Fundamental of Microbiology Notes Final

BVMLT – 103
Fundamentals of Microbiology
UNIT-1 Introductory microbiology: Introduction to and brief of microbiology, scope

and relevance of microbiology, modern developments in microbiology,
explain the types and methods of sterilization, use and types of microscopes;
bright microscope, field microscopy, dark field microscopy, phase contrast
microscopy, electron microscopy.
UNIT-2 Morphology and structure of microorganisms: Morphology and structure of

bacteria, fungi, actinomycete and algae etc., microscopic examination of
microorganisms, preparation of culture media, spread plates, pour plates,
types of selective and differential media, separation of pure cultures, principles
and uses of microbiology equipments and instruments.
UNIT-3 Stains used in microbiology: Introduction to stains; importance of stain in

microbiology; types of stains in detailed giving example-simple stain
differential stain, negative stain, impregnation method; special staining for
certain bacteria, bacterial spores, parasites and fungi; principle, procedure,
application and result, interpretation of gram staining and ziehl neelsen
staining.
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UNIT - 1
Learning objective: On completion of this unit student will able to:

 Introduce microbiology
 Define scope and relevance of microbiology,
 Define modern developments in microbiology,
 explain the types and methods of sterilization, use and types of microscopes; bright
microscope, field microscopy, dark field microscopy, phase contrast microscopy, electron
microscopy
Microbiology is the study of microorganisms (also known as microbes), which are unicellular or cell-
cluster organisms and infectious agents too small to be seen with the naked eye. This includes
eukaryotes (organisms with a nucleus), such as fungi and protists, and prokaryotes (organisms without
a nucleus), such as bacteria.
A fundamental understanding of how a cell works has come through the study of microorganisms.
Microbiologists study microbes at the level of the community (ecology and epidemiology), at the level
of the cell (cell biology and physiology) and at the level of proteins and genes (molecular biology).
Microorganisms are extremely important in our everyday lives. Some are responsible for a significant
proportion of the diseases. Still others play an essential role in industry, where their unique properties
have been harnessed in the production of food, beverages and antibiotics. Scientists also have learned
how to exploit microorganisms in the field of molecular biology, which makes an enormous impact
both industrially and medically. Microbiology also encompasses immunology, the study of the body‘s
ability to mount defenses against infectious microbes.
Because microbiology, by definition, studies organisms not visible to the naked eye, we can consider
late-17th-century Dutch scientist Antony van Leeuwenhoek the father of the discipline. Leeuwenhoek
was the first person to describe tiny cells and bacteria, and he invented new methods for grinding and
polishing microscope lenses that allowed for curvatures providing magnifications of up to 270
diameters, the best available lenses at that time. But while van Leeuwenhoek is cited as the first
microbiologist, the first recorded microbiological observation — the fruiting bodies of molds — were
made earlier, in 1665, by English physicist Robert Hooke.
Other notable people in the history of science who made fundamental discoveries about
microorganisms are 19th-century scientists Louis Pasteur and Robert Koch, who are considered the
founders of medical microbiology. Pasteur is most famous for his series of experiments designed to
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disprove the then-widely held theory of spontaneous generation, which solidified microbiology‘s
identity as a biological science. Pasteur also designed methods for food preservation (pasteurization)
and vaccines against several diseases, such as anthrax, fowl cholera and rabies. Koch is best known for
his contributions to the germ theory of disease, proving that specific diseases were caused by specific
pathogenic microorganisms. He developed a series of criteria that have become known as Koch‘s
postulates. Koch was one of the first scientists to focus on the isolation of bacteria in pure culture,
resulting in his description of several novel bacteria, including Mycobacterium tuberculosis, the
causative agent of tuberculosis.
Finally, some of the most important discoveries affecting public health occurred in the 20th century,
such as the discovery of penicillin by Alexander Fleming, which started a rush to find other natural,
and eventually synthetic, antibiotics; the development of vital vaccines, including those for polio and
yellow fever; and the birth of molecular biology, which happened in the 1940s with the study of
bacteria.
What Are Microbes?

A microbe, or microorganism, is a microscopic organism that comprises either a single cell
(unicellular); cell clusters; or multicellular, relatively complex organisms.
The study of microorganisms is called microbiology, a subject that began with Anton van
Leeuwenhoek‘s discovery of microorganisms in 1675, using a microscope of his own design.
A Drawing of Microbes: This is a drawing of what Arthur Hill Hassall saw under a microscope in a
sample of water taken from the River Thames at two locations. Hassall was able to identify many
microscopic organisms not perceptible to the unaided eye.
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(green algae); and animals such as rotifers and planarians. Some microbiologists also include viruses,
but others consider these as non-living. Most microorganisms are unicellular, but this is not universal,
since some multicellular organisms are microscopic. Some unicellular protists and bacteria, like
Thiomargarita namibiensis, are macroscopic and visible to the naked eye.
Microorganisms live in all parts of the biosphere where there is liquid
Microorganisms live in all parts of the biosphere where there is liquid water, including soil, hot
springs, on the ocean floor, high in the atmosphere, and deep inside rocks within the Earth‘s crust.
Most importantly, these organisms are vital to humans and the environment, as they participate in the
Earth‘s element cycles, such as the carbon cycle and the nitrogen cycle.
Microorganisms also fulfill other vital roles in virtually all ecosystems, such as recycling other
organisms‘ dead remains and waste products through decomposition. Microbes have an important
place in most higher-order multicellular organisms as symbionts, and they are also exploited by people
in biotechnology, both in traditional food and beverage preparation, and in modern technologies based
on genetic engineering. Pathogenic microbes are harmful, however, since they invade and grow within
other organisms, causing diseases that kill humans, animals, and plants.
The Pathogenic Ecology of Microbes
Although many microorganisms are beneficial, many others are the cause of infectious diseases. The
organisms involved include pathogenic bacteria, which cause diseases such as plague, tuberculosis,
and anthrax. Biofilms —microbial communities that are very difficult to destroy—are considered
responsible for diseases like bacterial infections in patients with cystic fibrosis, Legionnaires‘ disease,
and otitis media (middle ear infection). They produce dental plaque; colonize catheters, prostheses,
transcutaneous, and orthopedic devices; and infect contact lenses, open wounds, and burned tissue.
Biofilms also produce foodborne diseases because they colonize the surfaces of food and food-
processing equipment. Biofilms are a large threat because they are resistant to most of the methods
used to control microbial growth. Moreover, the excessive use of antibiotics has resulted in a major
global problem since resistant forms of bacteria have been selected over time. A very dangerous strain,
methicillin-resistant Staphylococcus aureus (MRSA), has wreaked havoc recently.
In addition, protozoans are known to cause diseases such as malaria, sleeping sickness, and
toxoplasmosis, while fungi can cause diseases such as ringworm, candidiasis, or histoplasmosis. Other
diseases such as influenza, yellow fever, and AIDS are caused by viruses.
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Food-borne diseases result from the consumption of contaminated food, pathogenic bacteria, viruses,
or parasites that contaminate food. ‖ Hygiene‖ is the avoidance of infection or food spoiling by
eliminating microorganisms from the surroundings. As microorganisms (bacteria, in particular) are
found virtually everywhere, the levels of harmful microorganisms can be reduced to acceptable levels
with proper hygiene techniques. In some cases, however, it is required that an object or substance be
completely sterile (i.e., devoid of all living entities and viruses). A good example of this is a
hypodermic needle.
Louis Pasteur
Louis Pasteur ForMemRS (/ˈluːi pæˈstɜːr/, French: [lwi pastœʁ]; December 27, 1822 – September 28,
1895) was a French biologist, microbiologist, and chemist renowned for his discoveries of the
principles of vaccination, microbial fermentation, and pasteurization. He is remembered for his
remarkable breakthroughs in the causes and prevention of diseases, and his discoveries have saved
many lives ever since. He reduced mortality from puerperal fever and created the first vaccines
for rabies and anthrax.
His medical discoveries provided direct support for the germ theory of disease and its application in
clinical medicine. He is best known to the general public for his invention of the technique of
treating milk and wine to stop bacterial contamination, a process now called pasteurization. He is
regarded as one of the three main founders of bacteriology, together with Ferdinand Cohn and Robert
Koch, and has been called a "father of bacteriology" and the "father of microbiology", though the latter
appelation has also been applied to Antonie van Leeuwenhoek.
Pasteur was responsible for disproving the doctrine of spontaneous generation. He performed
experiments that showed that, without contamination, microorganisms could not develop. Under the
auspices of the French Academy of Sciences, he demonstrated that in sterilized and sealed flasks,
nothing ever developed; and, conversely, in sterilized but open flasks, microorganisms could
grow. Although Pasteur was not the first to propose the germ theory, his experiments indicated its
correctness and convinced most of Europe that it was true.
Today, he is often regarded as one of the fathers of germ theory. Pasteur made
significant discoveries in chemistry, most notably on the molecular basis for the asymmetry of
certain crystals and racemization. Early in his career, his investigation of tartaric acid resulted in the
first resolution of what is now called optical isomers. His work led the way to the current
understanding of a fundamental principle in the structure of organic compounds.
He was the director of the Pasteur Institute, established in 1887, until his death, and his body was
interred in a vault beneath the institute. Although Pasteur made ground breaking experiments, his
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reputation became associated with various controversies. Historical reassessment of his notebook
revealed that he practiced deception to overcome his rivals.
Spontaneous generation is an obsolete body of thought on the ordinary formation of living organisms
without descent from similar organisms. Typically, the idea was that certain forms such as fleas could
arise from inanimate matter such as dust or that maggots could arise from dead flesh. A variant idea
was that of equivocal generation, in which species such as tapeworms arose from unrelated living
organisms, now understood to be their hosts.
Doctrines held that these processes were commonplace and regular. Such ideas were in contradiction
to that of univocal generation: effectively exclusive reproduction from genetically related parent(s),
generally of the same species. The doctrine of spontaneous generation was coherently synthesized by
Aristotle, who compiled and expanded the work of prior natural philosophers and the various ancient
explanations of the appearance of organisms; it held sway for two millennia.
Today spontaneous generation is generally accepted to have been decisively dispelled during the
19th century by the experiments of Louis Pasteur. He expanded upon the investigations of
predecessors, such as Francesco Redi who, in the 17th century, had performed experiments based on
the same principles.
Louis Pasteur‘s 1859 experiment is widely seen as having settled the question. In summary, Pasteur
boiled a meat broth in a flask that had a long neck that curved downward, like a goose. The idea was
that the bend in the neck prevented falling particles from reaching the broth, while still allowing the
free flow of air. The flask remained free of growth for an extended period. When the flask was turned
so that particles could fall down the bends, the broth quickly became clouded. In detail, Pasteur
exposed boiled broths to air in vessels that contained a filter to prevent all particles from passing
through to the growth medium, and even in vessels with no filter at all, with air being admitted via a
long tortuous tube that would not allow dust particles to pass. Nothing grew in the broths unless the
flasks were broken open, showing that the living organisms that grew in such broths came from
outside, as spores on dust, rather than spontaneously generated within the broth. This was one of the
last and most important experiments disproving the theory of spontaneous generation.
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Pasteur’s test of spontaneous generation: By sterilizing a food source and keeping it isolated from
the outside, Pasteur observed no putrefaction of the food source (top panel). Upon exposure to the
outside environment, Pasteur observed the putrefaction of the food source (bottom panel). This
strongly suggested that the components needed to create life do not spontaneously arise.
Despite his experiment, objections from persons holding the traditional views persisted. Many of these
residual objections were routed by the work of John Tyndall, succeeding the work of Pasteur.
Ultimately, the ideas of spontaneous generation were displaced by advances in germ theory and cell
theory. Disproof of the traditional ideas of spontaneous generation is no longer controversial among
professional biologists. Objections and doubts have been dispelled by studies and documentation of the
life cycles of various life forms. However, the principles of the very different matter of the original
abiogenesis on this planet — of living from non-living material — are still under investigation
Louis Pasteur is also known as father of microbiology. He has many contributions to

microbiology:
1. He has proposed the principles of fermentation for preservation of food
2. He introduced sterilization techniques and developed steam sterilizer, hot air over and
autoclave.
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Robert Koch
Robert Koch was born in Clausthal in the Harz Mountains, then part of the Kingdom of Hanover, as
the son of a mining official. He studied medicine at the University of Göttingen and graduated in 1866.
He then served in the Franco-Prussian War and later became district medical officer in Wollstein
(Wolsztyn), Prussian Poland. Working with very limited resources, he became one of the founders of
bacteriology, the other major figure being Louis Pasteur.
Robert Koch: An image of Robert Koch, a pioneering

microbiologist. Koch‘s research and methods helped link the
causal nature of microbes to certain diseases, including
anthrax.
After Casimir Davaine demonstrated the direct transmission of

the anthrax bacillus between cows, Koch studied anthrax more
closely. He invented methods to purify the bacillus from blood
samples and grow pure cultures. He found that, while it could
not survive outside a host for long, anthrax built persisting
endospores that could last a long time. These endospores,
embedded in soil, were the cause of unexplained ―spontaneous‖ outbreaks of anthrax. Koch published
his findings in 1876 and was rewarded with a job at the Imperial Health Office in Berlin in 1880. In
1881, he urged for the sterilization of surgical instruments using heat.
Probably as important as his work on tuberculosis, for which he was awarded a Nobel Prize in 1905,
are Koch‘s postulates. These postulates stated that to establish that an organism is the cause of a
disease, it must be found in all cases of the disease examined. Additionally, it must be absent in
healthy organisms prepared and maintained in a pure culture capable of producing the original
infection, even after several generations in culture retrievable from an inoculated animal and cultured
again. By using his methods, Koch‘s pupils found the organisms responsible for diphtheria, typhoid,
pneumonia, gonorrhoea, cerebrospinal meningitis, leprosy, bubonic plague, tetanus, and syphilis.
Perhaps the key method Koch developed was the ability to isolate pure cultures, explained in brief
here. Pure cultures of multicellular organisms are often more easily isolated by simply picking out a
single individual to initiate a culture. This is a useful technique for pure culture of fungi, multicellular
algae, and small metazoa. Developing pure culture techniques is crucial to the observation of the
specimen in question. The most common method to isolate individual microbes and produce a pure
culture is to prepare a streak plate. The streak plate method is a way to physically separate the
microbial population and is done by spreading the inoculate back and forth with an inoculating loop
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over the solid agar plate. Upon incubation, colonies will arise and single cells will have been isolated
from the biomass.
He investigated the anthrax disease cycle in 1876, and studied the bacteria that cause tuberculosis in
1882 and cholera in 1883. He discovered bacteria such as the anthrax bacilli, tubercle bacilli and
cholera bacilli. Koch observed the phenomenon of acquired immunity
Robert Koch provided remarkable contributions to the field of microbiology:
1. He used of solid media for culture of bacteria-Eilshemius Hesse, the wife of Walther hesse, one
of Koch‘s assistants had suggested the use of agar as solidifying agent.
2. Koch‘s phenomenon: Robert Koch observed that guinea pigs already infected with tubercle
bacillus developed a hypersensitivity reaction when injected with tubercle bacilli or its protein.
This reaction is called Koch‘s phenomenon.
Paul Ehrlich
In the first phase of his career (1878-1890), Ehrlich developed the principles of modern hematology
and immunology by describing various leukocyte subsets and their precursor cells. In ddition, Ehrlich
contributed to the field of microbiology and was involved in the classification of diverse microbes.
1. He was the first to report the acid-fast nature of tubercle bacillus.
2. He developed techniques to stain tissues and blood cells.
3. He proposed a toxin antitoxin interaction called as Ehrlich phenomenon and also introduced
methods of standardising toxin and antitoxin.
MICROSCOPY
A microscope (from the Ancient Greek: μικρός, mikrós, "small" and σκοπεῖν, skopeîn, "to look" or
"see") is a laboratory instrument used to examine objects that are too small to be seen by the naked
eye. Microscopy is the science of investigating small objects and structures using a
microscope. Microscopic means being invisible to the eye unless aided by a microscope.
There are many types of microscopes, and they may be grouped in different ways. One way is to
describe the method an instrument uses to interact with a sample and produce images, either by
sending a beam of light or electrons through a sample in its optical path, by detecting photon
emissions from a sample, or by scanning across and a short distance from the surface of a sample using
a probe. The most common microscope (and the first to be invented) is the optical microscope, which
uses lenses to refract visible light that passed through a thinly sectioned sample to produce an
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observable image. Other major types of microscopes are the fluorescence microscope, electron
microscope (both the transmission electron microscope and the scanning electron microscope) and
various types of scanning probe microscopes.
A good microscope should have three properties:
1. Good resolution: Resolution power refers to the ability to produce separate images of closely
placed objects so that they can be distinguished as two separate entities. The resolution power
of:
 Unaided human eye is about 0.2 mm (200 m)

 Light microscope is about 0.2 m
 Electron microscope is about 0.5 nm
Resolution depends on refractive index. Oil has a higher refractive index than air.
2. Good contrast: This can be further improved by staining the specimen.
3. Good magnification: This is achieved by use of concave lenses.
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Bright-Field or Light Microscope
The bright-field or light microscope forms a dark image against a brighter

background. Microbiology and bacteriology have always relied on the bright field technique.
Different stains and staining techniques are used depending upon the type of specimen and cell
structure being examined.
For example:
 Fuchsin is used to stain smooth muscle cells
 Gram stain is used on bacteria and gives rise to the name gram-negative or gram-positive bacteria
based on the reaction of the bacteria to the stain.
Dark Field Microscope
 Principal: In dark field microscope, the object appears bright against a dark background. This is
made possible by use of a special dark field condenser.
 Applications: It is used to identify the living, unstained cells and thin bacteria like spirochetes
which cannot be visualized by light microscopy.
 In 1830, J.J. Lister invented the dark field microscope in which the standard bright field condenser
is replaced with a single or double reflecting dark field condenser. In 1906 in Vienna, Karl
Landsteiner and Viktor Mucha were the first to use dark field microscopy to visualize T.pallidum
from syphilis lesions.
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Comparison between Bright Field and Dark Field Microscope
BRIGHT FIELD MICROSCOPE DARK FIELD MICROSCOPE

Light from a plane wave source is focused An opaque disc is put between the source and
through an object by a condenser. the condenser blocking out the middle of the
beam.
Some light is blocked (absorbed) by opaque The condenser focuses the beam onto the
parts of the object, or reflected away at the sample.
boundaries between components or materials of
different refractive indices.
The remainder passes through the objective lens No light enters the objective directly from the
to the observer. source. Light from the beam is scattered by the
sample- some scattered on to the objective.
This produces a bright background with object Only light scattered by the object enters the
details appearing darker in the image than their objective. This produces a dark background with
surroundings. The brightness of the image is sample details appearing brighter than
determined by the source brightness and block surroundings. The brightness of the brightest
size. parts of the image is determined by the amount
of light scattered by the object.
This result in poorer contrast compared to dark This results in superior contrast to bright field as
field as the dark areas are generally grey rather dark areas may be completely black while
than black. increasing the brightness of the source brightens
the areas.
Phase Contrast Microscope
The phase contrast microscope is used to visualise unstained living cells. Most of the stains or staining
procedures will kill the cells. Phase contrast microscopy enables the visualization of living cells and
life events.
The phase contrast microscope was developed by Dutch physicist Frits Zernike in early 1930s. the
invention of this microscope enables us to visualise live cells and cellular processes. The inventor was
awarded by Noble prize in physics in 1953.
Principle: When light passes through cells, small phase shifts occur, which are invisible to the human
eye. In a phase-contrast microscope, these phase shifts are converted into changes in amplitude, which
can be observed as differences in image contrast.
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Working of Phase contrast microscope:
1. Partially coherent illumination produced by the tungsten-halogen lamp is directed through a

collector lens and focused on a specialized annulus (labeled condenser annulus) positioned in
the substage condenser front focal plane.
2. Wavefronts passing through the annulus illuminate the specimen and either pass through
undeviated or are diffracted and retarded in phase by structures and phase gradients present in
the specimen.
3. Undeviated and diffracted light collected by the objective is segregated at the rear focal plane
by a phase plate and focused at the intermediate image plane to form the final phase-contrast
image observed in the eyepieces.
Parts of Phase contrast Microscope:
Phase-contrast microscopy is basically a specially designed light microscope with all the basic parts in
addition to which an annular phase plate and annular diaphragm are fitted.
The annular diaphragm
1. It is situated below the condenser.
2. It is made up of a circular disc having a circular annular groove.
3. The light rays are allowed to pass through the annular groove.
4. Through the annular groove of the annular diaphragm, the light rays fall on the specimen or
object to be studied.
5. At the back focal plane of the objective develops an image.
6. The annular phase plate is placed at this back focal plane.
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The phase plate
1. It is either a negative phase plate having a thick circular area or a positive phase plate having a
thin circular groove.
2. This thick or thin area in the phase plate is called the conjugate area.
3. The phase plate is a transparent disc.
4. With the help of the annular diaphragm and the phase plate, the phase contrast is obtained in
this microscope.
5. This is obtained by separating the direct rays from the diffracted rays.
6. The direct light rays pass through the annular groove whereas the diffracted light rays pass
through the region outside the groove.
7. Depending upon the different refractive indices of different cell components, the object to be
studied shows a different degree of contrast in this micro-scope.
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Applications of Phase contrast Microscopy:
To produce high-contrast images of transparent specimens, such as
 living cells (usually in culture),
 microorganisms,
 thin tissue slices,
 lithographic patterns,
 fibers,
 latex dispersions,
 glass fragments, and
 subcellular particles (including nuclei and other organelles).

Applications of phase-contrast microscopy in biological research are numerous.
Advantages:
 Living cells can be observed in their natural state without previous fixation or labeling.
 It makes a highly transparent object more visible.
 No special preparation of fixation or staining etc. is needed to study an object under a phase-
contrast microscope which saves a lot of time.
 Examining intracellular components of living cells at relatively high resolution. eg: The
dynamic motility of mitochondria, mitotic chromosomes & vacuoles.
 It made it possible for biologists to study living cells and how they proliferate through cell
division.
 Phase-contrast optical components can be added to virtually any brightfield microscope,

provided the specialized phase objectives conform to the tube length parameters, and the
condenser will accept an annular phase ring of the correct size.
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Limitations:
 Phase-contrast condensers and objective lenses add considerable cost to a microscope, and so
phase contrast is often not used in teaching labs except perhaps in classes in the health
professions.
 To use phase-contrast the light path must be aligned.
 Generally, more light is needed for phase contrast than for corresponding bright-field viewing,
since the technique is based on the diminishment of the brightness of most objects.
Electron Microscope
Electron microscopy (EM) is a technique for obtaining high resolution images of biological and non-
biological specimens. It is used in biomedical research to investigate the detailed structure of tissues,
cells, organelles and macromolecular complexes. The high resolution of EM images results from the
use of electrons (which have very short wavelengths) as the source of illuminating radiation. Electron
microscopy is used in conjunction with a variety of ancillary techniques (e.g. thin sectioning, immuno-
labeling, negative staining) to answer specific questions. EM images provide key information on the
structural basis of cell function and of cell disease. It was invented by Ernst Ruska in 1931.
There are two main types of electron microscope – the transmission EM (TEM) and the scanning EM
(SEM)
1. Transmission EM (MC type, examine the internal structure, resolution 0.5 nm, gives 2-dimensional
view)
2. Scanning EM (examine the surfaces, resolution 7 nm, gives 3-dimensional view)
Working Principle: An electron microscope uses an ‗electron beam‘ to produce the image of the
object and magnification is obtained by ‗electromagnetic fields‘; unlike light or optical microscopes, in
which ‗light waves‘ are used to produce the image and magnification is obtained by a system of
‗optical lenses‘.
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Transmission Electron Microscope (TEM): In this microscope, an electron beam from an
electron gun is transmitted through an ultra-thin section of the microscopic object and the image is
magnified by the electromagnetic fields. It is used to observe finer details of internal structures of
microscopic objects like bacteria and other cells.
The specimen to be examined is prepared as an extremely thin dry film or as an ultra-thin section on a
small screen and is introduced into the microscope at a point between the magnetic condenser and the
magnetic objective.(fig 4.13)
The point is comparable to the stage of a light microscope. The magnified image may be viewed on a
fluorescent screen through an airtight window or recorded on a photographic plate by an in-built
camera. Modern variants have facility to record the photograph by digital camera.
Scanning Electron Microscope (SEM): In a scanning electron microscope, the specimen is

exposed to a narrow electron beam from an electron gun, which rapidly moves over or scans the
surface of the specimen (Figure 4.13). This causes the release of a shower of secondary electrons and
other types of radiations from the specimen surface.
The intensity of these secondary electrons depends upon the shape and the chemical composition of
the irradiated object. These electrons are collected by a detector, which generates electronic signals.
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These signals are scanned in the manner of a television system to produce an image on a cathode ray
tube (CRT).
The image is recorded by capturing it from the CRT. Modern variants have facility to record the
photograph by digital camera. This microscope is used to observe the surface structure of microscopic
objects.
The limitations of electron microscopes are as follows:
(a) Live specimen cannot be observed.
(b) As the penetration power of electron beam is very low, the object should be ultra-thin. For this, the
specimen is dried and cut into ultra-thin sections before observation.
Sterilization:
Sterilization refers to any process that removes, kills, or deactivates all forms of life (in particular
referring to microorganisms such as fungi, bacteria, spores, unicellular eukaryotic organisms such
as Plasmodium, etc.) and other biological agents like prions present in a specific surface, object or
fluid, for example food or biological culture media.[1][2] Sterilization can be achieved through various
means, including heat, chemicals, irradiation, high pressure, and filtration. Sterilization is distinct
from disinfection, sanitization, and pasteurization, in that those methods reduce rather than eliminate
all forms of life and biological agents present. After sterilization, an object is referred to as being
sterile or aseptic.
Introduction
Sterilization is the process of killing all microorganisms (bacterial, viral, and fungal) with the use of
either physical or chemical agents. A disinfectant is a chemical substance that kills microorganisms on
inanimate objects, such as exam tables and surgical instruments. Skin can never be completely sterile.
Sterilization in the microbiological laboratory denotes sterilization process implemented in preparation
of culture media, reagents and equipment where the work warrants maintaining sterile condition.
Sterilization in microbiology laboratory is done by following methods
Physical method i.e., use of heat, filters, radiation
Chemical method i.e., by use of chemicals
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Heat sterilization
a. Dry heat sterilization. Dry heat sterilization Inoculation loops or needle are sterilized by heating to
'red' in Bunsen burner or spirit lamp flame. Sterilization in hot air oven is performed at a temperature
of 160C and maintained or holding for one hour. Spores are killed at this temperature and this is the
most common method of sterilization of glassware, swab sticks, pestle and mortar, mineral oil etc. Dry
heat sterilization causes protein denaturation, Oxidative damage, toxic effect of elevated electrolyte in
absence of water.
b. Wet heat or moist heat sterilization Moist heat sterilization is accomplished by
1). Boiling at 100°C for 30 minutes is done in a water bath. Syringes, rubber goods and surgical
instruments may be sterilized by this method. Almost all bacteria and certain spores are killed in this
method
2). Steaming at 100°C for 20 to 30 minutes under normal atmospheric pressure are more effective than
dry heat at the same temperature because bacteria are more susceptible to moist heat, Steam has more
penetrating power and sterilizing power as more heat is given up during condensation. Suitable for
sterilizing media which may be damaged at a temperature higher than 100°C
3).Tyndallization (Fractional Sterilization) is the steaming process performed at 100°C is done in

steam sterilizer for 20 minutes followed by incubation at 37°C overnight and this cycle is 12 repeated
for successive 2 days. Spores, if any, germinate to vegetative bacteria during incubation and are
destroyed during steaming on second and third day. Heat labile media containing sugar, milk, gelatin
can be sterilized using this method.
4). Autoclaving is done by steam under pressure. Steaming at temperature higher than 100°C is used
in autoclaving. This is achieved by employing a higher pressure. The autoclave is closed and made air-
tight for pressure development and at 15 lbs per sq. inch pressure, 121°C temperatures will be reached
and this temperature is given as sterilizing holding time for further 15 minutes. This process kill spores
and this works like a pressure cooker and one of the most common methods of sterilization.
5). Pasteurization is another one method of moist heat sterilization which works below 100°C heat.
This process is used in heating of milk and other liquid food. The product is held at temperature and
for a period of time to kill pathogenic bacteria that may be present in the product. This process does
not destroy complete organism including spores. All these moist heat sterilization causes denaturation
and coagulation of protein, breakage of DNA strands, and loss of functional integrity of cell
membrane.
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c). Filtration: This method of sterilization is used for media particularly heat labile in nature (e.g. sera
an media containing proteins or labile metabolites. If the study warrants bacteriafree filtrates it can be
obtained through 0.45micron sized filter membranes and if the study requires viral particle free
solution, then 0.22micron sized filter membranes are use. In earlier days absorptive filters of asbestos
or diatomaceous earth were replaced by unglazed porcelain or sintered glass are used. Nowadays these
are replaced by nitrocellulose membrane filters of graded porosity, PVDF etc.
d). Ultraviolet Radiation: at wavelength between 330nm and 400nm causes sterilizing effect. This
method is used in surface sterilization of laminar airflow, biosafety cabinet and in certain cases in
laboratory. In microbiology laboratory autoclaving, hot air oven sterilization, filtration and UV
radiation are commonly used.
Standard operating procedure for the setting up of autoclave
 Pack your media, reagents, plastic wares, in their appropriate autoclavable resistant polypropylene
or borosilicated glassware
 Screw the lid of the tube and leave one thread loose in case of closed containers or plastics
 Stick at random autoclavable indicators for each run in any of the items to be autoclaved
 Check for the water level in the autoclave machine
 Donot jam pack the items in the autoclave machine
 Switch on the machine
 Keep the lid of the machine tightly closed with one valve open until it reaches boiling
 Leave heated air to escape for few minute through valve
 Completely close the valve and wait to reach the temperature for 121 degree Celsius at 15lbs
pressure.
 Hold the sterilization cycle for 15 minutes
 Once the sterilization cycle end, switch off the heating and leave the machine to reach to 65
degree C
 Then open the lid and take out the items back after sterilization
Standard operating procedure for the setting up of hot air oven
 Pack all the glassware such as pipette with pipette can, glass petridishes, sample dish, test tubes,
pestle and mortar, mineral oil to be sterilized by hot air oven sterilization with suitable wrapping
 Switch on the hot air oven until to reach 160 degree C
 Hold on in that temperature for 1 hour
 Switch off the heating of hot air oven and open the door once come below 65 degree C
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Standard operating procedure for the setting up of filtration
 Once the bio safety cabinet is ready for filtration
 Switch on the blower
 Filtration unit should be inside the cabinet
 Vacuum or positive pump should be kept outside of the cabinet
 Filtration assembly should be with the suitable filters
 Pour the media or reagents to be sterilized in the top of the filtration assembly
 Connect the bottom assembly to vacuum pump or top of the assembly to the positive pump
It is a process by which an article, surface or medium is made free of all micro-organisms either in the
vegetative or spore form.
Disinfection means the destruction or removal of all pathogenic organisms or organisms capable of
giving rise to infection.Antisepsis, term used to indicate the prevention of infection usually by
inhibiting the growth of bacteria in wounds and tissues.
Table 1.2.1: Classification of sterilization/disinfection methods

A. Physical methods
1. Heat
Dry heat: Flaming, Incineration and Hot air oven Moist heat:
a. Temperature <1000C, e.g. pasteurization, water bath and inspissation
b. Temperature at 1000C, e.g. boiling, steaming and tyndallisation
c. Temperature >1000C, e.g. membrane filters
2. Filtration: Depth filters and membrane filters
3. Radiation
Ionizing radiation: Y-rays, X-rays and cosmic rays
Non-ionizing radiation: Ultraviolet (UV) and infrared rays
4. Ultrasonic vibration
B. Chemical methods
1. Alcohols: Ethyl alcohol, isopropyl alcohol
2. Aldehydes: Formaldehyde, glutaraldehyde, Ortho-phthaladehyde hexachlorophene
3. Phenolic compounds: Cresol, Lysol, chlorhexidine, chloroxylenol, hexachlorophene
4. Halogens: Chlorine, iodine, iodophors
5. Oxidising agents: Hydrogen peroxide, Peracetic acid
6. Salts: Mercuric chloride, copper salts
7. Surface active agents: Quaternary ammonium compounds and soaps
8. Dyes: Aniline dyes and acridine dyes
9. Gas sterilization: Low temperature steam formaldehyde, Ethylene oxide (ETO) and Beta-
Propiolactone (BPL)
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METHODS OF STERILISATION
1. Physical method
i) Sunlight: has an active germicidal effect due to its content of UV rays. It is natural method of
sterilisation in cases of water tanks, rivers and lakes.
ii) Heat: most reliable and commonly employed method of sterilisation. Two types of heat – dry and
moist heat.
DRY HEAT MOIST HEAT

Kills the organism by denaturation proteins, Kills the micro-organisms by denaturation and
oxidative damage and by toxic effect of elevated coagulation of proteins.
levels of electrolytes.
 Bacterial spores are killed by moist heat at 121 C for 15 min. most vegetative bacteria, fungi and
viruses are killed in 30 min at 65 C by moist heat.
 Materials containing organic substances require more time for sterilisation. Proteins, sugars, fats
and starch are some of the organic substances.
DRY HEAT STERILISATION:
 Red heat: inoculating loops, tips of forceps and needles are held in the flame of a Bunsen burner
till they become red hot.
 Flaming: glass slides, scalpels and mouth of culture tubes are passed through Bunsen flame
without allowing them to become red hot.
 Incineration: by this method, infective materials are reduced to ashes by burning. Soiled dressings,
animal carcasses, bedding and pathological materials are dealt with this method.
 Hot air oven.
MOIST HEAT STERILISATION: this method of sterilisation may be used at different temperatures
as follows-
 At temperature below 100 C.
i) Pasteurisation of milk: two types of method, holder method (63 C for 30 min) and flash method
(72 C for 20 sec followed by quickly cooling to 13 C or lower)are used. All non sporing pathogens
such as mycobacteria and Brucella are killed except Coxiella burnetti which being relatively heat
resistant may survive in holder method.
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ii) Inspissation: some serum and egg media such as LOwerstein Jensens and Loefflers serum, are
rendered sterile by heating at 80-85 C temperature for half an hour daily on three consecutive
days.At a temperature of 100 C.
i) Boiling: 10-30 min may kill most of the vegetative forms but many spores withstand for a
considerable time.
ii) Tyndallisation: Steam at 100 C for 20 min on three consecutive days is used.
iii) Steam steriliser at 100 C for 90 min: Kochs or Arnolds steam steriliser is usually used for
media which are decomposed at high temperature of autoclave.
 At temperature above 100 C.

i) Filtrations: This method is used for substances which get damaged by heat process eg sera,
sugars, antibiotic solution etc.
Uses:
 to sterile sera sugars and antibiotic solution
 Separation of toxins and bacteriophages.
 To obtain bacteria free filtrates of clinical samples of virus isolation.
 Sterilisation of hydatid fluid.
 Purification of water.
RADIATIONS
1. Ionising Radiations: include gamma rays , X- rays and cosmic rays. They are highly lethal to all
cells including bacteria. They damage DNA. Gamma radiations from cobalt 60 source are
commercially used for sterilisation of disposable items such as swabs, cultures, catheters, cannulas
plastic syringes. This method is known as cold sterilisation because3 there is no appreciable increase in
temperature.
2. Non ionising radiations: include UV radiation. Ultraviolet rays of wavelength 240- 280 nm have
marked bactericidal activity. It acts by denaturation of proteins and interfere with DNA replication.
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CHEMICAL METHODS OF STERILISATION
ALCOHOLS: ethyl alcohol and isopropyl alcohol are the most frequently used. They act by
denaturing bacterial proteins. They rapidly kill bacteria including tubercle bacilli but they have no
sporicidal or virucidal activity.
HIV (human immunodeficiency virus) is susceptible to 70% ethyl alcohol and 35% isopropyl alcohol
in the absence of organic matter.
ALDEHYDES: markedly bactericidal, sporicidal and virucidal.
Formaldehyde – it is used as both aqueous solution and gaseous solution. Used to prevent tissue for
histological examination.To sterile bacterial vaccines, to prepare toxoids from toxins.
Glutaraldehyde – effective against bacteria, fungi and viruses (including HIV, hepatitis B viruses and
enteroviruses), it also kills spores. Used for sterilisation of cystoscopes, endoscopes and
bronchoscopes. To sterilise plastic endotracheal tubes, face masks, corrugated rubber anaesthetic tubes
and metal instruments.
Ortho-phthalaldehyde (OPA) – is a high level disinfectant. More stable during storage and more
rapidly mycobactericidal than glutaraldehyde.
HALOGENS: chlorine and iodine are two commonly used disinfectants. These are bactericidal and
are effective against sporing bacteria and viruses. Chlorine is used in water supplies, swimming pools
and dairy industries. Iodine in alcoholic and aqueous solutions is used as skin disinfectant. It is actively
bactericidal with moderate action against viruses. Like chlorine it is inactivated by inorganic matter.
SALTS: salts of heavy metals have toxic effect on bacteria. The salts of copper, silver and mercury
are used as disinfectants. They are protein coagulants and act by combining with sulphydryl groups of
bacterial proteins and other essential intracellular compounds.
Copper salts are used as fungicides. Mercuric chloride once used as disinfectant, is highly toxic.
DYES: two groups of dyes, aniline dyes and acridine dyes have been used extensively as skin and
wound antiseptics. Both are bacteriostatic in high dilution but low bactericidal action. Aniline dyes
include crystal violet, brilliant green and malachite green –they are more active against Gram positive
bacteria than Gram negative bacteria.
Acridine dyes include acriflavine, euflavine, proflavine and aminacrine. They are affected very little
by the presence of organic material. They are also more active against Gram positive bacteria than
Gram negative bacteria but are not used as selective as the aniline dyes.
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OXIDISING AGENTS:
i) Hydrogen peroxide: is effective against most organisms at concentration of 3-6%, while it kills all
organisms including spores at higher concentration. Mode of action is by liberation of free hydroxyl
radical on decomposition of H2O2. These free radicals are the active ingredient in the disinfection
process. H2O2 is used to disinfect contact lenses, surgical prostheses and plastic implants. It is used for
high level disinfection and sterilisation.
ii) Peracetic Acid: is an oxidising agent. It is one of the high level disinfectants. It is effective in the
presence of organic matter. It is a more potent germicidal agent than hydrogen peroxide. The end
products of this agent are non-toxic. It is also used in plasma sterilisation procedure.
SURFACE ACTIVE AGENTS:
Quaternary ammonium compounds are the most important cationic surfactants. Although these
compounds are bactericidal for a wide range of organisms, Gram positive species are more susceptible.
They have no action on spores and tubercle bacilli. They are active against viruses with liquid
envelopes and much less against non-enveloped viruses.

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MCQ
1. Which of the microscopes below is usually good for use on unstained specimens?
(a) Phase contrast
(b) Fluorescence
(c) Bright field
(d) Scanning electron
2. Which of the microscope below form image in visible light?
(a) bright field
(b) Dark field
(c) Fluoscence
(d) A and B
3. The instrument that produce a bright image of the specimen against a dark background is called
______
(a) Phase contrast
(b) Transmission electron
(c) Bright field
(d) Dark field
4. If you were given a specimen of an active , mobile microorganism, which of the following types of
microscopy would be the most effective in visualizing the live micobe?
(a) Bright field microscopy
(b) Dark field microscopy
(c) Flucoschence microscopy
(d) Phase contrast microscopy
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5. Which put of the compound microscopy helps in gathering and focusing light rays on the specimen
to be viewed?
(a) Eyepiece long
(b) Objective lens
(c) Condenser lens
(d) Magnifying lens
6. Resolving power of a microscope is a function of ________________
(a) Wavelength of light used
(b) Numerical aperture of lens system
(c) Refractive index
(d) wavelength of light used and numerical aperture.
7. In phase contrast microscopy the rate at which light enters through objects is -----------------.
(a) constant
(b) inversely proportion to their refractive index.
(c) Directly proportion to their refractive index.
(d) Exponentially related to their refractive index.
8. removal and killing of all microorganism is known as
(a) Destruction
(b) Sterilization
(c) Pasteurization
(d) removal
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9. Sterilization is done by autoclave, consisting exposure to stream, about
(a) 120°C
(b) 170°C
(c) 121°C
(d) 116°C
10. In hospitals, surgical instruments and plastics are washed with
(a) Ethylene oxide
(b) Iodine
(c) Tincture
(d) chlorine
ANSWER KEY
1. A 2. D 3. D 4. A 5. C
6. D 7. B 8. B 9. C 10. A
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ONE WORD QUESTION
1. Scanning electron microscopy is most often used to reveal.
2. The instrument that produces a bright image of the specimen against a dark background is
called.
3. The microscopy that is most effective in visualizing the live microbe If you were given a
specimen of an active, motile microorganism.
4. A microscope that exposes specimens to ultraviolet and forms an image with the resulting light
emitted at a different wavelength is called.
5. A process that eliminates, removes, kills, or deactivates all forms of life.
6. A widely used method for heat sterilization that, sometimes called a converter or steam
sterilizer.
7. Temperature of Autoclaves use steam heated to under pressure.
8. The study of microscopic organisms, such as bacteria, viruses, archaea, fungi and protozoa.
9. Is a small infectious agent that replicates only inside the living cells of an organism.
10. The branch classified into applied sciences, or divided according to taxonomy, as is the case
with bacteriology, mycology, protozoology, and phycology is.
ANSWER KEY
1. Internal 2. Dark field 4. A fluroscence
3. Phase Contrast 5. Sterilization
structure microscope microscope
7. 121-1340C (250-
6. Autoclave 8. Microbiology 9. Virus 10. Microbiology
2730F)
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Short Questions
1. What is the use of dark field microscope?
___________________________________________________________________________________
___________________________________________________________________________________
2. Why is the field dark and the specimen bright when a dark field microscope is used to examine a
specimen?
___________________________________________________________________________________
___________________________________________________________________________________
3. What is the difference between bright field and dark field microscope?
___________________________________________________________________________________
___________________________________________________________________________________
4. What is the principle of bright field microscope?
___________________________________________________________________________________
___________________________________________________________________________________
5. What is the principle of sterilization?
___________________________________________________________________________________
___________________________________________________________________________________
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Long Questions
1. Describe microscope with suitable diagram.

2. What are the different types of microscope and their uses?
3. What is the principle of sterilization and its type?
4. What bacteria are gram positive and how they are differentiated from gram negative explain with
diagram?
5. Describe the structure and morphology of bacteria.

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UNIT 2

 Define Morphology and structure of microorganisms
 Morphology and structure of bacteria, fungi, actinomycete and algae
 Explain microscopic examination of microorganisms, preparation of culture media, spread
plates, pour plates, types of selective and differential media
 Explain separation of pure cultures, principles and uses of microbiology equipments and
instruments
MORPHOLOGY OF BACTERIA
 Bacteria are a type of biological cell that is prokaryotic and unicellular. Due to the lack of a
membrane-bound nucleus, these are simpler than other types of living organisms.
 Although only some of them can be seen by naked eyes while the rest are microscopic, they
display a wide range of shapes, sizes, and structures.
Bacterial Size
Figure : The relative sizes of various microscopic and non-microscopic objects.Note that a typical
virus measures about 100nm, 10 times smaller than a typical bacterium (~1µm), which is atleast 10
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times smaller than a typical plant or animal cell cell (~10–100 µm). An object must measure about 100
µm to be visible without a microscope. Image Source: Lumen Learning.
• The unit of measurement used in bacteriology is the micron (micrometer) which is one-thousandth
of a millimeter.
• Bacteria are, in general one-tenth the size of the eukaryotic cell. On average, the size of bacteria
ranges from 0.5 to 5 µm.
• However, they can be as tiny as 0.3 µm and as large as 0.7mm.
• The limit of resolution with the unaided eye is about 200 microns, and as many bacteria are
smaller than this size, they are not visible with naked eyes.
Among the largest bacteria is Thiomargarita namibiensis, which is up to half a millimeter long and
Epulopiscium fishelsoni which is 0.75 mm long.
The smallest bacteria are members of genus Mycoplasma which are only 0.3 µm, as small as the
largest viruses.
• The size of common bacteria like Escherichia coli ranges in size from 1.1 to 1.5 µm in diameter.
• It has been observed that the size of bacteria has a significant role in the survival of the organisms.
• Owing to their tiny size, they are capable of surviving and even thriving in various unlikely
environments like the vertical sediments in the marine environment.
• Since other organisms are absent in such an environment, bacteria can utilize the available
resources.
• Besides, the small size of bacteria favors parasitism and the ability to survive in areas with low
nutrition.
• The high surface area-volume ratio also allows the bacteria to take up all the nutrients required for
survival while allowing the steady growth and reproduction
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Table 1.1.6: Shape of bacteria and their Gram staining property
Cluster Staphylococcus
Chain Streptococcus
Pairs, lanceolate shaped Pneumococcus
Tetrads Micrococcus
Octate Sarcina
Pair or inj short chain, spectacle eyed shaped Enterococcus
Gram-positive bacilli arranged in
Chain (bamboo stick appearance Bacillus anthracis
Chinese letter arrangement c. diphtheriae
Pallisade arrangement Diphtheroids
Branching and filamentous form Actinomycetes
Pairs, lens shaped Meningococcus
Pairs, kidney shaped Gonococcus
Gram-negative bacilli arranged in
Pleomorphic (various shapes) Haemophilus, Proteus
Thumb print appearance Bordetella pertussis
Curved Campylobacter (Gull-wing shaped) and
Helicobacter
Spirally coiled, rigid Spirillum
Spirally coiled, flexible Spirochetes
Comma shaped (fish in stream) Vibrio cholerae
Chain Streptobacillus
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Bacterial shape
 Most of the bacteria have a rigid cell wall that provides a definite shape to the bacteria while
protecting the internal components.
 Even though this characteristic is valid for the majority of bacteria, they vary in shape that allows
them to be classified into different groups based on their forms.
 This wide variety of shapes is determined by the bacterial cell wall and cytoskeleton.
 Even though bacteria have a wide variety of shapes, anyone genus typically exhibits a limited
subset of morphologies, indicating that, with a universe of shapes to choose from, individual
bacteria adopt only those that are adaptive.
 Bacteria with different shapes present different physical features to the outside world, and these
features help cells cope with and adapt to external conditions.
 It has been observed that bacterial shape contributes a measure of survival value in the face of
nutrient acquisition, cell division, predators, attachment to surfaces, passive dispersal, active
motility, and internal or external differentiation.
The common categories of bacteria based on their shapes are:
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Cocci
 The bacteria that are oval or spherical in shape are included called cocci bacteria.
 These may either remain single or attached to one another in groups. They appear flattened when
placed in groups.
 It is assumed that coccoid forms were derived from rod-shaped organisms through evolutionary
time.
Bacilli (Rod-shaped)
 These are rod-shaped cells that also like cocci, remain either single or attached to other cells.
 Bacilli bacteria are among the first bacteria to have arisen, and this shape is said to be not as
advantageous as other shapes. This has been assumed upon the observation of the behavior of
filamentous E. coli cells which, though motile and chemotactic, move slowly and cannot tumble
to change direction.
Spiral
 This group includes bacteria that are either helical-shaped or curved (comma-shaped).
 The bacteria can range from slightly curved to corkscrew-like spiral.
Arrangement of Cocci
 Cocci bacteria can be arranged either singly, in pairs, in groups of four, in chains, in clusters or
cubes consisting of eight cells.
 These cells remain attached during cell division. Coccus
Coccus
 This group includes bacteria that are present as a single cell.
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Diplocooci
 This arrangement results when two bacterial cells occur as a pair (joined together).
 Some of the cells in this arrangement might remain spherical while some might appear flattened,
elongated, or bean-shaped.
 Examples: Streptococcus pneumonia, Moraxella catarrhalis, Enterococcus spp, Neisseria

gonorrhea.
Tetrad
 Tetrad bacteria are arranged in a group of four cells that remain attached and grow in the
attachment after cell division.
 This arrangement results when the cells divide into two planes.
 Examples: Aerococcus, Pediococcus, and Tetragenococcus.
Sarcina
 In this arrangement, the bacterial cells form a group of eight cells.
 This happens when the cells divide in a perpendicular plane.
 The common characteristic associated with these organisms is being strict anaerobe.
 Examples: Sarcina aurantiaca, Sarcina lutea, Sarcina ventriculi.
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Streptococci
 Here, the bacteria are arranged in long chains.
 These bacteria are present in family Streptococcaceae, which is characterized by a lack of

motility and Gram-positive bacteria.
 Examples: Streptococcus pyogenes, Streptococcus pneumonia, Streptococcus mutans.
Staphylococci
 This type includes bacteria that are arranged in grape-like clusters.
 This results from cell division in both the planes and are characterized by organisms which are
immotile and Gram-positive.
 Examples: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus aureus,

Staphylococcus capitis.
Arrangement of Bacilli
Bacillus
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 Bacilli are the bacteria which are rod-shaped and are present as single cells.
 These bacteria can form endospores and are facultative anaerobes.
 Examples: Salmonella enterica subsp, Bacillus cereus, and Salmonella choleraesuis.
Diplobacilli
 As in Diplococci, Diplobacilli also exists in pairs.
 After cell division, the two cells do not divide and grow in an attached arrangement.
 Examples: Coxiella burnetii, Klebsiella rhinoscleromatis, Moraxella bovis.
Streptobacilli
 In this group, bacteria are arranged in chains.
 This results from cell division in a single chain.
 Examples: Streptobacillus moniliformis, Streptobacillus Levaditi, Streptobacillus felis,

Streptobacillus hongkongensis.
Coccobacilli
 As the name suggests, coccobacilli resemble both cocci as well as bacilli.
 These are shorter in size and thus, appear stumpy.
 Examples: Chlamydia trachomatis, Haemophilus influenza, Gardnerella vaginalis.
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Pallisades
 Pallisades are the type of bacilli bacteria that resemble a picket fence structure as a result of the
bent at the point of division during cell division.
 They appear similar to Chinese letters.
 Example: Corynebacterium diphtheria that causes diphtheria.
Arrangement of Spiral
Vibrio
 These are the slightly curved bacteria resembling a comma shape.
 Examples: Vibrio mytili, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio cholera.
Spirochetes
 Spirochetes are spiral bacteria having a helical shape.
 These are flexible and have an axial filament which helps in motility. These filaments are
essential distinguishing character between spirochetes and other bacteria.
 These filaments run throughout the length of the bacteria and thus, help in twisting the motion of
the bacteria.
 Examples: Leptospiraspecies (Leptospira interrogans), Treponema pallidum, Borrelia recurrentis.
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Spirilla (Helical-shaped/Corkscrew form)
 These bacteria are similar in structure with spirochetes but are more rigid.
 They, too, have a flagellum but lack the endoflagella like in spirochetes.
 Examples: Campylobacter jejuni, Helicobacter pylori, Spirillum winogradskyi.
Other Shapes & Arrangement:
Appendaged Bacteria
 The bacteria that produce a unique structure like pillus or fimbriae are called appendaged
bacteria.
 These bacteria are more virulent than other bacteria that do not form these appendages.
 Example: Neisseria gonorrheae, the agent of Gonorrhea.
Box-shaped/ Rectangular Bacteria
 Box-shaped bacteria are rectangular in shape and resemble a box.
 Example: Haloarcula marismortui.
Club-shaped Rod Bacteria
 These bacteria are thinner on one side than the other.
 One of the classic examples of this group is Corynebacterium.
Filamentous Bacteria
 These are bacteria that are long, thin, and filament-shaped.
 They, sometimes, divide to form branches resembling strands of hair or spaghetti called
mycelium.
 Example: Actinomycetes.
Triangular-shaped Bacteria
 This group includes bacteria that are triangular in shape.
 Example: Haloarcula.
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Pleomorphic Bacteria
 The bacteria that do not have a specified shape are included in this group.
 They can change their shape, but in pure culture, they appear to have a definite form.
 Examples: Mycoplasma pneumoniae, M. genitalium.
Stalked Bacteria
 These are the bacteria that possess a stalk on one end of the cell.
 Examples: Caulobacter crescentus.
Star-shaped Bacteria
 The bacteria that look like stars or are star-shaped are included in this group.
 Examples: Stella humosa.
Bacterial Cell Wall

The cell wall is a tough and rigid structure, surrounding the bacterium. Apart from providing
protection and conferring rigidity, certain parts of cell wall (e.g. LPS) are immunogenic and act as
virulence factor.
 Peptidoglycan is the main component of the cell wall which makes it rigid. It is composed of
alternate units of N-acety1 muramic acid (NAM) and N-acetyl glucosamine (NAG) molecules;
cross linked to each other via tetrapeptide side chains and pentaglycine bridges.
 Gram-positive bacteria has a thicker peptidoglycan and contains teichoic acid.
 Gram-negative bacteria-peptidoglycan layer is thin, and it contains additional parts such as
(1) Outer membrane,
(2) Lipopolysaccharide (LPS) which in turn consists of
(i) Lipid A or endotoxin,
(ii) core polysaccharide,
(iii) O side chain
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Fig. Structure of Gram-positive cell wall
Nucleoid
Bacteria do not have a true nucleus; but the genetic material is located in an irregularly shaped region
called the nucleoid.
 There is no nuclear membrane or nucleolus and lacks basic proteins.
 Possess a single haploid chromosome, comprising of super coiled circular ds DNA (except two
chromosomes in vibrio)
 Bacterial DNA divides by simple binary fission.
 The nucleoid can be seen by electron microscopy or on staining with the Feulgen stain
 Bacteria also possess extra-chromosomal DNA called plasmids.
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Capsule and Slime Layer
Some bacteria possess a layer of amorphous viscid material lying outside the cell wall called
glycocalyx; which may be well organized (capsule) or unorganized loose material (slime layer). Some
bacteria may possess both capsule and slime layer as in streptococcus salivarius.
The capsule has various functions
 Acts by inhibiting phagocytosis and complement-mediated lysis
 Biofilm formation and thereby helps in adherence to damaged tissues and plastic surfaces (e.g.
medical devices
 Source of nutrients and energy for the bacteria
 Capsules as vaccine, e.g. Pneumococcus, Meningococcus and haemophilus nfuenzae serotype-b

and S. typhi Vi vaccine.
Flagella
Flagella are thread-like appendages, protruding from the cell wall, composed of protein subunits called
flagellin. It has three parts: filament, hook and basal body.
 Size-They measure 5-20 m in length and 0.01-0.02 in thickness

 They are organs of locomotion, confer motility to the bacteria.
Arrangement of flagella
 Size-They measure 5-20 m in length and 0.01-
 They are organs of locomotion, confer motility to the bacteria.
Arrangement of flagella
 Monotrichous (single polar flagellum) e.g. Vibrio cholerae, Pseudomonas and Campylobacter
 Lophotrichous (multiple polar flagella) e.g. spirillum.
 Peritrichous (over the entire cell surface e.g. Salmonella Typhi, Escherichia coli.
 Amphitrichous (single flagellum at both the ends) e.g. Alcaligenes faecalis, Spirillum
(Note: Spirillum has tuft of flagella at one or both the ends (Amphi>lophotrichous)
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Fimbriae or Pili
Many Gram-negative and some Gram-positive bacteria possess short, fine, hair-like appendages that
long and 10nm in thickness:
 Pili are made-up of protein called pilin.
 They are antigenic; however, the antibodies against fimbrial antigens are not protective.
 Functions: fimbriae are called the organ of adhesion. This property enhances the virulence of
bacteria.
 Certain fimbriae called sex pili also help in bacterial gene transfer.
 Fimbriae are not related to motility, can be found both in motile as well as in nonmotile
organisms.
Bacterial Spores
Spores are highly resistant resting (or dormant) stage of the bacteria formed in unfavourable
environmental conditions as a result of the depletion of exogenous nutrients.
ACTINOMYCETES
Actinomycetes are considered to be transitional forms between bacteria and fungi. Like fungi they
form a mycelial network of branching filaments but like bacteria they are thin, possess cell walls
containing muramic acid, have prokaryotic nuclei and are susceptible to antibacterial antibiotics.
Actinomycetes are related to mycobacteria and Corynebacterium. They are gram positive, non-motile,
non sporing, non-capsulated filaments that break up into bacillary and coccoid elements. Most are free
living particularly in the soil.
Actinomycetes Variants
There are two Actinomycetes variants such as, Actinomyces and Nocardia.
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Classic Actinomycetes have well-developed radial mycelium. According to the difference of

morphology and function, the mycelia can be divided into substrate mycelium and aerial mycelium
(fig 1.). .Some actinobacteria can form complicated structures, such as spore, spore chain, sporangia,
and sporangiospore.
Fig:
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FUNGI
The word fungus comes from the Latin word for mushrooms. Indeed, the familiar mushroom is a
reproductive structure used by many types of fungi. However, there are also many fungi species that
don‘t produce mushrooms at all. Being eukaryotes, a typical fungal cell contains a true nucleus and
many membrane-bound organelles. The kingdom Fungi includes an enormous variety of living
organisms collectively referred to as Ascomycota, or true Fungi. While scientists have identified about
100,000 species of fungi, this is only a fraction of the 1.5 million species of fungus probably present
on earth. Edible mushrooms, yeasts, black mold, and the producer of the antibiotic penicillin,
Penicillium notatum, are all members of the kingdom Fungi, which belongs to the domain Eukarya.
Fungi belong to their own kingdom (Kingdom Fungi). Compared to higher plants and animals, they
obtain their nutrition through a range of ways including degradation of organic material and symbiosis
(as lichen) among others.
As such, they are categorized as heterotrophic because they are unable to synthesize their own food
(they lack chlorophyll). They can reproduce sexually or asexually with a majority of fungi being spore
producers. Examples include:
 Molds
 Penicillin
 Yeast
 Truffles
 Mushrooms
Morphology and Structure of Fungi
There are a wide variety that range from the smallest unicellular fungi such as yeast to larger
multicellular capable of forming hyphal threads or false roots. For this reason, fungi are also classified
according to their morphologies.
The following are classifications of fungi based on morphology:
Yeast
Yeast are single celled fungi that can be found in a variety of environment from soil and plants to
animal and aquatic environments. Unlike bacteria, yeasts are also eukaryotic, which means that they
have different types of organelles that are common in the cells of higher animals.
The Saccharomyces cerevisiae is a good example of yeast that ranges between 1 and 7 micrometers in
size. When viewed under the microscope, these organisms may be pigmented on their surface.
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As with the cells or higher organisms, the S. cerevisiae contains such organelles as a membrane bound
nucleus, a vacuole, mitochondria and the Golgi apparatus as well as the E.R (endoplasmic reticulum).
The cell wall of these yeast is composed of glucan (a polysaccharide compound) and mannoproteins.
As for the genome, research has shown these yeast to carry a single, linear double stranded DNA that
consists of several repeated sequences. For yeast, the primary mode of reproduction is through
budding.
Following the copying of the genetic material, a bud is formed on the surface of the cell that ultimately
breaks off with its genetic material and grows to form a new cell.
Yeast-like Fungi
Yeast-like fungi are yeast that partly grow like normal yeast. However, they also attach to each other
to form what is known as a pseudo hyphae (not a true hyphae).
Candida albicans is one of the most common. When viewed under the microscope, these organisms
have been shown to consist of several layers that make up the cell wall.
As with yeast, the wall of C. albicans contains layers of mannoproteins, lipids and a beta glucon, a
chitin inner layer that strengthen the cell wall. Like yeast, C. albicans also appear spherical or ovoid in
shape and measure between 4 to 8 micrometers.
Since they also reproduce through budding, like yeast, C. albicans may end up creating an elongated
chain of cells as they continue dividing to form the pseudohyphae. However, some studies suggest
that some of the yeast-like fungi tend to form true hyphae in the process.
Yeast-like fungi such as C. albicans are also described as being polymorphic fungus. This is because
they present four types of morphology including the yeast cell, pseudohyphae, hyphae as well as
chlamydospores. As such, they are likely to be seen having varying appearance when viewed under the
microscope depending on such conditions as the availability of nutrition, pH and temperature among
others.
The type of morphological appearance of these cells has also been associated with the pathogenicity of
the organism. Given that they are not bacteria, some of these organisms (yeast-like fungi) have
characteristics associated with eukaryotic cells in that some have been shown to contain a nucleus and
other essential organelles.
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MOLDS
Mold (Mould) are a type of fungi that often grow well in favorable environments with warmth and
moisture. They can be found growing on various surfaces such as food surfaces from which they
obtain their nutrients.
Compared to yeast, molds are multicellular organisms. As such, they can be seen with the naked eye
without using a microscope. However, when viewed under the microscope, it is possible to observe
numerous filaments (Hyphae) that are collectively referred to as Mycelium.
While these organisms are microscopic, it is their numerous hyphae (that form the mycelium) that
make it possible to see mold as it grows on food surface (bread, oranges etc). There are two main types
of mycelium depending on their functions.
These include:
Vegetative mycelium - Vegetative mycelium include the hyphae that penetrates into the substrate and
absorbs nutrients for the continued growth and development of the organisms. These hyphae therefore
act like plant roots.
Aerial mycelium - Aerial mycelium are the hyphae that are located above the food substance. When
viewed closely under the microscope, aerial mycelium contain a spherical structure at the top of the
hyphae. These are known as the conidia and sere as the reproductive part of the mold. This part
produces spores (asexual reproduction) that can grow in favorable conditions.
Cell Structure and Function
Fungi are eukaryotes and have a complex cellular organization. As eukaryotes, fungal cells contain a
membrane-bound nucleus where the DNA is wrapped around histone proteins. A few types of fungi
have structures comparable to bacterial plasmids (loops of DNA). Fungal cells also contain
mitochondria and a complex system of internal membranes, including the endoplasmic reticulum and
Golgi apparatus.
Unlike plant cells, fungal cells do not have chloroplasts or chlorophyll. Many fungi display bright
colors arising from other cellular pigments, ranging from red to green to black. The poisonous
Amanita muscaria (fly agaric) is recognizable by its bright red cap with white patches. Pigments in
fungi are associated with the cell wall. They play a protective role against ultraviolet radiation and can
be toxic.
The poisonous Amanita muscaria: The poisonous Amanita muscaria is native to temperate and boreal
regions of North America.
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The rigid layers of fungal cell walls contain complex polysaccharides called chitin and glucans. Chitin,
also found in the exoskeleton of insects, gives structural strength to the cell walls of fungi. The wall
protects the cell from desiccation and predators. Fungi have plasma membranes similar to other
eukaryotes, except that the structure is stabilized by ergosterol: a steroid molecule that replaces the
cholesterol found in animal cell membranes. Most members of the kingdom Fungi are nonmotile.
CULTURE MEDIA
The basic constituents of culture media are:
 Peptone: Mixture of partially digested proteins
 Agar: It is used for solidifying the culture media. It has no nutritional property.
 It is prepared from seaweeds (red algae of species Gelidium and Gracilaria).

 Agar is preferred over gelatine, as it is bacteriologically inert, and it melts at 980C and usually
solidifies at 420C
 For solid agar: 1-2% (Japanese ager 2% or New Zealand agar 1.2%)
 For semisolid agar:0.5%
 For solid agar to inhibit proteus swarming: 6%
Others: Meat extract, Yeast extract, Blood and serum, Water and Electrolytes (NaCl).
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Simple/basal Media
They contain minimum ingredients that support the growth of non-fastidious bacteria. Examples
include:
1. Peptone water: it contains peptone (1%)+NaCl(0.5%)+water
2. Nutrient broth: It is made-up of peptone water + meat extract (1%)
3. Nutrient agar: It is made-up of nutrient broth +2% agar the basal media are used for:
 Testing the non-fastidiousness of bacteria
 They serve as the base for the preparation of any other media
 Nutrient broth is used for studying the bacterial growth curve
 Nutrient agar is the preferred medium for:
 Performing the biochemical tests such as oxidase, catalase and slide agglutination
 To study the colony character and Pigment demonstration.
Enriched Media
When a basal medium is added with additional nutrients such as blood, serum or egg, it is called
enriched medium. They also support the growth of fastidious bacteria, e.g.:
1. Blood agar: Prepared by adding 5-10% of sheep blood to the molten nutrient agar at 450C. It is
used to test the haemolytic property of the bacteria.
2. Chocolate agar: It is the heated blood agar; blood is added to the molten nutrient agar at 700c. It
is more nutritious than blood agar, and even supports Haemophilus influenzae.
3. Loeffler‘s serum slope is used for isolation of Corynebacterium diphtheriae.
4. Blood culture media: used for blood culture. They are of two types.
 Monophasic medium is made-up of brain heart infusion (BHI) broth

 Biphasic medium has a liquid phase (BHI broth) and a solid agar slope (BHI agar).
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Selective Media
Solid media that allow certain organism (pathogens) to grow and inhibit others (normal flora):
1. Lowenstein Jensen (LJ) medium is used for isolation of mycobacterium tuberculosis
2. Thiosulphate Citrate Bile salt Sucrose (TCBS) agar used for isolation of Vibrio species
3. For the isolation of enteric pathogens such as salmonella and shigella from stool.
 DCA (Deoxycholate Citrate Agar)

 XLD (Xylose Lysine Deoxycholate) agar
4. Potassium tellurite agar (PTA) is used for isolation of Corynebacterium diphtheriae.
5. Wilson Blair bismuth sulphite medium: It is used for isolation of salmonella typhi.
Differential Media
These media differentiate between two groups of bacteria by using an indicator.
1. MacConkey agar: It differentiates organisms into LF or lactose fermenters (produce pink

colonies, e.g. E. coli and Klebsiella) and NLF (produce color less colonies, e.g. shigella and
Salmonella)
2. CLED agar (Cysteine lactose electrolyte-deficient agar) This is similar to MacConkey agar,
differentiates between LF and NLF. It is used as an alternative to combination of blood agar
and MacConkey agar, for the processing of urine specimens.:
 Advantages over MacConkey agar: It is less inhibitory than MacConkey agar, supports gram
positive bacteria (except  haemolytic Streptococcus) and Candida.
 Advantage over blood agar: It can prevent the swarming of Proteus.
6. Pour-plate culture: Quantitative culture method, used to estimate viable bacterial count.
Synthetic or defined Media
These media are prepared from pure chemical substances and the exact composition of the medium is
known. These are used for various special studies such as metabolic requirements. Simple peptone
water medium, 1% peptone with 0.5% NaCl in water, may be considered a semi defined medium since
its composition is approximately known.
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Indicator Media
These media contain an indicator which changes colour when a bacterium grows in them, for example
incorporation of sulphite in Wilson and Blair medium. S. typhi reduces sulphite to sulphide in the
presence of glucose and the colonies of S. typhi have a black metallic sheen. Potassium tellurium by
the diphtheria bacillus to produce black colonies.
Methods of Isolating Pure Culture
1. Mixed Culture: A culture contain more than one species of microbes.

2. Pure culture: A culture containing only one species of microbes.
The following methods are used to isolate pure culture.
1. Streak plate technique.
2. Pour plate technique.
3. Spread plate technique.
1. Streak plate technique
Streaking is the process of spreading the microbial culture with an inoculating needle on the surface of
the media. Sterilizing the inoculating needle by flame to make red hot and allow it to cool for 30
seconds.
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2. Pour plate technique
The bacterial culture and liquid agar medium are mixed together. After mixing the medium, the
medium containing the culture poured into sterilized Petri dishes allowed solidifying and then
incubate. After incubation colonies appear on the surface.
Disadvantage of pour plate method: Method is tedious, time consuming and requires skills. The
micro-organisms are trapped beneath the surface of medium when it solidifies. Hence, surface as well
as subsurface colonies are developed and it is very difficult to isolate and count the subsurface
colonies.
The micro-organisms are subjected to hot shock because liquid medium is maintained at 45 C
temperature. This method is unsuitable for isolation of psychrophile bacteria.
Spread Plate technique
This is the best method to isolate the pure colonies. In this technique, the culture is not mixed with the
agar medium. Instead it is mixed with normal saline and serially diluted.
0.1ml of sample taken from diluted mixture, which is placed on the surface of the agar plate and spread
evenly over the surface by using L shaped glass rod call spreader.
Incubate the plates. After incubation, colonies are observed on the agar surface.
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Advantages of spread plate method: It is a simple method, only surface colonies are formed. Micro-
organisms are not exposed to higher temperature.
MICROBIOLOGY EQUIPMENTS AND INSTRUMENTS
Centrifuge:
 Centrifugation is a technique of separating substances which involves the application of

centrifugal force.
 A centrifuge is a device for separation of microorganisms from the suspended fluid using
centrifugal force (g-force).
 The particles are separated from a solution according to their size, shape, density, viscosity of the
medium and rotor speed.
 Centrifugation can only be used when the dispersed material is denser than the medium.
 In a solution, particles whose density is higher than that of the solvent sink (sediment) are in
influence of gravitational field. Movement of particle under the gravitational force is called
sedimentation and particles that are lighter than it float to the top.
 The centrifuge works using the sedimentation principle, when centrifugal force applied by the
centrifuge where the centripetal acceleration causes denser substances and particles to move
faster (> g) outward in the radial direction.
 The biological materials show drastic increase sedimentation when they undergo under
acceleration in centrifugal force.
 Relative centrifugal force (RCF) is expressed as a multiple of the acceleration (G) due to gravity
(g).
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 The biological materials show drastic increase sedimentation when they undergo under
acceleration in centrifugal force.
 Relative centrifugal force (RCF) is expressed as a multiple of the acceleration (G) due to gravity
(g).
Basic Principle of Sedimentation
 When a biological sample moves in centrifuge, it experiences an outward centrifugal force.
 Rate of sedimentation of biological sample is depending on the applied centrifugal field.
 The applied centrifugal force is determined by the radial distance of the particle from the axis of
rotation.
 If the angular velocity of particle ω and the radial distance of a particle r , applied centrifugal
field is G would be
G= ω2 r ……………………(1)
 If the mass of the particle m, centrifugal force F, F = mG = m ω2 r
Angular velocity ω = 2π s, Where s = frequency
 Frequency s can defined as numbers of revolutions (cycles) per second. We can express the
angular velocity in per minute then,
ω = 2π (rev per min)/ 60 ………… (2)
 Put value of ω from equation (2) to equation (1)
G= 4 π2 (rev per min) 2 / 3600 --------------------- (3)
 The centrifugal field is generally expressed in multiples of the gravitational field g (981cm/s 2).
Relative centrifugal force (RCF) is the ratio of the centrifugal acceleration (G) and gravitational
acceleration (g).
RCF= G/g
 Putting value of G from previous eq.(3),
RCF= 4 π 2 r (rev per min) 2 / 3600*981
RCF=1.12 x 10-5 (r.p.m.) 2 x r, RCF unit is dimensionless
So the relative centrifugal force (RCF) applied to the particle in centrifugation can be calculated.
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Types of Centrifuges
High Speed centrifuges Ultracentrifuges
- 15,000 – 20,000 RPM - 65,000 RPM (100,000‘s x g)
- Large sample capacity depending on - Limited lifetime & expensive

rotor.
- Normally refrigerated - Require special rotors
- Research applications - Care in use , balance critical
- Research applications
Microcentrifuges
 Microcentrifuges are used to process small volumes of biological molecules, cells, or nuclei.
 Microcentrifuge tubes generally hold 0.5 - 2 ml of liquid, and are spun at maximum angular
speeds
 of 12000–13000 rpm.
 Microcentrifuges are small enough to fit on a table-top and have rotors that can quickly change
speeds.
 They may or may not have a refrigeration function. This is the important process
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Small Bench Centrifuge
 Simplest centrifuges that are used to separate erythrocytes, Blood samples, coarse precipitates and
cells are known an bench or laboratory centrifuges.
 They have a speed ranging from 4000 – 6000 RPM and a relative centrifugal force of 3000 –
7000 g.
 Small samples are sedimented now a days with microfuge that after a speed of 8000 – 13000
RPM
 and relative RCF of approximately 10000 g.
 They sediment small volume (250 mm to 1.5 cm) of material in 1 or 2 min.
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Gradient Media:
There are several gradient materials used in centrifugation:-

Gradient media Their uses
Sucrose and Ficoll Helps in preserving the morphology and activity of Subcellular
fractions.
Cesium chloride Useful in isopycnic density gradient centrifugation technique.
Potassium bromide Useful in isopycnic density gradient centrifugation technique.
Percoll Because of its low osmolarity, low viscosity and large particle size, is
suitable for separating cells, bacteria, viruses and sub cellular
organelles.
Metrizamide For the isolation of membrane fractions by floatation
Nycodenz For the isolation of membrane fractions by floatation
Renografin For cell fractionation
Centrifugal fractionation of cell organelles
Cell Organelle Required centrifugal force
Nuclei 800 – 1000 g
Mitochondria 20,000 – 30,000 g
Chloroplasts 20,000 – 30,000 g
Lysosomes 20,000 – 30,000 g
Microbodies 20,000 – 30,000 g
Rough ER membranes (microsomes) 50,000 – 80,000 g
Plasma Membranes, Smooth ER membranes 80,000 – 1,00,000 g
Free Ribosome particles 1,50,000 – 3,00,000 g
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Corrosion:
 Many rotors are made from either titanium or aluminium alloy, chosen for their advantageous
mechanical properties.
 When corrosion occurs, the metal is weakened and less able to bear the stress from the
 centrifugal force exerted during operation.
 The combination of stress and corrosion causes the rotor to fail more quickly and at lower stress
levels than an un-corroded rotor.
Precautions:
A centrifuge user should strictly observe the following precautions:
1. Manufacturer‘s manual should be strictly followed.
2. Rotor should be stored in proper containers.
3. Attention should be given to imbalance detectors.
4. Rotor speed should not exceed the assigned speed.
5. Lid of the rotor chamber should remain locked during operation.
6. To avoid the rotor failure, manufactures instructions regarding rotor care and use should always be
followed.
Applications in Biological Sciences:
 To separate cellular and subcellular components
 Separating one cell type from another.
 Removing cells or other suspended particles from their surrounding milieu on either a batch or a
continuous-flow basis.
 Isolating viruses and macromolecules, including DNA, RNA, proteins, and lipids or establishing
physical parameters of these particles from their observed behaviour during centrifugation.
 To study the effects of centrifugal forces on cells, developing embryos, and protozoa.
 These techniques have allowed scientists to determine certain properties about cells, including
surface tension, relative viscosity of the cytoplasm, and the spatial and functional
interrelationship of cell organelles when redistributed in intact cells.
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Conclusion:
 The centrifugation is a modern & easy technique of separation and sedimentation on the basis of
shape, size and density of macromolecules and other particles.
 In the centrifugation there are different types of forces are applied like as centrifugal force,
gravitational force and centripetal force etc. and also different types of rotors are to be used that
is; Swinging Bucket Rotor and fixed angle rotors at different RPM/RCF.
Incubator:
Incubator, in microbiology, is an insulated and enclosed device that provides an optimal condition of
temperature, humidity, and other environmental conditions required for the growth of organisms.
An incubator is a piece of vital laboratory equipment necessary for the cultivation of micro-organisms
under artificial conditions. This medical laboratory equipment is available with digital temperature
controller with thermocouple sensor for better temperature accuracy.
An incubator can be used for the cultivation of both unicellular and multicellular organisms.
They are insulated enclosures that are thermostatically regulated to maintain a constant temperature. In
this type of incubator, environmental conditions, such as temperature and humidity can be controlled
for growing bacterial cultures (37°C), hatching eggs artificially / providing suitable conditions for a
chemical or biological reaction.
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Components / Parts of Incubator:
A microbial incubator is made up of various units, some of which are:
Cabinet
 The cabinet is the main body of the incubator consisting of the double-walled cuboidal enclosure
with a capacity ranging from 20 to 800L.
 The outer wall is made up of stainless steel sheets while the inner wall is made up of aluminum.
 The space between the two walls is filled with glass wool to provide insulation to the incubator.
 The insulation prevents heat loss and in turn, reduces the electric consumption, thereby ensuring
the smooth working of the device.
 The inner wall of the incubator is provided with inward projections that support the shelves
present inside the incubator.
Door
 A door is present in all incubators to close the insulated cabinet.
 The door also has insulation of its own. It is also provided with a glass that enables the
visualization of the interior of the incubator during incubation without disturbing the interior
environment.
 A handle is present on the outside of the door to help with the manoeuvring of the door.
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Control Panel
 On the outer wall of the incubator is a control panel with all the switches and indicators that
allows the parameters of the incubator to be controlled.
 The control panel also has a witch to control the thermostat of the device.
Thermostat
 A thermostat is used to set the desired temperature of the incubator.
 After the desired temperature is reached, the thermostat automatically maintains the incubator at
that temperature until the temperature is changed again.
Perforated shelves
 Bound to the inner wall are some perforated shelves onto which the plates with the culture media
are placed.
 The perforations on the shelves allow the movement of hot air throughout the inside of the
incubator.
 In some incubators, the shelves are removable, which allows the shelves to be cleaned properly.
Asbestos door gasket

 The asbestos door gasket provides an almost airtight seal between the door and the cabinet.
 This seal prevents the outside air from entering the cabinet and thus, creating an isolated hot
environment inside the cabinet without being interrupted by the external environment.
L-shaped thermometer
 A thermometer is placed on the top part of the outer wall of the incubator.
 One end of the thermometer provided with gradations remains outside of the incubator so that
temperature can be read easily.
 The next end with the mercury bulb is protruded slightly into the chamber of the incubator.
HEPA filters
 Some advanced incubators are also provided with HEPA filters to lower the possible
contamination created due to airflow.
 AN air-pump with filters creates a closed-loop system so that the air flowing inside the incubator
generates less contamination.
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Humidity and gas control
 The CO2 incubators are provided with a reservoir underneath the chamber that contains water.
 The water is vapourised to maintain the relative humidity inside the chamber.
 Similarly, these incubators are also provided with gas chambers to give the desired concentration
of CO2 inside the incubator.8K
Principle / Working of Incubator:
 An incubator is based on the principle that microorganisms require a particular set of parameters
for their growth and development.
 All incubators are based on the concept that when organisms are provided with the optimal
condition of temperature, humidity, oxygen, and carbon dioxide levels, they grow and divide to
form more organisms.
 In an incubator, the thermostat maintains a constant temperature that can be read from the outside
via the thermometer.
 The temperature is maintained by utilizing the heating and no-heating cycles.
 During the heating cycle, the thermostat heats the incubator, and during the no-heating period, the
heating is stopped, and the incubator is cooled by radiating heat to the surrounding.
 Insulation from the outside creates an isolated condition inside the cabinet, which allows the
microbes to grow effectively.
 Similarly, other parameters like humidity and airflow are also maintained through different
mechanisms that create an environment similar to the natural environment of the organisms.
 Similarly, they are provided with adjustments for maintaining the concentration of CO2 to
balance the pH and humidity required for the growth of the organisms.
 Variation of the incubator like a shaking incubator is also available, which allows for the
continuous movement of the culture required for cell aeration and solubility studies.
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Procedure for running an Incubator:
Once the cultures of organisms are created, the culture plates are to be placed inside an incubator at the
desired temperature and required period of time. In most clinical laboratories, the usual temperature to
be maintained is 35–37°C for bacteria.
The following are the steps to be followed while running an incubator:
1. Before using the incubator, it should be made sure that no remaining items are present in the
incubator from the previous cycles. However, in some cases, if the same incubator is being used
for multiple organisms, and they require the same set of parameters, they can be placed together in
the same incubator.
2. The door of the incubator is then kept closed, and the incubator is switched on. The incubator has
to be heated up to the desired temperature of the growth of the particular organism. The
thermometer can be used to see if the temperature has reached.
3. In the meantime, if the organism requires a particular concentration of CO 2 or a specific humidity,

those parameters should also be set in the incubator.
4. Once all the parameters are met, the petri dish cultures are placed on the perforated shelves upside
down, i.e., media uppermost. This is necessary because if the plates are incubated normally,
condensation collects on the surface of the medium and prevents the formation of isolated
colonies.
5. If it is necessary to incubate Petri dish cultures for several days, the plates are sealed with adhesive
tapes or are placed in plastic bags or plastic food containers.
6. Now, the door is locked, and the plates are kept inside for the required time before taking them
out.
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Types of Incubator:
Figure: Some Incubators used in Microbiology Lab.
On the basis of the presence of a particular parameter or the purpose of the incubator, incubators are
divided into the following types:
Benchtop incubators
 This is the most common type of incubator used in most of the laboratories.
 These incubators are the basic types of incubators with temperature control and insulation.
CO2 incubators
 CO2 incubators are the special kinds of incubators that are provided with automatic control of
CO2 and humidity.
 This type of incubator is used for the growth of the cultivation of different bacteria requiring 5-
10% of CO2 concentration.
 For humidity control, water is kept underneath the cabinet of the incubator.
Cooled incubators
 For incubation at temperatures below the ambient, incubators are fitted with modified
refrigeration systems with heating and cooling controls.
 This type of incubator is called the cooling incubator.
 In the cooling incubator, the heating and cooling controls should be appropriately balanced.
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Shaker incubator
 A thermostatically controlled shaker incubator is another piece of apparatus used to cultivate
microorganisms.
 Its advantage is that it provides a rapid and uniform transfer of heat to the culture vessel, and its
agitation provides increased aeration, resulting in acceleration of growth.
 This incubator, however, can only be used for broth or liquid culture media.
Portable incubator
 Portable incubators are smaller in size and are used in fieldwork, e.g. environmental microbiology
and water examination.
Uses of Incubator:
Incubators have a wide range of applications in various areas including cell culture, pharmaceutical
studies, hematological studies, and biochemical studies.
Some of the uses of incubators are given below:
1. Incubators are used to grow microbial culture or cell cultures.
2. Incubators can also be used to maintain the culture of organisms to be used later.
3. Some incubators are used to increase the growth rate of organisms, having a prolonged growth
rate in the natural environment.
4. Specific incubators are used for the reproduction of microbial colonies and subsequent
determination of biochemical oxygen demand.
5. These are also used for breeding of insects and hatching of eggs in zoology.
6. Incubators also provide a controlled condition for sample storage before they can be processed in
the laboratories.
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Precautions:
The following precautions are to be followed while running an incubator:
1. As microorganisms are susceptible to temperature change, the fluctuations in temperature of the

cabinet by repeatedly opening the door should be avoided.
2. The required parameters growth of the organism should be met before the culture plates are placed
inside the cabinet.
3. The plates should be placed upside down with the lid at the bottom to prevent the condensation of
water on to the media.
4. The inside of the incubators should be cleaned regularly to prevent the organisms from settling on
the shelves or the corners of the incubator.
5. While running the incubator for an extended period of time, sterile water should be placed
underneath the shelves to prevent the culture media from drying out.
Autoclave:
An autoclave is a machine that provides a physical method of sterilization by killing bacteria, viruses,
and even spores present in the material put inside of the vessel using steam under pressure.
Autoclave sterilizes the materials by heating them up to a particular temperature for a specific period
of time.
The autoclave is also called a steam sterilizer that is commonly used in healthcare facilities and
industries for various purposes.
The autoclave is considered a more effective method of sterilization as it is based on moist heat
sterilization.
Autoclaves provide a physical method for disinfection and sterilization. They work with a combination
of steam, pressure and time. Autoclaves operate at high temperature and pressure in order to kill
microorganisms and spores. To be effective against spore forming bacteria and viruses, autoclaves
need to have steam in direct contact with the material being sterilized (i.e. loading of items is very
important).
Create vacuum in order to displace all the air initially present in the autoclave and replacing it with
steam.
Implement a well-designed control scheme for steam evacuation and cooling so that the load does not
perish.
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The efficiency of the sterilization process depends on two major factors. One of them is the thermal
death time, i.e. the time microbes must be exposed to at a particular temperature before they are all
dead. The second factor is the thermal death point or temperature at which all microbes in a sample are
killed.
Autoclave Parts/ Components
The simplest form of the autoclave is the pressure cooker types or laboratory bench autoclaves. The
following is the detailed description of different components/ parts of an autoclave:
A) Pressure Chamber
 The pressure chamber is the main component of a steam autoclave consisting of an inner chamber
and an outer jacket.
 The inner chamber is made up of stainless steel or gunmetal, which is present inside the out
chamber made up of an iron case.
 The autoclaves used in healthcare laboratories have an outer jacket that is filled with steam to
reduce the time taken to reach the sterilization temperature.
 The inner chamber is the case where the materials to be sterilized are put.
 The size of the pressure chamber ranges from 100 L to 3000 L.
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B) Lid/ Door
 The next important component of an autoclave is the lid or door of the autoclave.
 The purpose of the lid is to seal off the outside the atmosphere and create a sterilized condition on
ht inside of the autoclave.
 The lid is made airtight via the screw clamps and asbestos washer.
 The lid consists of various other components like:
Pressure gauge
 A pressure gauge is present on the lid of the autoclave to indicate the pressure created in the
autoclave during sterilization.
 The pressure gauge is essential as it assures the safety of the autoclave and the working condition
of the operation.
Pressure releasing unit/ Whistle
 A whistle is present on the lid of the autoclave is the same as that of the pressure cooker.
 The whistle controls the pressure inside the chamber by releasing a certain amount of vapor by
lifting itself.
Safety valve
 A safety valve is present on the lid of autoclave, which is crucial in cases where the autoclave
fails to perform its action or the pressure inside increases uncontrollably.
 The valve has a thin layer of rubber that bursts itself to release the pressure and to avoid the
danger of explosion.
C) Steam generator/ Electrical heater
 An electrical steam generator or boiler is present underneath the chamber that uses an electric
heating system to heat the water and generate steam in the inner and the outer chamber.
 The level of water present in the inner chamber is vital as if the water is not sufficient; there are
chances of the burning of the heating system.
 Similarly, if the water is more than necessary, it might interfere with the trays and other
components present inside the chamber.
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D) Vacuum generator (if applicable)
 In some types of autoclaves, a separate vacuum generator is present which pulls out the air from
the inside of the chamber to create a vacuum inside the chamber.
 The presence of some air pockets inside the chamber might support the growth of different
microorganisms. This is why the vacuum chamber is an important component of an autoclave.
E) Wastewater cooler
 Many autoclaves are provided with a system to cool the effluent before it enters the draining
pipes.
 This system prevents any damage to the drainage pipe due to the boiling water being sent out of
the autoclave.
Autoclave Principle/ Working:
Figure: Autoclave Principle or Working.
 The autoclave works on the principle of moist heat sterilization where steam under pressure is
used to sterilize the material present inside the chamber.
 The high pressure increases the boiling point of water and thus helps achieve a higher
temperature for sterilization.
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 Water usually boils at 100°C under normal atmospheric pressure (760 mm of Hg); however, the
boiling point of water increases if the pressure is to be increased.
 Similarly, the high pressure also facilitates the rapid penetration of heat into deeper parts of the
material, and moisture present in the steam causes the coagulation of proteins causing an
irreversible loss of function and activity of microbes.
 This principle is employed in an autoclave where the water boils at 121°C at the pressure of 15
psi or 775 mm of Hg.
 When this steam comes in contact on the surface, it kills the microbes by giving off latent heat.
 The condensed liquid ensures the moist killing of the microbes.
 Once the sterilization phase is completed (which depends on the level of contamination of
material inside), the pressure is released from the inside of the chamber through the whistle.
 The pressure inside the chamber is then restored back tot eh ambient pressure while the
components inside remain hot for some time.
Procedure for running an autoclave:
 In general, an autoclave is run at a temperature of 121° C for at least 30 minutes by using

saturated steam under at least 15 psi of pressure. The following are the steps to be followed while
running an autoclave:
1. Before beginning to use the autoclave, it should be checked for any items left from the previous
cycle.
2. A sufficient amount of water is then put inside the chamber.
3. Now, the materials to be sterilized are placed inside the chamber.
4. The lid is then closed, and the screws are tightened to ensure an airtight condition, and the
electric heater is switched on.
5. The safety valves are adjusted to maintain the required pressure in the chamber.
6. Once the water inside the chamber boils, the air-water mixture is allowed to escape through the
discharge tube to let all the air inside to be displaced. The complete displacement can be ensured
once the water bubbles cease to come out from the pipe.
7. The drainage pipe is then closed, and the steam inside is allowed to reach the desired levels (15
lbs in most cases).
8. Once the pressure is reached, the whistle blows to remove excess pressure from the chamber.
9. After the whistle, the autoclave is run for a holding period, which is 15 minutes in most cases.
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10. Now, the electric heater is switched off, and the autoclave is allowed to cool until the pressure
gauge indicates the pressure inside has lowered down to that of the atmospheric pressure.
11. The discharge pipe is then opened to allow the entry of air from the outside into the autoclave.
12. Finally, the lid is opened, and the sterilized materials are taken out of the chamber.
Types of autoclave
There are different types of autoclaves present in the market, some of which are:
Figure: Types of Autoclave.
Pressure cooker type/ Laboratory bench autoclaves (N-type)
 These, as domestic pressure cookers, are still in use in many parts of the world.
 The more modern type has a metal chamber with a secure metal lid that can be fastened and
sealed with a rubber gasket.
 It has an air and steam discharge tap, pressure gauge, and safety valve. There is an electric
immersion heater in the bottom of the chamber.
Gravity displacement type autoclave
 This is the common type of autoclave used in laboratories.
 In this type of autoclave, the steam is created inside the chamber via the heating unit, which then
moves around the chamber for sterilization.
 This type of autoclave is comparatively cheaper than other types.
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Positive pressure displacement type (B-type)
 In this type of autoclave, the steam is generated in a separate steam generator which is then
passed into the autoclave.
 This autoclave is faster as the steam can be generated within seconds.
 This type of autoclave is an improvement over the gravity displacement type.
Negative pressure displacement type (S-type)
 This is another type of autoclave which contains both the steam generator as well as a vacuum
generator.
 Here, the vacuum generator pulls out all the air from inside the autoclave while the steam
generator creates steam.
 The steam is then passed into the autoclave.
 This is the most recommended type of autoclave as it is very accurate and achieves a high sterility
assurance level.
 This is also the most expensive type of autoclave.
Uses of autoclave:
Autoclaves are important devices to ensure the sterilization of materials containing water as they
cannot be sterilized by dry heat sterilization. Besides, autoclaves are used for various other purposes.
1. They are used to decontaminate specific biological waste and sterilize media, instruments, and
labware.
2. Regulated medical waste that might contain bacteria, viruses, and other biological materials are
recommended to be inactivated by autoclaving before disposal.
3. In medical labs, autoclaves are used to sterilize medical equipment, glassware, surgical
equipment, and medical wastes.
4. Similarly, autoclaves are used for the sterilization of culture media, autoclavable containers,
plastic tubes, and pipette tips.
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Precautions:
Although autoclaves are pretty simple to use, there are certain rules of precautions to be followed
while operating an autoclave. Some of the important precautions to be followed while running an
autoclave are:
1. Autoclaves should not be used to sterilize water-proof or water-resistant substances like oil or
powders.
2. The autoclave should not be overcrowded, and the materials should be loaded in a way that
ensures sufficient penetration of articles by the steam.
3. The items to be autoclaved should always be placed in a secondary container.
4. Only autoclavable bags are to be used to autoclave packaged waste.
5. To ensure sufficient penetration, articles should be wrapped in something that allows penetration
by steam, and materials like aluminum foils should not be used.
6. The items placed inside the chamber should not touch the sides or top of the chamber.
7. The wastes and clean items should be autoclaved separately.
8. Attempts to open the lid when the autoclave is working should never be made.
9. Liquid components should never be autoclaved in sealed containers.
10. The liquid inside the containers should only be filled 2/3rd of the total volume to prevent the
spilling of the liquid.
11. Plastic or polyethylene trays or containers should not be used as they might melt and damage the
autoclave.
12. Besides, never autoclave flammable, reactive, corrosive, toxic or radioactive materials, household
bleach, or paraffin-embedded tissue.
13. The paper should not be placed directly inside an autoclave as it is a combustible substance. It
should be autoclaved in a waste bag on a bio bag setting to prevent fire.
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Hot Water Bath:
A hot water bath or Laboratory Hot water bath is one of the essential instruments of a laboratory. It‘s
normally used for incubation of test samples underwater at constant temperature (hot or cold) over a
long period of time.
A hot water bath features a large container with heated water. The design configurations, sizes, and
dimensions of a hot water bath always varies. The container size of a laboratory water bath varies
from12 liters to 32 liters for a standard model and 50 -100 liters for a large size water bath.
Its mainly used in clinical and microbiology laboratories, university‘s lab, environmental research, and
even food technology for warming reagents, sample thawing, corrosion tests and bacteriological
examinations etc.
A water bath can heat a small amount of liquid sample for over a long period of time without changing
the concentration of constituents by evaporation.
There are present different types of laboratory water baths and they are used depending on the
applications.
When you require balanced high-temperature heating that no more than 100℃, a water bath is a good
choice.
Figure: Laboratory Hot water bath
Definition of Hot Water Bath
A hot water bath or Laboratory hot water bath is one of the essential instruments of a laboratory, which
contains a large container with heated water. It‘s normally used for incubation of test samples
underwater at constant temperature (hot or cold) over a long period of time without changing the
concentration of constituents by evaporation.
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Working Principle of a Hot Water Bath
Laboratory water bath has a Cu50 temperature sensor, which transfers water temperature to resistance
value, and amplified and compared by an integrated amplifier. Then output the control signal, and
efficiently control the average heating power of the electric heating tube and maintain water in
constant temperature.
Components of a Laboratory Hot Water Bath
1. Container or Tank Bath: In the container, the test samples are kept in hot water for a long period
of time. The container of a Laboratory Water Bath is made up of insulated metal such as stainless
steel.
2. Container Led: The lid helps to keep covering the container, so that water does not evaporate out
of it. It‘s mainly made up of heat resistant glass or insulated metal.
3. Heater: A laboratory water bath contains a Cu50 temperature sensor, which helps to generate heat.
4. Thermometer: This helps to check the temperature of the water bath. It can be inbuilt or placed
individually.
5. Thermostat or regulator: A thermostat helps to maintain the temperature of a water bath at a

constant level.
6. Propeller or stirrer device: It helps to circulate the water inside the water bath (Found in
Circulating water baths).
7. Outlet: It helps to get the water out of the container.
8. Indicator light: All water bath should contain an indicator light. When the light is on the water
bath is heating. If the water bath reaches the required temperature the light will be turn off to
maintain the constant temperature.
Controls of a Hot Water Bath
Temperature controller: All water baths contain a temperature controller it should be digital or dial.
Safety Controler: Most of the water baths contain a safety controller, which is mainly located above
the temperature controller or associated with the indicator light. A Safety controller helps to set a
maximum temperature which the water bath should attain. If somehow the water bath is able to reach
the temperature which is set by the safety controller, then the safety light will be turn on. It is
impossible for a water bath to reach the temperature higher than the safety settings even the
temperature setting is higher.
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Shaking Controller: A shaking controller only found in a shaking water bath. This may allow us to
speed up and stop or turn the shaker on.
Types of Water Hot Bath
There are present three types of water bath. They are divided based on their applications.
1. Shaking water bath
 This type of water bath has an extra control for shaking, which help in the movement of hot water
and liquid test sample.
 This shaking features of a shaking water bath can be turned on or off.
 In microbiological laboratories, a shaking water bath helps in the incubation of a growing culture
with proper air circulation.
 A shaking Water bath has some key benefits such as,
 User-friendly operation via keypad.
 It has convenient bath drains.
 It has a controller to adjust the shaking frequencies.
 It contains a bright LED-display with good visibility.
 It has an optional lift-up bath cover.
 Power switch integrated in keypad and warning and cut-off protection for low/high temperature.
2. Circulating water bath
Stirrers or circulating water bath is used for enzymatic and serologic experiments. In the circulating
water bath, the hot water is thoroughly circulated throughout the bath, which is resulting in a more
uniform temperature.
3. Non-circulating water bath
Non-circulating water baths rely primarily on convection instead of water being uniformly heated,
which results in a less accurate in terms of temperature control.
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Operating Procedure of a Hot Water Bath:
1. Before use make sure water at the desired level in water bath. If not, fill it with tap water at the
desired level and then turn on the switch.
2. After that set the desired temperature by using the temperature controller and allow the water to
warm to that temperature.
3. Monitor the temperature from the thermometer.
4. After reaches the desired temperature, now insert your test samples in it for incubation.
Limitation of Water Bath:
 Keep Changing water daily and keep clean from the inside to prevent the encrustation of
important components in a water bath.
 When using the water bath, keep the lid closed so that the water does not evaporate.
 Measure the inside and outside temperature of the water bath once a week.
 Make sure, the thermometer does not stick to the wall of the water bath.
Application of Water Bath:
 Used for incubation of cell culture.
 Water Bath also used as a heat source for flammable chemicals.
 It is used to facilitates chemical reactions.
 Used to heat up chemical reagents.
 Used for the melting of some substance.
 It is used to increase the solubility of some insoluble substances.
Biological Safety Cabinets:
Biological safety cabinets (BSCs) are among the most effective primary containment devices used in
laboratories working with infectious agents. They act as primary barriers to prevent the escape of
biological aerosols into the laboratory environment.
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BSCs are designed for:
 Personnel Protection: Protects personnel from harmful agents inside the BSC.
 Product Protection: Protects the work, product, experiment, or procedure performed in the BSC
from contaminants in the laboratory environment or from cross contamination inside the cabinet.
 Environmental Protection: Protects the environment from contaminants contained in the BSC.
Inoculating loops and Needles: Inoculating loops are used to transfer micro-organisms to grow media
and staining slides. The wire forms a small loop with a diameter of about 5mm. the loop of wire at the
tip may be made of platinum or nichrome. Needles are straight wires used to pick up bacteria from
closely packed colonies or to inoculate in a much defined area. Needles are commonly used to
inoculate semi soft media.
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MCQ
1. Isolation of pure culture refers to ____
(a) Purification of culture
(b) Introduction of inoculum
(c) sporation of a single colony
(d) 10 Grow micioorganism on a surface
2. Enrichment media allows the growth of a large number of valied bacterial species.
(a) True
(b) False
3. In pour plate method, the medium should be maintained at what temperature?
(a) 370C (b) 670C (c) 450C (d) 00C
4. Which of the following method can be used to determine the number of bacteria quantitatively?
(a) Streak – Plate (c) Pour plate
(b) Spread plate (d) Pour plate & Sprewell plate
5. Which of the following is a function of cry protective agents?
(a) for long – term preservation of cultures
(b) Prevents cell damage due to ice crystal formation
(c) Prevents formation of ice
(d) to trap the liquid nitrogen
6. Ni chrome 100p wire is used in which to the following technique?
(a) Pour plate (c) Spread plate
(b) Streak plate (d) Roll tube technique
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7. In the classification of bacteria according to shape, which among the following refer to cuboidal
arrangement of bacterial cells?
(a) Tetrad (c) Sarcinae
(b) Staphylococcal (d) Streptococci
8. When rod shaped bacteria appears in pairs, it is known as?
(a) Diplobacilli (c) diplococcic
(b) Styreptobalilli (d) Staphylococci
9. Bacteria with less than a complete twist or comma shaped is known as?
(a) spirilla
(b) Helical
(c) vibrioid
(d) Spirochetes
10. Peptidoglycan layer are present in large quantity in?
(a) Gram +Ve bacteria
(b) Gram –Ve bacteria
(c) fungi
(d) Algae
ANSWER KEY
1. C 2. B 3. C 4. D 5. B
6. B 7. C 8. A 9. C 10. A
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ONE WORD QUESTIONS
1. A medium is a solid, liquid or semi-solid designed to support the growth

of microorganisms or cells, or small plants.
2. A defined medium that has just enough ingredients to support growth is called.
3. A media is used for the growth of only selected microorganisms.
4. A media distinguishes one microorganism type from another growing on the same medium.
5. A media contains the nutrients required to support the growth of a wide variety of organisms,
including some of the more fastidious ones.
6. A successful plate will have a countable number of isolated bacterial colonies evenly
distributed on the plate is.
7. The molten agar is poured on to the inoculum during the preparation of the.
8. The bacterial cell wall differs from that of all other organisms by the presence of which is
located immediately outside of the cytoplasmic membrane.
9. Gram-negative cell walls are thin and unlike the gram-positive cell walls, they contain a
thin layer adjacent to the cytoplasmic membrane is.
10. A whip-like structures protruding from the bacterial cell wall and are responsible for
bacterial motility.
ANSWER KEY
1. Culture 2. A Minimal 3. Selective 4. Differential or 5. Enriched
medium Media media indicator
6. Spread Plate 7. Pour Plate 8. Peptidoglycan 9. Peptidoglycan 10. Flagella
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Short Questions
1. What is the importance of sterilization? 2020
_______________________________________________________________________________
_______________________________________________________________________________
2. What are the basic morphological features of bacteria?
_______________________________________________________________________________
_______________________________________________________________________________
3. What are five characteristics of bacterial colony morphology? 2020
_______________________________________________________________________________
_______________________________________________________________________________
4. What is the difference between bacterial morphology and colony morphology?
_______________________________________________________________________________
_______________________________________________________________________________
5. What are the main characteristics of the kingdom fungi?
_______________________________________________________________________________
_______________________________________________________________________________
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Long questions
1. Describe the structure and morphology of bacteria.

2. Describe the structure and morphology of fungi.
3. What is gram staining and their procedure describe it with steps?
4. What is the principle of culture media and their types?
5. What is the principles and uses of microbiology equipments and instruments?
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UNIT -3

 Define Stains used in microbiology
 Explain types of stains in detailed giving example-simple stain differential stain, negative stain,
impregnation method
 Define special staining for certain bacteria, bacterial spores, parasites and fungi; principle,
procedure, application and result.
 interpretation of gram staining and ziehl neelsen staining.
STAINING TECHINIQUES
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains
and dyes are frequently used in histology (the study of tissue under the microscope) and in
the medical fields of histopathology, hematology, and cytopathology that focus on the study
and diagnoses of disease at a microscopic level. Stains may be used to define biological
tissues (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying
different blood cells), or organelles within individual cells.
In biochemistry it involves adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a

substrate to qualify or quantify the presence of a specific compound. Staining and fluorescent
tagging can serve similar purposes. Biological staining is also used to mark cells in flow cytometry,
and to flag proteins or nucleic acids in gel electrophoresis.
Staining is necessary to produce colour contrast and thereby increase the visibility of the object. Before
staining, the fixation of the smear to the slide is done:
 Heat fixation is usually done for bacterial smears by gently flame heating an air-dried film of
bacteria
 Chemical fixation such as ethanol, acetic acid, mercuric chloride, formaldehyde, methanol and
glutaraldehyde. This is useful for examination of blood sears.
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Staining Techniques Used in Microbiology
1. Simple Stain: basic dyes such as methylene blue or basic fuchsin are used as simple stains.
They provide the colour contrast, but impart the same colour to all the bacteria in a smear. It is
used to compare morphological shapes and arrangements of bacterial cells.
i)Loeffler‘s methylene blue :
 Saturated solution of methylene blue in alcohol (30 ml).

 KOH, 0.01 % in water – 100ml.
 Dissolve the dye in water and filter.
 For smear stain for 3 inch.
 For section stain for 5 inch.
ii)Dilute Carbol Fuschin :

 Made by diluting Z-N stain with 10-15 times its volume of water.
 Stain for 20-25 seconds, wash with water.
Use : To demonstrate the morphology of vibrio cholera.
2. Negative staining, e.g. India ink or nigrosine. The background gets stained black whereas
unstained bacteria stand out in contrast. This is very useful in the demonstration of bacterial
capsules which do not take up simple stains
Use: To demonstrate the capsule of Cryptococcus neoformans and Streptococcus pneumoniae.
3. Impregnation methods (e.g. silver(: Used for demonstration of thin structures like bacterial
flagella and spirochetes.
4. Differential stain: Two stains are used which impart different colors which help in
differentiating bacteria, e.g. Grams Stain and Acid fast stain.
i) Gram’s stain:
 Originally devised by Hans Christian Gram in the late 19th century.
 Differentiates bacteria into Gram-positive (appear violet) and Gram-negative (appear pink)
 groups.
 Also be used for the detection of fungal hyphae and yeast cells.
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Fig. Principle and procedure of Gram staining
Smear is allowed to dry and it is viewed under oil immersion objective lens.
This procedure shows Gram positive bacteria resists decolourisation and appears violet and Gram
negative bacteria which are decolourised by organic solvents take up the counter stain and appear red.
ii) Acid-fast stain or Ziehl Neelsen stain:
 It was discovered by Ehrlich and modified by Ziehl Neelsen.
 It differentiates acid fast and non-acid fast organisms.
Procedure
 Make a thin smear of the material for study and heat fix by passing the slide 3-4 times through the
flame of a Bunsen burner or use a slide warmer at 65-75 C. Do not overheat.
 Place the slide on staining rack and pour carbol fuschin over smear and heat gently underside of
the slide by passing a flame under the rack until fumes appear (without boiling!). Do not overheat
and allow it to stand for 5 minutes.
 Rinse smears with water until no color appears in the effluent.
 Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until the slide
appears light pink in color (15-20 sec).
 Wash well with clean water.
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 Cover the smear with methylene blue or malachite green stain for 1–2 minutes.
 Wash off the stain with clean water.
 Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not
blot dry).
 Examine the smear microscopically, using the 100x oil immersion objective.
Result :
iii) Albert stain:
 Special staining for Corynebacterium diphtheria.
 To demonstrate the metachromatic granules.
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Procedure:
 Prepare the smear, dry in air and fix by heat.
 Cover the slide with Alberts stain for 3-5 min.
 The slide is washed off with distilled water.
 Cover the slide with Albert‘s iodine for 1-2 min.
 Wash with water and blot dry.
 Examine under oil immersion objective of light microscope.
Using Alberts stain, metachromatic granules of C. Diphtheria appears blue black, while the body of
the bacillus appears green or bluish green.
Result :
iv) Leishman’s stain:
 Used to demonstrate blood parasites.
 Made by dissolving 0.15g of Leishman‘s powder in 100 ml pure methyl alcohol.
Procedure :
 2ml of undiluted stain is poured on to the unfixed film and allowed to stand for 1min.
 4 ml of distilled water is added to dilute the stain on the slide or to prevent it from drying; this is
allowed to stand for 10-15 min.
 The slide is then washed with running tap water until the blood film appears bright pink in colour;
the preparation is then dried in air.
 The smear is examined under the oil immersion objective lens of the light microscope.
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Result :
Blood smear of Plasmodium falciparum parasite.
v) Giemsa Stain :
 Giemsa powder 3.8 gm.
 Glycerol 250 ml.
 Methanol 250 ml.
 Add methanol to Giemsa powder and dissolve.
 Add glycerol and place in water bath at 60 C for 3 hours with intermittent shaking.
Procedure :
 Prepare the smear.
 Fix with pure methanol for 3-5 min.
 Diluted stain (5ml) is added and allowed to dry for 30-45 min.
 Smear is then washed with running water.
 Dry it in vertical position.
 Observe under oil immersion lens.
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Result :
5. Other special Staining Methods:
 Spore staining: Acid fast stain (using 0.25% sulfuric acid) and Malachite green stain (Schaeffer
and Fulton method modified by Ashby) methods are used; however, phase contrast microscope
of unstained wet film is the best method.
In vivo vs In vitro
In vivo staining (also called vital staining or intravital staining) is the process of dyeing living tissues.
By causing certain cells or structures to take on contrasting colour(s), their form (morphology) or
position within a cell or tissue can be readily seen and studied. The usual purpose is to reveal
cytological details that might otherwise not be apparent; however, staining can also reveal where
certain chemicals or specific chemical reactions are taking place within cells or tissues.
In vitro staining involves colouring cells or structures that have been removed from their biological
context. Certain stains are often combined to reveal more details and features than a single stain alone.
Combined with specific protocols for fixation and sample preparation, scientists and physicians can
use these standard techniques as consistent, repeatable diagnostic tools. A counterstain is stain that
makes cells or structures more visible, when not completely visible with the principal stain.
 Crystal violet stains both Gram positive and Gram negative organisms. Treatment with alcohol
removes the crystal violet colour from gram negative organisms only. Safranin as counterstain is
used to colour the gram negative organisms that got decolorised by alcohol.
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While ex vivo, many cells continue to live and metabolize until they are "fixed". Some staining
methods are based on this property. Those stains excluded by the living cells but taken up by the
already dead cells are called vital stains (e.g. trypan blue or propidium iodide for eukaryotic cells).
Those that enter and stain living cells are called supravital stains (e.g. New Methylene
Blue and brilliant cresyl blue for reticulocyte staining). However, these stains are eventually toxic to
the organism, some more so than others. Partly due to their toxic interaction inside a living cell, when
supravital stains enter a living cell, they might produce a characteristic pattern of staining different
from the staining of an already fixed cell (e.g. "reticulocyte" look versus diffuse "polychromasia"). To
achieve desired effects, the stains are used in very dilute solutions ranging
from 1:5000 to 1:500000 (Howey, 2000). Note that many stains may be used in both living and fixed
cells.
Microscopy in Staining
A common microscope used in staining is the bright-field microscope. The bright-field microscope is
categorized as a light microscope because of the illuminator which produces light to create a bright
background. These microscopes are usually binocular, meaning that there are two eyepieces which are
typically at ten times magnification. When viewing stained organisms at one hundred times
magnification, oil is needed to help create a clearer view due to the distortion of resolution from light
refraction which becomes incredibly pronounced at high magnification.
Preparation
The preparatory steps involved depend on the type of analysis planned; some or all of the following
procedures may be required.
Wet Mounts- wet mounts are used to view live organisms and can be made using water and certain
stains. The liquid is added to the slide before the addition of the organism and a coverslip is placed
over the specimen in the water and stain to help contain it within the field of view.[1]
Fixation: which may itself consist of several steps–aims to preserve the shape of the cells or tissue
involved as much as possible. Sometimes heat fixation is used to kill, adhere, and alter the specimen so
it accepts stains. Most chemical fixatives (chemicals causing fixation) generate chemical
bonds between proteins and other substances within the sample, increasing their rigidity. Common
fixatives include formaldehyde, ethanol, methanol, and/or picric acid. Pieces of tissue may be
embedded in paraffin wax to increase their mechanical strength and stability and to make them easier
to cut into thin slices.
Mordant: These are chemical agents which have power of making dyes to stain materials which
otherwise are unstainable
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Mordants are classified into two categories:
a) Basic Mordant: React with acidic dyes e.g. alum , ferrous sulfate , cetylpyridinium chloride etc .
b) Acidic Mordant : React with basic dyes e.g. picric acid , tannic acid etc.
Direct Staining: Carried out without mordant.
Indirect Staining: Staining brought by the aid of a mordant.
Table represents Indirect Staining Techniques and mordants applied in each:
Sr No. Name of Indirect Staining Technique Name of mordant applied
1.) Gram's Staining Gram's iodine
Cell Wall Staining

10% Tannic acid
2.) a.) Ringer's method 0.34% C.P.C
b.)Dyar's method
Flagella Staining
Tannic acid in Leifson's stain
3.) a.) Leifson's method
Loeffler's mordant (20%Tannic acid )
b.) Loeffler's method
Spirochete Staining
Fontana's mordant(5%Tannic acid)
4.) a.) Fontana's method
Fontana's mordant(5%Tannic acid)
b.) Becker's method
Permeabilization involves treatment of cells with (usually) a mild surfactant. This treatment
dissolves cell membranes, and allows larger dye molecules into the cell's interior.
Mounting usually involves attaching the samples to a glass microscope slide for observation and
analysis. In some cases, cells may be grown directly on a slide. For samples of loose cells (as with a
blood smear or a pap smear) the sample can be directly applied to a slide. For larger pieces of tissue,
thin sections (slices) are made using a microtome; these slices can then be mounted and inspected.
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Standardization
Most of the dyes commonly used in microscopy are available as BSC-certified stains. This means that
samples of the manufacturer's batch have been tested by an independent body, the Biological Stain
Commission (BSC), and found to meet or exceed certain standards of purity, dye content and
performance in staining techniques ensuring more accurately performed experiments and more reliable
results. These standards are published in the Commission's journal Biotechnic &
Histochemistry. Many dyes are inconsistent in composition from one supplier to another. The use of
BSC-certified stains eliminates a source of unexpected results.
Some vendors sell stains "certified" by themselves rather than by the Biological Stain Commission.
Such products may or may not be suitable for diagnostic and other applications.
Negative staining
Example of negative staining
A simple staining method for bacteria that is usually successful, even when the positive
staining methods fail, is to use a negative stain. This can be achieved by smearing the sample onto the
slide and then applying nigrosin (a black synthetic dye) or India ink (an aqueous suspension of carbon
particles). After drying, the microorganisms may be viewed in bright field microscopy as lighter
inclusions well-contrasted against the dark environment surrounding them.[6] Negative staining is able
to stain the background instead of the organisms because the cell wall of microorganisms typically has
a negative charge which repels the negatively charged stain. The dyes used in negative staining are
acidic.[1] Note: negative staining is a mild technique that may not destroy the microorganisms, and is
therefore unsuitable for studying pathogens.
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Positive staining
Unlike negative staining, positive staining uses basic dyes to color the specimen against a bright
background. While chromophore is used for both negative and positive staining alike, the type of
chromophore used in this technique is a positively charged ion instead of a negative one. The
negatively charged cell wall of many microorganisms attracts the positively charged chromophore
which causes the specimen to absorb the stain giving it the color of the stain being used. Positive
staining is more commonly used than negative staining in microbiology. The different types of positive
staining are listed below.[1]
Simple Staining versus Differential Staining
Simple Staining is a technique that only uses one type of stain on a slide at a time. Because only one
stain is being used, the specimens (for positive stains) or background (for negative stains) will be one
color. Therefore, simple stains are typically used for viewing only one organism per slide. Differential
staining uses multiple stains per slide. Based on the stains being used, organisms with different
properties will appear different colors allowing for categorization of multiple specimens. Differential
staining can also be used to color different organelles within one organism which can be seen
in endospore staining.
Types of Staining Techniques
Sr. Staining
Preparation Application Result
No. Technique
Used to highlight
Smear stain with single dye . microbes and illustrate Organisms are
Simple cellular stained in the color
1.
(Monochrome) eg. Methylene blue , Safranin
shapes and arrangements of applied stain
etc
.
Smear mixed with Nigrosin Organism is

Negative and spread stained, the
2. Study cell morphology
(Relief) background is
into thin film black
Primary stain: Crystal violet Gram positive

Characterizes bacteria in
applied to film then treated appears purple in
one of two groups, Gram
3 Gram with iodine (mordant), color Grams
positive or Gram
alcohol (decolourizer) and negative appears
negative
counter stained with safranin pink in color
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Separate non-decolorized Acid fast
Film stained with hot
Acid fast (Ziehl- acid fast bacteria that are bacteria:Red
Z.N.C.F. decolourized (acid-
4 Neelsen not decolorized from
alcohol) and counter stain Non acid fast:
technique) colorized non-acid fast
with methylene blue Blue
bacteria
Primary stain Malachite

Endospore Detects the presence of Endospores: Green
green heat fixed to penetrate
5 (Dornor's endospores in six genera Vegetative cells:
spores; vegetative cells are
method) of bacteria Red
counterstained with Safranin
Capsule Capsule: Light

Smear stained with Hiss stain Capsules can be violet/ pale mauve
A: Hiss method following treatment with observed as clear zones color
(Positive copper sulphate surrounding cells of Bacteria: Purple
6 technique)
Bacterial suspension smeared capsulated bacteria and capsule, bacterial
B: Manevals's along with congo red and the are used to demonstrate cell, Stands out
technique Maneval's stain is applied the presence of capsules. against dark
(Negative) background
Smear treated with C.P.C.

which dissociates to form
positively charged cetyl
Cell wall pyridinium and negatively Stains cell wall of Cell wall: Red
7
(Dyar's method) charged chloride ions. bacterium Cytoplasm: Blue
Positively charged ions are
adsorbed on negatively
charged cell wall
Mordant acts to thicken

Flagella flagella before staining and Flagella: Red
Demonstrates presence
8 (Leifson's increases visibility Vegetative cells:
of flagella
method) microscopically when Blue
stained with Leifson stain
Smear is treated for

To demonstrate the
hydrolysis to release purines
presence of DNA in cell. Nuclear material-
from DNA, purines to cause
Nuclear material But for detection of the pinkish purple,
shift form furanose to
9 (Feulgen DNA, RNA should be
aldehyde. Aldehyde groups Cytoplasm-
technique) selectively destroyed by
are available to react with colorless
acid hydrolysis without
schiff's reagent to form
affecting DNA
addition compounds.
Metachromatic The smear is first treated The granules show the Granules: Bluish
10 granules with chloroform to remove typical monochromatism black, Cytoplasm:
(Alberts's fats . Smear applied with nature, this is used to Green
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method) Alberts stain which contains demonstrate granules
cationic dyes such as
toluidine blue amd malachite
green. Toluidine blue
preferentially stains granules
while malachite green stains
cytoplasm.
Lipids are stained with fat

soluble dyes like Sudan To detect the presence of Lipid granules:
Intracellular black. On application of lipids in cell wall, cell Deep blue,
11 lipids (Burdon's Sudan black-B dyes move membrane or fat
method) into lipids and are retained globules (PHB in Cytoplasm: Light
there while cytoplasm is cytoplasm) pink
counter stained with safranin.
Polysaccharide is oxidized
with periodate to form
Polysaccharide polyaldehyde which reacts Detects the accumulation Polysaccharide:
12 (Hotch kuss with Schiff's reagents to red of polysaccharide Red
method) color, while cytoplasm is granules in the cells Cytoplasm: Green
counter stained with
malachite green
Specific techniques:
Gram staining
Gram staining is used to determine gram status to classifying bacteria broadly based on the
composition of their cell wall. Gram staining uses crystal violet to stain cell walls, iodine (as a
mordant), and a fuchsin or safranin counterstain to (mark all bacteria). Gram status, helps divide
specimens of bacteria into two groups, generally representative of their underlying phylogeny. This
characteristic, in combination with other techniques makes it a useful tool in clinical microbiology
laboratories, where it can be important in early selection of appropriate antibiotics.
On most Gram-stained preparations, Gram-negative organisms appear red or pink due to their
counterstain. Due to the presence of higher lipid content, after alcohol-treatment, the porosity of the
cell wall increases, hence the CVI complex (crystal violet – iodine) can pass through. Thus, the
primary stain is not retained. In addition, in contrast to most Gram-positive bacteria, Gram-negative
bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of
lipopolysaccharide.
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Endospore staining
Endospore staining is used to identify the presence or absence of endospores, which make bacteria
very difficult to kill. Bacterial spores have proven to be difficult to stain as they are not permeable to
aqueous dye reagents. Endospore staining is particularly useful for identifying endospore-forming
bacterial pathogens such as Clostridium difficile. Prior to the development of more efficient methods,
this stain was performed using the Wirtz method with heat fixation and counterstain. Through the use
of malachite green and a diluted ratio of carbol fuchsin, fixing bacteria in osmic acid was a great way
to ensure no blending of dyes. However, newly revised staining methods have significantly decreased
the time it takes to create these stains. This revision included substitution of carbol fuchsin with
aqueous Safranin paired with a newly diluted 5% formula of malachite green. This new and improved
composition of stains was performed in the same way as before with the use of heat fixation, rinsing,
and blotting dry for later examination. Upon examination, all endospore forming bacteria will be
stained green accompanied by all other cells appearing red.
Ziehl-Neelsen stain
A Ziehl–Neelsen stain is an acid-fast stain used to stain species of Mycobacterium tuberculosis that do
not stain with the standard laboratory staining procedures such as Gram staining.
This stain is performed through the use of both red coloured carbol fuchsin that stains the bacteria and
a counter stain such as methylene blue.
Haematoxylin and eosin (H&E) staining
Microscopic view of a histologic specimen of human lung tissue stained with hematoxylin and eosin.
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Haematoxylin and eosin staining is frequently used in histology to examine thin tissue
sections. Haematoxylin stains cell nuclei blue, while eosin stains cytoplasm, connective tissue and
other extracellular substances pink or red. Eosin is strongly absorbed by red blood cells, colouring
them bright red. In a skillfully made H&E preparation the red blood cells are almost orange, and
collagen and cytoplasm (especially muscle) acquire different shades of pink.
Papanicolaou staining
Papanicolaou staining, or PAP staining, was developed to replace fine needle aspiration cytology
(FNAC) in hopes of decreasing staining times and cost without compromising quality. This stain is a
frequently used method for examining cell samples from a variety of tissue types in various organs.
PAP staining has endured several modifications in order to become a ―suitable alternative‖ for FNAC.
This transition stemmed from the appreciation of wet fixed smears by scientists preserving the
structures of the nuclei opposed to the opaque appearance of air dried Romanowsky smears. This led
to the creation of a hybrid stain of wet fixed and air dried known as the ultrafast papanicolaou stain.
This modification includes the use of nasal saline to rehydrate cells to increase cell transparency and is
paired with the use of alcoholic formalin to enhance colors of the nuclei. The papanicolaou stain is
now used in place of cytological staining in all organ types due to its increase in morphological
quality, decreased staining time, and decreased cost. It is frequently used to stain Pap
smear specimens. It uses a combination of haematoxylin, Orange G, eosin Y, Light Green SF
yellowish, and sometimes Bismarck Brown Y.
PAS staining
PAS diastase showing the fungus Histoplasma.
Periodic acid-Schiff is a histology special stain used to mark carbohydrates (glycogen, glycoprotein,
proteoglycans). PAS is commonly used on liver tissue where glycogen deposits are made which is
done in efforts to distinguish different types of glycogen storage diseases. PAS is important because it
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can detect glycogen granules found in tumors of the ovaries and pancreas of the endocrine system, as
well as in the bladder and kidneys of the renal system. Basement membranes can also show up in a
PAS stain and can be important when diagnosing renal disease. Due to the high volume of
carbohydrates within the cell wall of hyphae and yeast forms of fungi, the Periodic acid -Schiff stain
can help locate these species inside tissue samples of the human body.
Masson's trichrome
Masson's trichrome is (as the name implies) a three-colour staining protocol. The recipe has evolved
from Masson's original technique for different specific applications, but all are well-suited to
distinguish cells from surrounding connective tissue. Most recipes produce red keratin and muscle
fibers, blue or green staining of collagen and bone, light red or pink staining of cytoplasm, and
black cell nuclei.
SPECIAL STAINING
(Endospore / Capsule / Flagella/ Metachromatic granules )
Spore Staining:
If spore bearing organisms are stained with ordinary dyes, or by Gram‘s stain, the body of the bacillus
is deeply coloured, whereas the spore remains unstained. The vegetative bacterial cells are stainable
with aqueous dyes, but endospores possess permeability barrier that prevents stain/dyes from entering
the spore coat unless the barrier is destroyed by heating, UV light, mechanical rupture or by treatment
of acid. The tough spore coat is formed to protect the bacterial cells DNA and important proteins from
adverse environmental conditions (excessive heating, short of nutrients, drying etc.)Spore coat is a
complex multilayered structure containing high calcium ions and dipicolinic acid which makes the
structure more tough .
Below spore coat, lies the peptidoglycan. Once the protective tough spore coat is penetrated, the
stain/dye interacts with peptidoglycan to produce the desired effect of staining.
There are other staining methods to introduce dye into the substance of the spore. When thus stained,
the spore tends to retain the dye and resist decolourization. Several methods such as Acid Fast Staining
Method for Spores (Spores stained bright and protoplasm of the bacillus stains blue), Hansen‘s
Method,Dorner‘s method and Schaeffer and Fulton‘s Method are widely applied methods for staining
spores in proper.

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Commonly used staining methods for endospores includes:
1. The Schaeffer-Fulton method – Most common method used to stain endospores.
2. Dorner method
3. Hansens method
1. Schaeffer-Fulton method for staining endospores
Malachite green stain (0.5% (wt/vol) aqueous solution) 0.5 g of malachite green
100 ml of distilled water
Decolorizer Tap water,
Safranin counterstain Stock solution (2.5% (wt/vol) alcoholic solution) 2.5 g of

safranin O 100 ml of 95% ethanol Working solution 10 ml of stock
solution 90 ml of distilled water
Procedure:
Schaeffer-Fulton method:
1. Fix the air dried smear by passing over the flame 2-3 times.
2. Flood smear with malachite green and heat for 5 min. Do not boil.
3. Allow to cool and wash with water.
4. Counter stain with dilute safranin (working solution) for 1 min.
5. Wash the smear and air dry it.
Spore stains:Endospore takes bright green and bacterial cells are brownish red to pink.
Dorner method for staining endospores
Spore stains: Bacterial cells are colorless, endospores are red, and the background is black. Carbol
Fuchsin – primary stain and counterstain Nigrosin .
Hansen’s Method
Material: Bacterial smear, Cocn. carbol fuchsin, 5% Acetic acid, Loeffer‘s methylene blue.

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Procedure:
 Fix the air dried smear by passing over the flame 2-3 times.
 Stain the smear as follows,
 Flood smear with Conc. carbol fuchsin and heat for 5 min. Do not boil.
 Allow to cool and wash with water.
 Decolorize with 5% Acetic acid for 1 min. and wash with water.
 Counter stain with Loeffer‘s methylene blue for 3 min.
 Wash the smear and air dry the smear and observe under microscope.
Spore stains: spores stain red and the vegetative cells blue.
ii) Capsular Staining
The best way to demonstrate capsules of bacterial cells is to stain them by some procedure, which
differentiates them from the bacterial cell itself. Anthony‘s method (with Tyler‘s modification) to
stain capsule is the simplest method.
The best way to demonstrate capsules of bacterial cells is to stain them by some procedure, which
differentiates them from the bacterial cell itself. Anthony‘s method (with Tyler‘s modification) to
stain capsule is the simplest method.
Anthony’s method (With Tyler’s modification)
Staining solution:
 Acetic crystal violet
 Crystal violet (35% dye content)——— 0.1 g
 Glacial acetic acid ———————- 0.25 ml
 Distilled water —————————–100 ml

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Procedure:
Anthony‘s method (With Tyler‘s modification)
 Prepare a smear of bacterial culture on the slide.
 Dry it in the air.
 Stain for 4-7 minutes in the ‗acetic crystal-violet‘ solution.
 Wash with 20% aqueous copper sulphate (CuSO4, 5H2O)
 Dry with blotting paper and examine.
Capsules stains blue violet; Bacterial cell stains dark blue.
iii) Flagellar Staining
Robert Koch published staining procedure for bacterial flagella in 1877. Subsequently several
modifications and methods were developed for staining flagella were developed. In 1930, Leifson
published a simple flagella stain. Many modifications or alternative methods includes a wet-mount
procedure of Mayfield and Innis and a more traditional dried-smear preparation ,combination of the
wet-mount technique of Mayfield and Innis and the stain of Ryu suggested by Kodaka et al. ,overcame
most difficulties in staining flagella.Presque Isle Cultures flagella stain – ready made staining
method available commercially.
A silver-plating stain for flagella was developed in 1958 and simplified in 1977. Recently a
fluorescent protein stain, NanoOrange from Molecular Probes (Eugene, OR), is being applied to screen
for bacteria possessing flagella by light microscopy .
Materials 12-16 Hrs incubated bacterial culture, Microscope slides, 95% ethanol
 Micorpipette with sterile disposable tips, Distilled water. Leifson flagella stain
 Solution A: Sodium chloride 1.5 g
 Distilled water 100 ml
 Solution B: Tannic acid 3.0 g
 Distilled water 100 ml
 Solution C: Pararosaniline acetate 0.9 g Paraosaniline hydrochloride 0.3 g Ethanol, 95%

(vol/vol) 100 ml Take equal volumes of solutions A and B and then add 2 volumes of the
mixture to 1 volume of solution C. The resulting solution may be kept refrigerated for 1 to 2
months. (Ryu method – reagent stable at room temperature)

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Preparation:
Bacterial cultures incubated for 12-16 Hrs can be used for flagellar staining. Collect small quantity of
growth from agar medium and emulsify in 100 ml of distilled water. Take in a micro-centrifuge tube
mix by gentle vortexing. Avoid too much of inoculums.
If culture is used from incubated broth, centrifuge the culture, remove spent medium.
Resuspend in 100 ml of distilled water by gently vortexing, again centrifuge, and remove supernatant.
Finally, resuspend in 200 ml of distilled water and prepare slightly cloudy emulsion to be used for
staining.
Preparation of smear:
1. Take ethanol treated clean new microscope slide and flame to dry before use.
2. Cool the slide, place 5 to 10 ml of the culture emulsion on one end of the slide and spread it with
the help of pipette.
3. Dry at room temperature. Do not heat fix.(Heating destroys the flagella)

Staining procedure:

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Leifson flagella staining method:
1. 1.Take a prepared slide and mark an area of 1×1.5 inch2 with grease pencil.
2. Flood Leifson dye solution on the slide within the marked area.
3. Incubate at room temperature for 7 to 15 minutes or allow to act till formation of fine
precipitate.(Golden film develops on the dye surface).
4. Remove the stain by gentle wash with water steam and air dry.
5. Observe under oil immersion
Bacterial body and flagella will stain red.
iv) Staining of Metachromatic Granules – Albert’s Method
Metachromatic granules:
Special stains (Albert, Neisser) pick out the volutin granules and give the bacilli a beaded or barred
appearance; the granules are polar in short bacilli. Volutin staining reactions are best seen in young
cultures.
(See Albert Staining Procedure in upper section)


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MCQ Questions
1. The dye eosinate of methylene blue belongs to which group?
(a) Acidic dye
(b) Basic dye
(c) Neutral dye
(d) oxazine dye
2. which of the correct order of staining reagents in Gram-Straining
(a) Crystal violet, alcohol, iodine solution , safrauin
(b) Crystal violet, iodine solution, alcohol, saframen
(c) Crystal violet, safrauin , alcohol, iodne solution
(d) Iodine solution , Crystal violet, alcohol, safravin.
3. which bacteria appears purple-violet colour after staining?
(a) Gram + Ve
(b) Gram –Ve
(c) Both Gram +Ve and Gram –Ve
(d) Neither Gram +Ve and Gram –Ve
(4) gram positive bacteria are usually more susceptible to ?
(a) Strepomylin (c) Penicillin
(b) Tetralyclin (d) Ampicillin
(5) Which of the staining techniques helps in demonstrating spore structure in bacteria as well as free
spores?
(a) Acid – fast stain (c) capsule stain
(b) Endospore stain (d) Hagella stain

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(6) In Gram staining , iodine is used as a __________________
(a) fixative (c) Solublizer
(b) Mordant (d) Stain
7. Which staining method is used for the identification of bacterial pathogen clostridium diffcile
(a) Gram staining (c) Ziehi – Neelsen staining
(b) Endospore Staining (d) Differential stating
8. Mycobacterium tuberculosis is stained by which method :-
(a) Gram staining
(b) endospore staining
(c) Ziehl – Neelsen staining
(d) Differential staining
(9) Counter stain used ziehl – Neelsen staining is :-
(a) Carbol fuschin (c) Methylene blue
(b) Saffranim (d) Haematoxylin stains
10. Identify which statements are correct.
A. All bacteria have peptidoglycan in their cell walls.
B. All fungi have chitin in their cell walls.
C. All algae have cellulose in their cell walls
D. All protozoans have protein in their cell walls.
ANSWER KEY
1. B 2. B 3. A 4. C 5. B
6. B 7. A 8. C 9. C 10. B

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One Word Questions
1. A technique used to enhance contrast in samples, generally at the microscopic level.
2. A staining is used to stain species of Mycobacterium tuberculosis.
3. Gram staining uses to stain cell walls.
4. On most Gram-stained preparations, organisms appear red or pink because they are counterstained
Is.
5. The stain used in Ziehl-Neelsen staining that stains the bacteria and a counter stain such
as Methylene blue is.
6. A staining is particularly useful for identifying endospore-forming bacterial pathogens such

as Clostridium difficile.
7. A bacteria stain dark blue or violet.
8. The most basic method to enhance visualization of the cell or certain cellular components under a
microscope.
9. A stains animal cells to make nuclei more visible.
10. Is used to determine gram status to classify bacteria broadly. It is based on the composition of
their cell wall.
ANSWER KEY
1. Staining 2. Ziehl-Neelsen 3. Crystal violet 4. Gram-negative 5. Carbol fuchsin
6. Endospore 7. Gram-positive 8. Staining 9. Methylene 10. Gram
method Blue staining


Fundamental of Microbiology Notes Final

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BVMLT – 103

UNIT-1 Introductory microbiology: Introduction to and brief of microbiology, scope

UNIT-2 Morphology and structure of microorganisms: Morphology and structure of

UNIT-3 Stains used in microbiology: Introduction to stains; importance of stain in

Learning objective: On completion of this unit student will able to:

What Are Microbes?

Microorganisms live in all parts of the biosphere where there is liquid

The Pathogenic Ecology of Microbes

Louis Pasteur is also known as father of microbiology. He has many contributions to

1. He has proposed the principles of fermentation for preservation of food

Robert Koch: An image of Robert Koch, a pioneering

After Casimir Davaine demonstrated the direct transmission of

Robert Koch provided remarkable contributions to the field of microbiology:

1. He was the first to report the acid-fast nature of tubercle bacillus.

2. He developed techniques to stain tissues and blood cells.

A good microscope should have three properties:

 Unaided human eye is about 0.2 mm (200 m)

2. Good contrast: This can be further improved by staining the specimen.

3. Good magnification: This is achieved by use of concave lenses.

The bright-field or light microscope forms a dark image against a brighter

Dark Field Microscope

BRIGHT FIELD MICROSCOPE DARK FIELD MICROSCOPE

Phase Contrast Microscope

Working of Phase contrast microscope:

1. Partially coherent illumination produced by the tungsten-halogen lamp is directed through a

Parts of Phase contrast Microscope:

The annular diaphragm

1. It is situated below the condenser.

2. It is made up of a circular disc having a circular annular groove.

5. At the back focal plane of the objective develops an image.

6. The annular phase plate is placed at this back focal plane.

3. The phase plate is a transparent disc.

To produce high-contrast images of transparent specimens, such as

 living cells (usually in culture),

 thin tissue slices,

 glass fragments, and

 subcellular particles (including nuclei and other organelles).

 It makes a highly transparent object more visible.

 Phase-contrast optical components can be added to virtually any brightfield microscope,

 To use phase-contrast the light path must be aligned.

2. Scanning EM (examine the surfaces, resolution 7 nm, gives 3-dimensional view)

Scanning Electron Microscope (SEM): In a scanning electron microscope, the specimen is

The limitations of electron microscopes are as follows:

(a) Live specimen cannot be observed.

Physical method i.e., use of heat, filters, radiation

Chemical method i.e., by use of chemicals

b. Wet heat or moist heat sterilization Moist heat sterilization is accomplished by

3).Tyndallization (Fractional Sterilization) is the steaming process performed at 100°C is done in

Standard operating procedure for the setting up of autoclave

Standard operating procedure for the setting up of hot air oven

 Switch on the hot air oven until to reach 160 degree C

 Hold on in that temperature for 1 hour

Table 1.2.1: Classification of sterilization/disinfection methods

DRY HEAT MOIST HEAT

DRY HEAT STERILISATION:

 Hot air oven.

media which are decomposed at high temperature of autoclave.

 At temperature above 100 C.

 to sterile sera sugars and antibiotic solution

 Separation of toxins and bacteriophages.

 To obtain bacteria free filtrates of clinical samples of virus isolation.

 Sterilisation of hydatid fluid.

ALDEHYDES: markedly bactericidal, sporicidal and virucidal.

SURFACE ACTIVE AGENTS: