agglutination on D side the blood group is Rh(D) negative.
Diagram: ( any one depending on the results)
Observation: (will vary with the type of blood group an example is given below)
Agglutination is seen on A, B and D side
Result: The blood group of the blood sample was determined by slide agglutination test
and was found to be AB Rh positive.
1. BLOOD STAINING
3
AIM
To make a blood smear ,stain it using Field’s stain and observe the erythrocytes and
leucocytes.

Blood smears are used to determine leukocyte differentials, to evaluate erythrocyte,
platelet and leukocyte morphology, and, if necessary, to estimate platelet and leukocyte
counts. It is also used for diagnosis of parasites like plasmodium in the blood.
Field’s Stain is a romanowsky stain, used for rapid processing of blood specimens
and is used to stain thick and thin films. It consists of two differential stain.Field stain A
298

which is methylene blue and Azure dissolved in a phosphate buffer solution.It is the basic
component of the stain and Field stain B made up of Eosin Y in a buffer solution which
is the acidic component of the stain.These basic and acidic dyes induce several colours when
applied to cells.The fixator, methanol, does not allow any additional changes to the slide. The
basic component of peripheral white blood cell( cytoplasm) is stained with acid dye and the acid
component that is nucleic acid of the nucleus takes on the basic dye and is stained blue to violet.
The neutral components of the cells are stained by both dyes(Field’s stain A and B solution).
REQUIREMENTS
■■ Cotton
■■ Spirit
■■ Blood sample
■■ Clean grease free slides
■■ Methanol fixative
■■ Field’s stain A and Field’s stain B.
PROCEDURE
1. Finger Prick under aseptic condition.
2. Place a small drop of blood, on one side about 1-2 cm from one end of a slide.
3. Without delay place another slide at an angle of 45° to make contact with the drop.
4. Spread it over an area of about 2 cm2(The film should be distributed so thinly that it
appears transparent.
5. After air drying the thin blood film,immerse or fix the smear in methanol for 1
minutes.
6. Flood or dip the slide in Field’s Stain A for 2-3 seconds.
7. Wash it with distilled water,
8. Flood or dip the slide in Field’s Stain B for 2-3 seconds and wash with distilled water.
9. Now air dry the smear and observe under microscope.
DIAGRAM
299

OBSERVATION
COLOUR OF COLOUR OF COLOUR OF

TYPE OF CELL CYTOPLASM NUCLEUS GRANULES
RBC pink - -
WBCs(leucocytes)
Neutrophil pink blue lilac
Eosinophil pink blue orange
Basophil pink blue Dark blue black
lymphocyte blue violet -
RESULTS
The blood smear was stained using field’s stain and erythrocytes and leucocytes were
observed under microscope.
5. Test for Catalase
AIM
To test whether the given culture is catalase positive by the catalase test
THEORY AND PRINCIPLE

Catalase test demonstrates the presence of catalase, an enzyme that catalyses the release
of oxygen from hydrogen peroxide (H2O2). It is used to differentiate those bacteria that
produces an enzyme catalase, such as staphylococci, from non-catalase producing bacteria
such as streptococci.
The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and
water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum
is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs.
The lack of catalase is evident by a lack of or weak bubble production. The culture should
not be more than 24 hours old.
REQUIREMENTS
■■ Slides
■■ Nichrome loop or toothpick
■■ 24hour old culture
■■ 3%hydrogen peroxide
300

■ Dropper
PROCEDURE
Slide Method
1. Use a loop or sterile wooden stick to transfer a small amount of colony growth in the
surface of a clean, dry glass slide.
2. Place a drop of 3% H2O2 in the glass slide.
3. Observe for the evolution of oxygen bubbles.
DIAGRAM
OBSERVATION (any one to be reported depending on the culture)
Positive: Copious bubbles produced, active bubbling

Examples: Staphylococci, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Pseudomonas.
Negative: No or very few bubbles produced.
Examples: Streptococcus and Enterococcus sps.
Result
The given culture was found to be catalase positive as determined by the catalase slide test.
301

6. WIDAL TEST (slide test)
AIM
To carry out the widal test for the given blood sample and to determine the presence of
antibodies against salmonella antigens.
THEORY AND PRINCIPLE

Widal test is a serological test which is used for the diagnosis of enteric fever or typhoid
fever. Typhoid or enteric fever is caused by a gram negative bacteria Salmonella enterica
(Salmonella Typhi or Salmonella Paratyphi). Salmonella possess O antigen on their cell
wall and H antigen on their flagella. On infection, these antigen stimulates the body to
produce specific antibodies which are released in the blood. The Widal test is used to
detect these specific antibodies in the serum sample of patients suffering from typhoid
using antigen-antibody interactions. These specific antibodies can be detected in the
patient’s serum after 6 days of infection (fever).
Salmonella Typhi possesses O antigen on the cell wall and H antigen on flagella.
Salmonella Paratyphi A and S. Paratyphi B also possess O antigen on their cell wall and
but have AH and BH antigen on their flagella respectively.
Widal test is an agglutination test in which specific typhoid fever antibodies are
detected by mixing the patient’s serum with killed bacterial suspension of Salmonella
carrying specific O, H, AH and BH antigens and observed for clumping ie. Antigen-
antibody reaction. The main principle of Widal test is that if homologous antibody is
present in patient’s serum, it will react with respective antigen in the suspension and gives
visible clumping on the test slide.
Requirements
Fresh serum
The complete kit containing five vials containing stained Salmonella antigen
■■ S. Typhi O antigen
■■ S. Tyhhi H antigen
■■ S. Paratyphi AH antigen
■■ S. Paratyphi BH antigen
Widal positive control
Widal test card or slide
v) Applicator stick
Procedure
■■ Widal test can be done in two ways-one is rapid test on slide and another is tube test
in which result may be obtained after one night of incubation.
302

Rapid slide test:
1. Clean the glass slide or test card supplied in the kit well and make it dry.
2. Label the circles (1, 2, 3, 4, 5 and 6) in the test card as O, H, AH, BH, Negative control
and Positive control
3. Place a drop of undiluted test serum in each of the four labelled circle (1, 2, 3 and 4) ie
O, H, AH and BH and place a drop of Negative control serum in circle 5 and Positive
control in circle 6.
4. Place a drop of antigen O, H, AH and BH in circle 1, 2, 3, and 4 respectively and no
antigen in circle 5 and O/H antigen in circle 6.
5. Mix the content of each circle with a separate wooden applicator stick and spread to
fill the whole area of the individual circle.
6. Rock the test card for a minute and observe for agglutination.
DIAGRAM
OBSERVATION
Agglutination was observed in O and H side within a minute which indicates the presence
of antibodies in the serum sample against Salmonella typhi antigens.
Proceed for quantitative slide test or tube test for the quantitative estimation of the
titre of the antibody.
Result
Qualtative widal test was carried out using rapid slide agglutination method. Antibodies
against O and H antigens of Salmonella typhi were detected in the serum.
303

7. Demonstration of rhizobium from root nodules and its isolation
Aim:
To demonstrate the presence of rhizobium in root nodules by gram staining and isolate
them on a nutrient medium.

Leguminous plants like cowpea, red gram , black gram contain root nodules formed
by rhizobium.Rhizobium in the soil enter into the roots of leguminous plant and form
nodules and establish symbiotic association. Bacteria derive nutrients from the plants.
The rhizobacteria fix nitrogen which is beneficial to the plant. Rhizobium is a symbiotic
N2 fixer found to occur as bacteroids in the root nodules of leguminous plants. They can
be easily isolated and cultured in vitro.
Based on gram staining reaction bacteria can be divided into two large groups called
gram positive and gram negative. Gram positive bacteria have thicker peptidoglycan
compared to the gram negative bacteria. The lipid content of the cell wall of gram negative
bacteria is higher than that of gram positive bacteria. Both gram positive and gram
negative bacteria take up the primary stain crystal violet and stain red. When decolorized
the porosity and permeability of the gram negative bacteria increases due to which the
crystal violet iodine complex is given out by the gram negative bacteria. Further the gram
negative bacteria takes up the counterstain safranin and stains red. Hence when bacteria
are observed under microscope after gram staining the gram positive cells appear violet
and gram negative cells appear red. Rhizobia are Gram- negative rods which are motile
with bi-polar, sub-polar and peritrichous flagella.
Rhizobium grows well on Yeast Extract Mannitol Agar (YEMA). Congo red added
to the medium differentiates rhizobia that stand out as white, translucent, glistening
elevated, small colonies with entire margin, in contrast to the red stained colonies of
Agrobacterium and other bacteria.
Requirements:
1. Root nodules (pink) of any leguminous plant
2. Congo red, Yeast Extract, Mannitol Agar (pH 6.8 – 7.0):
304

Congo red (1% aqueous)
2.5 ml (1.0 g in 100 ml)
Distilled water 1000.0 ml
3. Inoculation loop
4. Bunsen burner/laminar clean air flow hood.
5. Slides and glass rod.
6. Petri plates with YEMACR medium.
7. Sterile distilled water.
8. 95% alcohol and 0.1% HgCl2.
Procedure:
1. Wash the root system under a slow stream of running tap water, taking care to see that
the nodules are intact.
2. Select pink nodules and remove them
3. Wash and keep the nodules in 95% ethanol for a minute, wash and transfer them to
0.1% HgCl2.
4. Remove after five minutes and wash the nodules about four to five times with sterile
distilled water.
5. Place the nodule on a sterile slide in a drop of sterile distilled water and crush it either
with a sterile glass rod or a flat tipped forceps.
6. Remove a loopful of this cloudy suspension and streak inoculate on YEMACR plates
and label.
7. Incubate in dark at 28°-30°C for 2-3 days and observe the colonies.
8. Make a smear of the remaining crushed material and gram stain and observe the gram
negative bacilli. Even samples from the colonies can be gram stained.
Diagram:

305

Observation
Gram’s stain
Colour of Colour of
Organism Morphology Arrangement cytoplasm Background Inference
Rhizobium from Rod (bacilli) Singles red colourless Gram
root nodule. negative
Colony characteristics of rhizobium on YEMA after incubation for 2-3 days at room
temperature
Size – 2-4 mm
Shape- circular
Colour – White
Margin - entire
Elevation – convex, raised
Opacity - semitranslucent
Texture – creamy
Consistency – mucilaginous
Gram nature – gram negative
Motility – actively motile
Results: Gram staining of the root nodule exudate revealed the presence of gram negative
rods.
The colony characteristics of rhizobia were studied after isolation on YEMA medium.
White, creamy, mucoid colonies were obtained.
306

Spotters
II A) SPECIMEN
1. Root nodules of leguminous plant
■■ Leguminous plants like cowpea, red gram contain root nodules formed by rhizobium.
■■ Rhizobium in the soil enter into the roots of leguminous plant and form nodules and
establish symbiotic association.
■■ Bacteria derive nutrients from the plants.
■■ The rhizobacteria fix nitrogen which is beneficial to the plant.
2. Tikka leaf spot of groundnut plant
307

■ Tikka leaf spot disease is a kind of fungal disease seen in groundnut leaf.
■ This disease is caused by Cercaspora personata.
■ Brown spots surrounded by a yellow halo appear on the upper surface of the leaf.
■ The fungal spores can be demonstrated if the leaf is processed and observed under
microscope.
3. Mushroom
■ Mushroom is a saprophytic fungus.

■ Primary mycelium grows from basidiospores.
■ It has high protein content and edible mushrooms are used as food.
■ Example: Agaricus species and Pleurotus species.
4. Sand fly
308

■ Bite of an infected sandfly transmits leishmania donovani infection.
■ Female sandfly during a blood meal ingest free as well as intracellular amastigotes in
the blood.
■ In the midgut these are transformed to flagellated promastigote.
5. Ascaris
■ The adult worm of ascaris lives in the small intestine of humans

■ They are large cylindrical worms with tapering ends, the anterior end being thinner
than the posterior end
■ The adult male worm is smaller than female worms.
IIB) Slide
6. Cyst of Entamoeba histolytica
■ Cyst is one of the three forms of entamoeba histolytica

■ A mature cyst is a quadrinucleate spherical body.
■ Mature cysts are passed in the stool of infected person
■ Direct examination of wet mount of stool for cysts is diagnostic of intestinal amoebiasis
309

7. Penicillium species
■ Colony of penicillium are initially white and fluffy and later produce pigmented spores
and turn into shades of green or blue green
■ Hyphae are hyaline and septate
■ Condiophores are long, give rise to branching phialids
■ Phialids branch and give the appearance of brush or penicillins
■ They produce sterigmata bearing chain of conidia (spores) which are oval or spherical
and measure 1-2micrometer.
8. Microfilariae
■ Filariasis is caused by nematodes (roundworms) like Wuchereria bancrofti that inhabit
the lymphatics and subcutaneous tissues.
■ The female worms release the first stage larvae called microfilariae, which are detected
in the peripheral blood.
■ Identification of microfilariae by microscopic examination is the most practical
diagnostic procedure.
■ The blood sample can be a thick smear, stained with Giemsa.
■ The larva measures about 290microns in length and 6-7micron in breath.
310

9. Egg of Ascaris lumbricoides
■■ These are passed in stool of the infected host.

■■ Brownish due to bile pigment.
■■ Fertilised eggs are rounded and have a thick shell (chitinous).
■■ Unfertilised eggs are elongated and larger than fertile eggs.
■■ When ingested through water or contaminated food by human it causes Ascariasis.
■■ Microscopic identification of eggs in the stool is the most common method for
diagnosing intestinal ascariasis.
10. Heterocysts of Nostoc
■■ Heterocysts are specialized structures having thick cell wall formed in some filamentous
blue green algae like Nostoc, Anabena.
■■ They may be terminal or found in between the vegetative cells attached to it by means
of pores.
■■ They are sites of atmospheric nitrogen fixation.
■■ They serve as a store house of food material.
311

11. Acid fast bacilli
■ Acid fast bacilli contains mycolic acid in their cell walls hence do not get stained easily,
however once stained cannot be decolourised easily.
■ Special method like Ziehl- Neelson’s Carbol fuchsin is used to stain acid fast bacilli.
■ The acid- fast bacilli are stained red in colour while the non acid fast cells appear blue
when counterstained with methylene blue.
■ Mycobacterium tuberculosis is and acid fast bacilli.
IIC) SPOTTER
12. Antibiotic sensitivity plate set up by Kirby Bauer technique
■ Kirby Bauer technique is used to determine the susceptibility of the organism to
various antimicrobial agents.
■ Standard suspensions of rapidly growing test bacterium is inoculated on the surface of
muller hinton agar plates.
■ Antibiotic discs are pressed on the surface of the seeded plates.
■ The zone of inhibition or the zone of growth determines the degree of susceptibility of
the organism towards antibiotic.
312

13. Sugar fermentation tube showing acid and gas production
■ Carbohydrate broth with bromocresol purple as indicator is used for testing the ability
of pure bacterial culture to ferment a specific sugar like lactose, xylose, mannitol and
other sugars.
■ Acid production is indicated by colour change of the indicator from purple to yellow
■ Gas production is indicated by an air bubble in the durham’s tube.
■ Escherichia coli ferments lactose producing acid and gas.
14. Agarose gel electrophoresis apparatus.

■ Electrophoresis refers to the movement of charged molecules in an electric field.
■ The negatively charged molecules move towards the positive electrode while the
positively charged molecules migrate towards the negative electrode.
■ Gel electrophoresis is a routinely used analytical technique for the separation and
purification of specific DNA fragments.
■ As the DNA is negatively charged, DNA fragments move through the gel towards the
positive electrode. The rate of migration of DNA is dependent on the size and shape.
313

15. Spoiled food
■ Spoilage is a process in which food deteriorates such that its quality of edibility is
reduced.
■ Food poisoning may result on eating contaminated or spoiled food.
■ Foods spoil due to attacks from enzymes, oxidation and microorganisms.
■ These include bacteria, mold, yeast, moisture, temperature and chemical reaction.
314

Microbiology – Class XII
List of Authors and Reviewers
Domain Expert Authors
Dr. Elanchezhiyan Manickan Gajalakshmi S
Professor & Chairman PGT., KBC GGHSS.,
Dr. ALM PG IBMS, Redhills,
University of Madras, Chennai. Thiruvallur District.
Reviewers Ramachandran N
PGT., GHSS.,
Mrs. C. Initha Lebanon Ebency Kovur,
Principal and Co ordinator of Academic Affairs, Kancheepuram District.
St. Paul’s College of Arts and Science for Women
and St. Paul’s Matriculation Hr. Sec. School, Manimegalai K
Eden Garden, St. Paul’s Nagar, PGT., Govt. Model HSS.,
Thadagam Road, Coimbatore. Saidapet, Chennai.
Mrs. B. Manjula Devi
HOD., Asst. Professor, Pameela Fathima H
Dept of Microbiology, PGT., JGGHSS.,
Bhaktavatsalam Memorial College for Women, Thiruvottiyur,
Korattur, Chennai. Thiruvallur District.
Ms. C. Banu Rekha Parvin Zeenath Anwar

Asst. Professor, PGT., St. Mary’s Girls, HSS.,
Dr. MGR Janaki College, Perambur,
Chennai. Chennai.
Mrs. G. Sangeetha Jaya Priya D
HOD., Asst. Professor, PG Asst., GHSS.,
Dept of Microbiology, Nandhivaram,
Mahalashmi Women’s College of Arts and Science, Kancheepuram.
Avadi, Chennai.
Academic Coordinator
Angeline Ruby G
Assistant Professor
SCERT, Chennai.
QR Code Management Team

S. Albert Valavan Babu
B.T. Asst.,
GHS., Perumal Kovil, Paramakudi,
Art and Design Team Ramanathapuram.
V. Padmavathi
B.T. Asst.,
Layout GHS., Vetriyur,
Jaishree Anbalagan Ariyalur.
Winmac Solutions, Chennai.
A. Devi Jesintha
Wrapper Design B.T. Asst.,
Kathir Arumugam GHS., N.M. Kovil,
Vellore.
QC
Arun Kamaraj Palanisamy This book has been printed on 80 G.S.M.
Elegant Maplitho paper.
Co-ordination
Ramesh Munisamy Printed by offset at:
Typist
Suresh Mani
315
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NOTES
316
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GOVERNMENT OF TAMIL NADU

HIGHER SECONDARY SECOND YEAR

A publication under Free Textbook Programme of Government of Tamil Nadu

Department of School Education

TN_GOVT_XII_Micro_Biology_FM.indd 1 04-03-2019 14:01:49

First Edition - 2019

Published under New Education Syllabus

NOT FOR SALE

State Council of Educational Research

Printing & Publishing

Tamil NaduTextbook and Educational

TN_GOVT_XII_Micro_Biology_FM.indd 2 04-03-2019 10:47:34

Chapter 1 Developments in Microbiology ............................................. 01

E-Book Assessment Digi-Link

Lets use the QR code in the text books ! How ?

TN_GOVT_XII_Micro_Biology_FM.indd 3 04-03-2019 10:47:34

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

To motivate the students to further explore the content

Evaluation Assess students to pause, think and check their understanding

Career corner List of professions particular to that chapter

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TN_GOVT_XII_Micro_Biology_CH01.indd 1 27-02-2019 15:14:45

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TN_GOVT_XII_Micro_Biology_CH01.indd 4 27-02-2019 15:14:47

•• A recombinant vaccine to prevent

The traditional monoclonal antibody •• Adult tissue (adult stem cells).

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Figure 1.4: Stem cells

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TN_GOVT_XII_Micro_Biology_CH01.indd 8 27-02-2019 15:14:49

1.6.2 DNA Sequencing System

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TN_GOVT_XII_Micro_Biology_CH01.indd 10 27-02-2019 15:14:50

4. Discuss on Emerging microbes. 9. Brief note on sequencing methods.

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TN_GOVT_XII_Micro_Biology_CH02.indd 12 27-02-2019 13:44:07

Figure 2.1: Components of phase contrast microscope

TN_GOVT_XII_Micro_Biology_CH02.indd 13 27-02-2019 13:44:07

Bacterlum Ray devlated by Deviated ray is Deviated and

Figure 2.2: Production of contrast in phase contrast microscopy by phase plate

Figure 2.3: Optical path of Phase contrast microscopy

remain non-deviated (no phase change).

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TN_GOVT_XII_Micro_Biology_CH02.indd 15 27-02-2019 13:44:10

•• It is used to examine various properties of the light microscope with

2.2 Fluorescence Microscope

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Some substances have the property

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S.No Light microscope Electron microscope

1. Light is the illuminating source The beam of electrons is the

examine objects. Electrons are considered

­resolving power of the electron microscope •• Transmission electron microscopes

TN_GOVT_XII_Micro_Biology_CH02.indd 20 27-02-2019 13:44:13

Figure 2.8: Interaction between electron beams with specimen

TN_GOVT_XII_Micro_Biology_CH02.indd 21 27-02-2019 13:44:14

resolving power of the electron microscope •• Transmission electron microscopes

5. Which of the following refers to a c.