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NEHRU ARTS AND SCIENCE COLLEGE
DEPARTMENT OF BIOTECHNOLOGY
E-LEARNING
: I MSC BIOTECH
: MICROBIOLOGY
UNIT-I
CLASS
SUBJECT
History of Microbiology
Discovery of the microbial world: Controversy over spontaneous generation; discovery of role of
microbes in causation of disease, first pure culture, development of culture media by koch and his
school, Microscopy: Light, phase contrast, fluorescent, TEM, SEM and scanning tunneling
Microscopy, Methods of staining and Microscopic observation of Bacteria and Fungi.
Define Microbiology?
(Section-A)
Micro – Small
Bio – Life
Logy – Study
 Microbiology in the study of microorganisms (some times called microbes)- living things
that are too small to be seen by the unaided eye.
 Through history, microorganisms have had a huge impact on human affairs, mostly
negative.
 Before the development of microbiology as a science, infectious disease controlled
human events.
 Microorganisms are closely associated with the health of human beings; some
microorganisms are beneficial and others are harmful .Eg. Microorganisms are involved
in the making of cheese, wine and in the production of antibiotics – (Penicillin, interferon
and alcohol).
 Microorganisms can cause disease, spoil food and deteriorate materials like iron pipes,
glass lenses and wood pilings, etc.
Write a note on formation of microbes?
(Section-A)
Microbes are of three forms.

Unicellular
Eg: Protozoa and bacteria.

Multicellular Eg:Algae and Fungi

Non-Cellular Eg:Virus
Characteristics of microorganisms?

(Section-A)
Ability to reproduce.
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
Ability to ingest food substances and metabolize them for energy and
growth.

Ability to excrete waste products.

Ability to react to changes in their environment.

Susceptibility to mutation.
Scope of microbiology and microbiology today:
(Section-B&C)
 Microorganisms are usually divided into six subgroups:
1. Bacteria
2. Archaea
3. Algae
4. Fungi
5. Protozoa
6. Viruses
 Microbiology is a cohesive science because of its methodology and approach to
problems, not because of the relatedness of the organisms it studies.
 Bacteria and archaea are prokaryotes; they lack internal membrane-bound structures.
 Algae, fungi and protozoa are eukaryotes; their organelles are membrane-bound.
 Viruses are acellular.
1. Bacteria:
 Bacteria are prokaryotes.
 Bacteria are extremely small, even for microorganisms.
 Bacteria vary in shape, motility and how they get energy. There are species that can
withstand freezing, boiling, and extreme acidity or alkalinity.
 Some bacteria cause disease, but others our environment in life-sustaining balance.
2. Archaea:
 Archae were discovered as a separate group of microorganisms in the 1970’s.
 At first they were called archaebacteria.
 Being prokaryotes and small, they resemble bacteria superficially, but they are as
distantly related to bacteria as they are to eukaryotes.
 Many but not all archae live in extremely hostile environments.
3. Algae:
 Algae are eukaryotic organisms that carry out photosynthesis.
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 Some algae are unicellular and microscopic. Others consist of so many cells they are
macroscopic.
 Algae are not important medically, but they are critically important to global ecology.
4. Fungi:
 Fungi include mushrooms, yeasts, and molds. Fungi are eukaryotic and nonphotosynthetic. Some are microscopic, others are macroscopic.
 A few fungi are pathogenic to humans, and many are pathogenic to plants, causing for
example, corn smut, wheat rust, and potato blight.
5. Protozoa:
 Protozoa are eukaryotic microorganisms that are superficially animal-like,
nonphotosynthetic and usually motile.
 Examples of protozoa are the amoeba, flagellates and ciliates.
 The study of protozoan (and helminthes-caused) diseases is called parasitology.
6. Viruses:
 Viruses are particles of nucleic acid (either RNA or DNA), usually enclosed in a
protein coat and some-times surrounded by a membrane.
 Viruses are obligate intracellular parasites.
 Viruses are extremely small, even compared to bacteria.
 Viruses can infect animals, plants, and microorganisms.
 Prions are even smaller infectious agents than viruses. They are composed entirely of
protein. Their method of reproduction is not completely understood.
Various fields of microbiology are listed out briefly in the forth-coming paragraphs:
Medical Microbiology:
 Medical microbiologists identify the causative agents responsible for infectious diseases
and in eliminating the disease.
Public Health Microbiology:
 Public health microbiologists try to control the spread of communicable diseases in the
society.
 They monitor the community food establishments and water supplies to keep them safe
and free from infectious diseases. It is very important to keep the people away from the
food or water – borne diseases.
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Immunology:
 It is concerned with the immune system that protects the body from pathogens and the
response of infectious agents.
Agricultural Microbiology:
 It is concerned with the impact of microorganisms on agriculture.
 Agricultural microbiologists try to combat plant diseases that attack important food crops,
work on methods to increase the soil fertility and crop yields etc.
Microbial Ecology:
 Microbial ecologists study the relationship between microorganisms and their habitats.
 Contributions of microorganisms to the carbon, nitrogen and sulphur cycles in soil and in
fresh water.
 The study of pollution effects on microorganisms is also very important.
Soil Microbiology:
 Study of particular microorganisms that use soil as a terrestrial habitat.
 Biodegradation – the biological breakdown of various compounds in soil.
Veterinary Microbiology:
 It deals with the harmful effects of disease agents on domestic animals and poultry.
Food Microbiology:
 Food microbiologists prevent microbial spoilage of food and transmission of food borne
diseases.
 They identify the causes for spoilage and prevent the economic loss.
Dairy Microbiology:
 It deals with manufacturing of cheese, butter, yoghurt, creams, etc.
Aero Microbiology:
 It deals with the study of atmospheric environment which includes microorganisms.
 Air sanitation which would be an efficient method to make the atmosphere free from the
microbes.
Aquatic Microbiology:
 It deals with the study of microorganisms and their activities in fresh and marine
water.
Petroleum Microbiology:
 Microorganisms are involved in the formation and recovery of petroleum.
 Microbes are useful in removing oil spills and reduce the problem of oil pollution.
Microbial Biotechnology:
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 Application of technology to living systems.
 Any technique in which a living organisms are used to produce a useful product or a
desirable chemical changes.
Genetic engineering:
 Engineered microorganisms are used to make hormones, vaccines and other products.
 New genes can be inserted to plants and animals.
Industrial Microbiology:
 Microorganisms are used to make products such as antibiotics, vaccines, steroids,
alcohols, vitamins, amino acids and enzymes.
HISTORY AND RECENT DEVELOPMENT? (OR) WRITE SHORT NOTE ON
MILESTONE OF MICROBIOLOGY? (Section-C)
Microorganisms were discovered more than 300 years ago, but we didn’t know
much about them until the mid-1800, when microbiology became an experimental science.
Microbial Ecology and soil microbiology :
 Martinus Beijerinck and Sergius Winogradsky studied the importance of microorganisms
in various biogeochemical cycles – Carbon, Nitrogen and Sulphur cycles.
Contribution of S. Winogradsky:
 He discovered chemoautotrophic bacteria in soil.
 They obtain energy from inorganic compounds – Nitrogen and Sulphur and use Carbon-dioxide as the carbon source.
 He isolated sulphur bacteria – Thiobacillus sp. and nitrifying bacteria – Nitrobacter and iron
bacteria – Ferrobacillus sp.
 He also isolated anaerobic nitrogen fixing bacteria – Azotobacter.
 He described the usefulness of these organisms in promoting the soil fertility and also as
biofertilizers.
 For the study of these bacteria, they developed the enrichment culture technique and the use
of selective enrichment media.
Plant Pathology:
 Plant pathology is the study of plant diseases.
 T.J.Burrill found that in pears, a disease known as fire blight was caused by a bacterium.
Microbiology in 20th century:
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 During the early part of 20th century, Microbiologists were concerned with the agents of
infectious disease, the immune response and the search for chemotherapeutic agents for
treatment.
 Cell biologists studied the cell organization, reproduction and development and the
mechanism of hereditary.
 The use of chemicals would kill or interfere with the growth of the pathogen without
damaging the infected individual is known as chemotherapy.
 Newer approaches include antitumor antibiotics and antiviral agents.
 At present interferon and interleukins are found to be very effective against cancers.
 Azidothymidine and Dideoxyinosine are found for the AIDS treatment.
 With the help of genetic engineering, therapeutic proteins – insulin and enzymes – Urokinase
for the problems of health has been discovered.
Discovery of Animal, Plant and Bacterial viruses (Bacteriophages) :
 In 1898, F.J.Loeffler and Paul Frosch proved that foot and mouth disease is caused by an
agent that is too small to be seen through the microscope and that passes the bacteria
retaining filters.
 In 1886, Adolf Mayer demonstrated the infectious nature of tobacco mosaic disease.
 In 1917, Felix d’ Herelle discovered the bacteriophages which means bacteria eating.
Major bacterial pathogens:
S.No
Disease
Causative Agent
Discoverer
Year
1
Anthrax
Bacillus anthracis
Koch
1876
2
Gonorrhea
Neisseria gonorrhoeae
Neisser
1879
3
Typhoid fever
Salmonella typhi
Eberth
1880
4
Malaria
Plasmodium sp.
Laveran
1880
5
Tuberculosis
Mycobacterium tuberculosis
Koch
1882
6
Cholera
Vibrio cholerae
Koch
1883
7
Diphtheria
Corynebacterium
Klebs and Loeffler
1883 – 1884
8
Tetanus
diphtheriae
Nicolaier
1885
9
Diarrhea
Clostridium tetani
Escherich
1885
10
Pneumonia
Escherichia coli
Fraenkel
1886
11
Meningitis
Streptococcus pneumoniae
Weichselbaum
1887
6
12
Undulant fever
Neisseria meningitidis
Bruce
1887
13
Gas gangrene
Brucella sp.
Welch & Nuttal
1892
14
Plague
Clostridium perfringens
Kitasato & Yersin
1894
15
Botulism
Yersinia pestis
Van Ermengem
1896
16
Dysentry
Clostridium botulinum
Shiga
1898
17
Syphilis
Shigella dysenteriae
Schaudin&Hoffmann
1905
18
Whooping cough
Treponema pallidum
Bordet & Gengou
1906
19
Rocky mountain spotted
Bordetella pertussis
Ricketts
1909
Ricketts & Wilder
1910
McCoy & Chapin
1912
fever
20
Typhus fever
21
Tularemia
Rickettsia rickettsii
Rickettsia prowazekii
Francisella tularensis.
Molecular biology and biotechnology:
 In 1944, Avery, Macleod and McCarty described bacterial transformation by DNA.
 In 1953, Watson and Crick described the structure and functions of DNA.
 In 1960, Jacob and Monad described the synthesis and regulation of proteins.
 In 1960, Nirenberg and Khorana worked out the genetic code.
 In 1970, new discoveries led to the development of recombinant DNA technology and
genetic engineering.
Development of Microbiology:
Year
Microbiological History
1546
Fracastoro suggests that invisible organisms cause disease
1676
Leeuwenhoek discovers “Animalcules”
1688
Redi publishes work on spontaneous generation of maggots
1799
Edward Jenner introduced vaccination for smallpox
1838 – 1839
Schwann and Schleiden, the cell theory
1835 – 1844
Bassi discover that a Silkworm disease is caused by fungi and proposed that many
diseases are microbial in origin.
1847 – 1850
Semmelweis shows that childhood is transmitted by physicians and introduces the use
of antiseptics to prevent the disease.
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1857
Pasteur shows that lactic acid fermentation is due to a microorganism.
1861
Pasteur shows that microorganisms do not arise by spontaneous generation.
1867
Lister publishes his work on antiseptic surgery.
1869
Meischer discovers nucleic acids.
1876 – 1877
Koch demonstrates that anthrax is caused by Bacillus anthracis.
1881
Koch cultured bacteria on gelatin and Pasteur develops anthrax vaccine.
1882
Koch discover tubercle bacillus
1884
Koch’s postulates first published – Metchnikoff describes phagocytosis – Gram stain
developed.
1885
Pasteur develops rabies vaccine.
1887
Petridish
1887 – 1890
Winogradsky studied Sulphur and nitrifying bacteria
1888
Beijerinck isolated root nodule bacteria.
1890
Von Behring prepared antitoxins for diphtheria and tetanus.
1892
Ivanowsky provided evidence for virus causation of Tobacco mosaic disease.
1895
Bordet discovers complement
1897
Buchner prepares yeast extract that ferments – Ross showed that malarial parasite is
carried by the mosquito
1899
Beijerinck proves that a virus particle causes the tobacco mosaic disease.
1900
Reed proves that yellow fever is transmitted by the mosquito.
1902
Landsteiner discovers blood groups.
1903
Wright and others discover antibodied in the blood of immunized animals.
1906
Schaudinn and Hoffmann showed Treponema pallidum causes syphilis – Wassermann
develops complement fixation test for syphilis – Ricketts showed that Rocky
mountain spotted fever is transmitted by ticks.
1910
Ehrlich developed chemotherapeutic agent for syphilis.
1915 – 1917
D’Herelle and Twort discovered bacterial viruses.
1923
First edition of Bergey’s manual.
1928
Griffith discovered bacterial transformation.
1929
Flemming discovered penicillin.
1933
Ruska developed first transmission electron microscope
1935
Stanley crystallizes the Tobacco mosaic virus – Domagk discovered sulpha drugs.
1941
Beadle and Tatum – one gene one enzyme hypothesis
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1944
Avery showed that DNA carries information during transformation.
1946
Lederberg and Tatum described bacterial conjucation.
1950
Lwoff induces lysogenic bacteriophages.
1952
Hershey and Chase showed that bacteriophages inject DNA into host cells – Zinder
and Lederberg discovered generalized transduction – Walsmann discovered
Streptomycin.
1953
Phase contrast microscope developed – Medawar discovered immune tolerance –
Watson and Crick proposed the double helix structure for DNA.
1955
Jacob and Wollman discovered the F factor is a plasmid
1961
Jacob and Monad proposed the operon model of gene regulation.
1961 – 1966
Nirenberg, Khorana and others elucidate the genetic code.
1970
Discovery of restriction endonucleases by Arber and Smith – Discovery of reverse
transcriptase in retro virus by Temin and Baltimore.
1971
Gel electrophoresis.
1972
Recombinant DNA technology – Berg.
1973
Ames developed bacterial assay for the detection of mutagens and carcinogens.
1975
Milstein and Kohler discovered hybridoma monoclonal antibodies – Lyme disease
discovered.
1977
Recognition of Archaebacteria as a distinct microbial group.
1979
Insulin synthesized using recombinant DNA technology.
1980
Development of scanning microscope.
1982
Recombinant hepatitis B vaccine developed.
1983 – 1984
HIV isolated and identified by Gallo and Montagnier – PCR developed by Mullis.
1986
First vaccine (Hepatitis) produced by genetic engineering approved for human use.
1987
First successful experiment in gene transplantation – Mulligan.
1990
Human gene therapy
1992
First human trials of anti – sense therapy.
1995
Chicken pox vaccine approved for US use – Haemophilus influenza genome
1996
sequenced.
Yeast genome sequenced.
1997
E.coli genome sequenced – Discovery of Thiomargartia namibiensis, the largest
bacteria.
2000
Vibrio cholerae has two separate chromosomes was discovered.
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Spontaneous generation:
(Section-C)
 While Leeuwen hoek was observing microorganisms in Holland, Francesco Redi, a
physician in Italy, was making ground breaking discoveries of his own.
 In 1665 Redi did an experiment with jars of covered and uncovered meat.
 By showing that maggots developed only in meat that flies could reach to lay eggs on, he
provided powerful additional evidence that spontaneous generation (formation of living
things from inanimate matter) did not occur, at least in the case of flies.
 Instead, flies and by extension, all living things come only from preexisting living things.
Needham versus Spallanzani:
 For 80 years the debate continued.
 Then the proponents of spontaneous generation seemed to gain ground when, in 1745, an
English cleryman named John Needham did a well-publicized experiment.
 Everyone knew boiling killed microorganisms.
 So he boiled chicken broth, put it in a flask, and sealed it.
 Microorganisms could develop in it only by spontaneous generation.
 Indeed, microorganisms did appear. But an Italian priest and professor named Lazzaro
Spallanzani was not convinced.
 May be microorganisms entered the broth after boiling but before sealing. So Spallanzani
put broth in a flask, sealed it, and then boiled it.
 No microorganisms appeared in the cooled broth still the critics were not persuaded.
 Spallanzani didn’t disprove Spontaneous generation, they said, he just proved that
spontaneous generation required air.
Pasteur’s Experiment:
 The controversy continued another 100 years. Finally in 1859, the French Academy of
science sponsored a competition to prove or disprove the theory of spontaneous
generation.
 A young French chemist named Louis Pasteur entered.
 To counter the argument that air was necessary to spontaneous generation, Pasteur used
barriers that allowed free passage of air but not microorganisms.
 In his most famous experiment, Pasteur boiled meat broth in flask and then drew out and
curved the neck of the flask in flame.
 No microorganisms developed in the flask.
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 But when he titled the flask so some broth flowed into the curved neck and then tilted it
back so the broth was returned to the base of the flask, the broth quickly became cloudy
with the growth of microbial cells.
 Gravity had caused the microbial cells that had entered the flask to settle at the low point
of the neck.
 They never reached the broth in the base until they were washed into it.
 Thus Pasteur showed that spontaneous generation of microorganisms does not occur even
in the presence of air.
 He concluded that the growth of microorganisms causes broth to spoil and, by extension,
dead plant and animal matter to decompose.
 Pasteur was brilliant and lack. Many early experiments to disprove spontaneous
generation failed because the samples contained endospores, highly heat-resistant
structures that are not killed by boiling.
 Had Pasteur done his experiments with vegetable rather than meat broth, they too
certainly would have failed because plant materials usually carry bacterial endospores.
Meat broths rarely do.
BIOGENESIS CONTRIBUTIONS OF LEEUWENHOEK, LOUIS PASTEUR, ROBERT
KOACH, ELIE METCHINKOFF AND FLEMING: (Section-C)
Biogenesis: Living organisms could develop from other living organisms.
(i) Antony Van Leeuwenhoek:
 The first person to observe and describe microorganisms
was Antony Van Leeuwenhoek (1632 – 1723), who
constructed simple microscopes composed of double convex
glass lenses held between two silver plates.
 Magnification was around 50 – 300 times and has
illuminated the liquid specimens by placing them between
two pieces of glass.
 This forms dark – field illumination and made bacteria
clearly visible.
 He described the “very little animalcules” which we
recognize as free – living protozoa.
 He saw both bacteria and protozoa.
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 Pasteur first filtered air through cotton and found that objects
resembling plant spores had been trapped.
 If a piece of cotton was placed in sterile medium after air had
been filtered through it, microbial growth appeared.
 Next, he placed nutrient solutions in flasks, heated their
necks in a flame and drew them out into variety of curves,
while keeping the ends of the necks open to the atmosphere.
 Pasteur then boiled the solutions for a few minutes and
allowed them to cool.
 “No growth took place even though the contents of the flasks
were exposed to the air”.
 No growth occurred because dust and germs had been
trapped on the walls of the curved necks.
 If the necks were broken, growth commenced immediately.
 Pasteur had shown how to keep the solutions sterile.
Louis Pasteur (1822
– 1895):
Louis Pasteur (1822 – 1895):
The Germ theory of fermentation: (Section-B)
 Louis Pasteur (1856 – 1857), worked on ethanol fermentation was facing the problem of sour
wine.
 During his study of spoiled beer, lactic acid bacteria had contaminated the wine and produced
sour taste in the wine.
 As a control measure, Pasteurization was applied for the wine preservation for long time.
 Pasteurization means heating of milk or other liquids to moderate temperature for a definite
time which kills most pathogenic bacteria – 62.8 0 C – 30 mins.
 This technique was applied in dairy industry to preserve milk and milk products.
Protection against infection:
Immunity:
 Pasteur makes discoveries concerning the cause and prevention of infectious diseases.
 During his work on chicken cholera, Pasteur discovered that older cultures of bacterium were
attenuated i.e., they had lost their ability to cause the disease.
 If the chickens were injected with these attenuated cultures, they remained healthy but
developed the ability to resist the disease.
 He called the attenuated culture, a vaccine (Latin Vacca means Cow)
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(i)
Pure culture of Chicken
Cholera Bacteria – 8 weeks old
Chicken A inoculated
Chickens remain
some week interval
(ii)Fresh virulent culture of chicken cholera bacteria
Chicken A inoculated,
Chicken not previously
reinoculated.
Inoculated.
Chicken remains healthy
Chicken dies
 Pasteur applied this principle to the prevention of anthrax.
 Edward Jenner used vaccination with material from cowpox lesions to protect people against
smallpox.
Rabies vaccine:
 Pasteur next prepared rabies vaccine.
 The pathogen was attenuated by growing in the rabbit.
 After infected, rabbits had died; their brains and spinal cords were removed and dried.
 Injection of a mixture of this material and glycerin stimulates dogs to develop resistance to
rabies.
 Pasteur tried vaccination with a nine year old boy (Joseph meister) who had been bitten by a
rabid dog and he survived after vaccination.
Saliva from
Mad dog
Injected into
rabbit
Contracts
Extract of brain and
rabies & dies
spinal cord attenuated
Patient
Injected into rabies
Recovers
patient
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Fig – Rabies vaccine.
Robert Koch (1843 – 1910):
 The role of bacteria as causative agent of disease was given by
Robert Koch, a German physician during his studies on
etiology of anthrax disease.
 Koch used the criteria proposed by his former teacher Jacob
Henle, to establish the relationship between pathogenic
microbe and disease.
 In his experimental work, Koch injected healthy mice with
material from diseased animals, and the mice became ill.
 After transferring anthrax by inoculation through a series of 20
mice, he incubated in beef serum, a piece of spleen containing
the anthrax bacillus.
 The bacilli grew, reproduced and produced spores.
 When the isolated bacilli or spores were injected into mice, anthrax developed.
 His criteria for proving the causal relationship between a microorganism and a specific
disease are known as Koch’s postulates :
 A specific microorganism can always be found in association with a given disease.
 The organism must be isolated and grown in a pure culture.
 The disease must result when the isolated organism is inoculated into a healthy host.
 The same organism must be again isolated from the diseased host.
 Koch’s proof that Bacillus anthracis caused anthrax was confirmed by Pasteur and his
coworkers.
 They discovered that after burial of dead animals, anthrax spores survived and were brought
to the surface by earth worms.
 Healthy animals then ingested the spores and became ill.
Staining:
 Koch was refining methods for the study of bacteria.
 He found that by smearing bacteria on a glass slide and adding certain dyes to them,
individual cells could be seen with the microscope.
Gelatin:
 He added gelatin and other solidifying materials such as agar to media in order to obtain
isolated growth of organisms known as colonies.
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 First Koch used potato surfaces. On sterile moist surfaces of boiled potatoes, the bacteria
were cultured.
 But moistened surface that spreads the growth of organism but prevents the formation of
isolated colony.
 So, he used gelatin as the solidifying agent.
 But gelatin was not an ideal solidifying agent because it was digested by many bacteria and
melted when the temperature arose above 28 degree C.
Nutrient Media:
 Nutrient media includes the preparation of several nutrient combinations for the growth.
 Meat extracts and protein digests also used as nutrient sources.
 The result was the development of nutrient broth and nutrient agar.
 In 1882, Koch used these techniques to isolate the bacillus that caused tuberculosis.
Use of agar as a solidifying agent:
 Mrs. Frau Herse, wife of Walter Hesse found an alternate solidifying agent than gelatin.
 The substance was agar-agar which is extracted from red marine algae called Gelidium.
 It is liquid at 100oC but becomes gel at 42oC.
Petriplate:
 Koch’s assistant named Richard.J.Petri introduced the Petridish, an effective container for
solid media.
 Koch also discovered pour plate method for isolation of bacteria in pure culture form.
Alexander Flemming:
 He is called as “Father of Antibiotics”. Antibiotics are defined as a chemical substance
produced by a microorganism, which has the ability to inhibit the growth of or to kill
other microorganisms.
 Alexander Fleming in 1928, observed fungal contamination that killed the colonies of
Cocci.
 Fungus is Penicillium notatum and it produced antibiotic called penicillin.
15
Elie Metchinkoff (1845-1916):
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MICROSCOPY
Explain about different types of microscope and its working?
(Section - C)
INTRODUCTION:
 The study of microbiology involves the use of a number of instruments.
 Microbiological instruments may be basically categorized into two-those that are used
for visual observation and those that are used for analytical purpose.
 Visual observation of microbes is made possible by the use of microscopes while a
wide array of instruments such as spectrophotometers, centrifuges, autoclaves, etc., are
used for various analytical purposes.
MICROSCOPY:

The most important tool of a biologist is the microscope. The invention of light
microscope in the sixteenth century is perhaps the single most important contribution
ever made to the advancement of biology. The present day microscope used in
biological laboratories has been developed over the course of several centuries.

Antony Van Leeuwenhoek (1632-1723), used his single lens microscope to observe
several minute objects like insects, mold, pond water, saliva etc.

It was Robert Hooke (1665), an Englishman who developed an instrument that could
truly be referred to as the forerunner of the modern day microscope.

Microscope is an optical instrument, consisting of a lens, or combination of lenses, for
making enlarged or magnified images of minute objects.
THE WORKING PRINCIPLE OF A MICROSCOPE:
The main function of the microscope is to produce a large and clear image of the
object. "Clarity is very often referred to as the resolving power of the microscope". The eye
cannot resolve details of an object, less than 5um in size.
SIMPLE MICROSCOPE:
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The simple microscope is nothing but a single convex lens or a combination of more than
one lenses functioning like a single lenses. This type of lens when placed between an object and
the eye allows the object to come closer, thus forming an enlarged image on the retina. The
superior quality lens can be constructed by combining two or more lenses into a single unit.
MODERN MICROSCOPES:
Modern microscopes, generally called compound microscopes have a complex system of
arrangement of lenses that help in higher magnification and better resolution.
Basically, the modern microscopes are of two types - light microscopes and electron
microscopes. In a light (optical) microscope, the source of illumination of the object is visible
light while in an electron microscope, the source of illumination is a beam of electrons.
Light microscopes are of the following types:
* Bright field (light) microscope
* Dark field microscope
* Phase contrast microscope
* Fluorescence microscope.
Electron microscopes are of two types:
* Transmission Electron Microscope (TEM)
* Scanning Electron Microscope (SEM)
BRIGHT FIELD (LIGHT) MICROSCOPE:
(12 MARKS)
The bright field microscope or light microscope or the compound microscope is the most
common and the most indispensable instrument in the microbiology laboratory.
Basically the microscope has two types of parts.
* Mechanical parts and
* Optical parts
The mechanical parts provide the structural frame work of the microscope and support the
optical parts. The optical parts are involved in the magnification of the object. Now we will
discuss about the detailed description of various parts of a light microscope.
BASE, PILLAR AND INCLINATION JOINT:
The base and the attached pillar serve to support the entire microscope. The base is
generally 'U' (horse shoe) shaped or sometime 'V' shaped. Usually the pillar and the base are
made heavy in order to minimize vibration. The inclination joint joins the arm of the
18
microscope to the pillar. The joints permits the lifting of the arm of the microscope to any
degree as desired by the observer.
ARM AND BODY TUBE:
The arm is a slightly bent solid piece of iron which is attached at one end to the pillar
by the inclination joint. The other end of the arm has the body tube to which the principle
optical systems are attached. The body tube is a cylindrical structure and at the point of
attachment to the arm there is a graduated rack which help in the movement of the body tube up
and down.
DRAW TUBE, REVOLVING NOSE PIECE:
The draw tube is at the upper end of the body tube. The ocular lens fits into the draw
tube. The purpose of the draw tube is to adjust the tube length so that the objective less and the
ocular are at a specified distance.
The revolving nose piece is a circular rotating disc attached to the body tube. Objective
lenses are attached to this.
ADJUSTMENT KNOBS:
The body tube can move up and down on the rack and pinion and the movement is
regulated by two sets of knobs - the coarse adjustment knob and the fine adjustment knob.
The coarse adjustment knob brings in a greater degree of movement while the fine
adjustment knob is used for slight degree of movement. The adjustment knobs are meant to bring
in the object into proper focus. While the range of course adjustment knob is greater, that of the
fine adjustment knob is very limited.
STAGE:
This is a small platform attached to the arm of the microscope. This is meant to keep the
objects (on sides) for the purpose of observation. It has a hole in the center exactly in line with
the body tube and the condenser below to allow the length to pass through. In most of the modern
microscopes, the stage is fitted with a mechanical device called mechanical stages to help in
the vertical and horizontal movement of the slide.
ILLUMINATION:
In order to collect and reflect the light beams there are two kinds of devices attached to
the base of the microscope. These are - mirror on an artificial illumination.
The mirror collects the light either from a natural source or an artificial source and
reflects the rags into the microscope. The mirror has both sides reflective - one side is plain, the
other is concave. Usually concave side is used when natural light is focused and plain side is
used while focusing artificial light.
19
SUB STAGE CONDENSER; IRIS DIAPHRAGM:
The light either reflected by the mirror or by a built in source will be diffuse and does
not have sufficient density. In order to condense the light and focus it on to the object, there is a
sub stage condenser. This consists of a condensing lens. The condensing lens may be fixed or in
some microscopes it can also move up and down for suitable adjustment.
The condenser helps to focus all the light on the object. Beneath the condenser there is a
diaphragm which regulates the quantity of light entering into the condenser. The iris diaphragm
may be closed and opened with the help of a lever to regulate the entry of light.
OPTICAL PARTS:
There are two sets of optics are lenses in a compound microscope. These are eye
pieces (ocular) and objectives.
EYE PIECES:
The ocular or eye piece is a short tube with 2 lenses which fits into the upper part of the
draw tube. The main function of the eye piece is to magnify the image of the object formed by
the objective eye pieces are marked 5x, 10x, 15x, etc.
The eye piece commonly used is the 10x - huygenian eye piece monocular tube. These
eye pieces are also referred to as negative oculars. The positive ocular or Ramsden eye pieces
are constructed with convex surface of both lenses facing inwards.
OBJECTIVE LENSES:
These are attached to the revolving nose piece and are very important as they affect the
quality of the image seen by the observer. Based on their type of construction objectives are
classified into achromatic, apochromatic and fluorite.
The first one is simple in construction and less expensive, while the other two are
complicated are more expensive and used in costly microscopes.
The objectives are of the following four types based on their magnification:
10x - low
40x - medium
60x - high
100x - oil immersion.
The oil immersion lens is used for very high magnification. The lens is so named
because a deep of a highly refractive liquid (refractive index same as glass) is added on the slide
while viewing through an oil immersion objective.
The main functions of the objective lenses are:
20
* Concentration of light rays coming from the object.
* Forming the image of the specimen and
* Magnifying the image.
MAGNIFICATION:
A microscope is primarily used to enlarge or magnify the object that is being viewed
which cannot otherwise be seen by the naked eye.
"Magnification may be defined as the degree of enlargement of an object provided by the
microscope". Magnification by a microscope is the product of the individual magnifying activity
of the oculars and the objectives.
For eg: if the ocular is 10x, and the objective is 40x, the specimen is magnified 400
times. If an oil immersion objective (100x) is used along with a 10x ocular, the specimen is
magnified 1000 times.
The following factors play an important role in magnification.
* Optical tube length
* Focal length of the objective lens.
* Magnifying ability of the ocular.
Total magnification can be calculated as follows:
Optical tube length
Total magnification = ------------------------- x magnification of ocular
Focal length of the
Objective.
RESOLVING POWER:
By using high powered lenses a magnification up to 3000 can be obtained but the image
will be blurred and details are not clear. This is due to the fact that in a microscope not only the
lenses, but the wave length of the illumination is also important and this decides the resolving
power of the microscope.
Resolving power of a microscope is defined as its "ability to distinguish between two
particles situated very close".
In a magnified image the object should not only be larger but the details should also be
clear. This is possible when a microscope has the ability to see two points situated very close at
two distinct entities.
In other words, resolving power may be said to be the minimum distance at which two
structural entities of an object can be visualized as discrete individual structures even in the
magnified image.
21
The resolving power of a microscope depends on two factors: the wavelength of the light
used for illumination and the numerical aperture (NA) of the objectives.
* In the optical light microscope, the wave length of the light used falls in the visible
range (400 -750nm). Within this range if lights of shorter wave length are used, the resolution
will be higher.
For eg: blue light has a shorter wave length than red light. Greater resolution can be
achieved by using a blue light as a source of illumination than a red light.
* “The numerical aperture (NA) is defined as the property of a lens that decides the
quantity of light that can enter into it". It depends on two factors.
1. Refractive index of the medium that fills the space between the specimen and the front of
the objective lens.
2. The angle of the most objective rays that can enter the objective lens.
LIMIT OF RESOLUTIONS:
This may be defined as the shortest distance between two objects when they can still be
distinguished as two separate entities. Highest resolution in a light microscope is possible with
the shortest wave length light (visible) and with the objective having highest NA.
PRINCIPLE OF OIL IMMERSION LENS:
The maximum resolving power of this microscope is approximately 0.27mm, about
one thousand th the thickness of a human hair. To achieve this level of resolution, however
immersion oil must be placed between the specimen and the 100x objective lens. Immersion oil
and glass have the same refractive index. Filling the air space between the glass lens and the
glass slide with immersion oil directs the light rays into the lens, there-by increasing resolution.
Light traveling from glass to air is bent because of the different light refractory
properties of air and glass. Since immersion oil and glass have virtually identical light refracting
properties, light is not bent as it goes from one into the other.
(Diagram)
Why we use oil immersion lens?
(Section- B)
Preventing light from refracting as it travels through the slide - specimen lens pathway
improves the quality of the image.
TYPES OF CONDENSERS:
Three types of condensers are in use in microscopes. They are:
* ABBE'S CONDENSOR:
22
This is the simplest one fitted to a microscope. There are two lenses in this, but both are
not corrected for any chromatic or spherical aberration. This type of condenser is good only for
low power viewing. At higher magnifications the images of the specimens get blurred due to
improper
focusing:
* APLANATIC CONDENSER:
In this, there is a third lens added to the original two, to correct the spherical aberration
only and not chromatic aberration. Aplanatic condenser gives a good image when used with
monochromatic light.
* ACHROMATIC CONDENSER:
This focuses most of the "incident" light on the object and allows very little to escape
and produce glare. The NA of the condenser generally, equals that of the objective. In their
condenser both spherical and chromatic aberrations are corrected.
ABERRATIONS IN THE OBJECTS:
The surface of objective lenses are spherical and such surfaces can not form perfect
images. This is due to variations in refraction and focus by the peripheral parts of the objectives.
These things cause defects or aberrations in the images. Aberrations in objectives are of two
types,
namely,
* Spherical aberrations and
* Chromatic aberrations.
Aberrations may be corrected by using better quality lenses or by the combination of
lenses with varying dispersive qualities.
BRIGHT FIELD MICROSCOPE
It is inexpensive and simple to align and use.
Only stained specimens can be seen directly .
Most biological specimens lack these characteristics and be stained with dyes before they can be
seen.
A microscope in which light from the illuminating source is caused to converge on the specimen
by sub stage condenser.
There by forming a cone of bright light that can enter the objective lens.
The cone of illuminating light is seen as a bright background against which the images of
specimen must be contrasted.
23
(Diagram)
EXPLAIN ABOUT DARK FIELD MICROSCOPE?
(Section- B)
There are several alternate types of microscopes other than bright field microscope that
increase the contrast between the background and the specimen without staining, thus allowing
for visualization of living specimens. One such microscope is dark field microscope.
(Diagram)
PRINCIPLE:
It depends on the ability of specimen to alter the characteristic of light rays that pass through it .
Only the rays that are diffracted by the specimen are used to form the image.
This is done by using an objective lens with a small aperture that allows only the rays diffracted
by the specimen to enter.
The image therefore appears bright against dark background.
APPLICATIONS:
Dark field microscopy is particularly valuable for the examination of unstained
microorganisms suspended in fluid - net amount and hanging drop preparations.
EXPLAIN ABOUT FLUORESCENE MICROSCOPE?
(Section- C)
FLUORESCENCE : it is defined as chemical compound is said to fluorescence if it is capable
of adsorbing ultraviolet light and remitting the energy of visible light .
Some are naturally occurring and some are synthetic compounds eg.Rhodamine,Rhodamine
labeled phalloidin.
 A fluorescent microscope has an exciter filter between the light source and condenser lens
that transmits only ultraviolet light .
 The condenser lens the focuses the uv light on the specimen, causing the fluorescent
compounds in specimen to emit visible light .
 Both UV light from the illumination and visible gathered by fluorescent compound in
specimen then pass through the objective lens .
 As the light passes through the objective lens , then it hits the barrier filter that
specifically removes the UV light .
 This lenses only of visible light to form the final image, which is therefore appears
 bright against dark background.
24
 Fluorescent dyes can be used to gain insight into functions and metabolism of specimen
understudy.
 Antibody molecule can be labeled or tagged with fluorescent dyes, there by making it
possible to visualize the Ab molecules with fluorescent microscope.
 When Ab recognize and bind to appropriate Ag molecules within the cell, they in effect
label the Ag molecules with fluorescent “tag” that will cause that region of the cell to
“light up” when illuminated with UV light .
PARTS OF FLUORESCENCE MICROSCOPE:
The fluorescent microscope is differs from light microscope as with the components of
light source, heat filter, exciter filter, condenser and barrier filter. But the principle of
magnification and resolution of fluorescent microscopy is analogous to the ordinary light
microscope.
LIGHT SOURCE:
This is provided by a bright mercury vapor are lamp. As against the normal
incandescent bulb, this lamp provides shorter wave length light rays such UV, blue and violet.
Mercury vapor lamps produce light rays in a range of 200 - 400 mm and visible rays in
the range of above 780nm.
Mercury vapor lamps are not only expensive but also harmful. Hence several
precautions are necessary.
HEAT FILTER:
Infrared rays produced by the lamp generate considerable heat, besides they are of no use
in fluorescence. In order to eliminate these rays a heat filter is placed in front of the lamp and
before the condenser to absorb heat. Heat filter however doesn't prevent the transmission of UV
and other
visible rays.
EXCITER FILTER:
The light cooled down by the heat filter next passes through the exciter filter which
absorbs all but short waves that are needed to excite the fluorochrome due coated specimen on
the slide. These filters which are dark allow only green, blue, violet or UV rays.
CONDENSER:
For best results always a dark field condenses is used, because in a dark background
even mild fluorescence can be easily detected. Another advantage of this condenser is that it
deflects majority of the UV rays thus protecting the observer's eyes. In order to achieve this the
NA of the objective is always kept at 0.05 less than that of the condenser.
25
BARRIER FILTER:
This is situated in the body tube of the microscope between the objectives and the eye
piece to remove all remnants of the exciting light so that only the fluorescence is seen. When
excitation is by UV, the exciter filters are dark and barrier filters will be almost colorless. On
the other hand, if blue excitation is used, the barrier filters are either yellow or deep orange in
color.
EXPLAIN ABOUT PHASE CONTRAST MICROSCOPE?
(Section- C)
Introduction :

It was developed by Zernike in 1993. It is a method for controlling the contrast in image,
so that
unstained living cells and cytological details within them becomes visible.

Fixing & staining makes a difference in size and also leads to the shrinkage of bacterial
cell. Unstained microorganisms show sharp outline under this microscope.

Accurate measurement of living cells is also made possible. A fixed stained cell can show
the structure of the cell at one instance of time as it is killed.

But phase contrast microscope can reveal the natural changes that occur in a single cell
over a period of hours or days.

Chromosome movement during cell division and cytoplasmic streaming have been
captured by attaching a camera to the phase contrast microscope.
(Diagram)
PRINCIPLE :

Objects in the microscopic field can be categorised into two : 1. Amplitude objects and 2.
Phase objects.

Amplitude objects show up as dark object under the microscopic view because of the
reduction in the intensity of the rays that pass through phase objects which are transparent
allow the light rays to pass through without any reduction in the intensity.

But when the light rays are passing through an transparent object, some rays will have
retardation by about one quarter wavelength.

This retardation is called phase – shift. The one-fourth wavelength phase shift is utilized
in phase – contrast microscope to create image contrast.

Light rays passing through an transparent object emerge out in two different ways –
Direct and diffracted.

The rays passing through the object in a straight line are called direct rays.
26

The rays that are bent and slowed down as they pass through due to the differences in
density of the medium are called as diffracted rays..

If the direct rays and diffracted rays of an object can be brought into the same phase with
each other, the resultant increases in amplitude is due to the sum of both the converged
rays is coincidence. So, the objective look very bright in the field.

If the direct and diffracted rays are out of phase is the reverse phase & the object looks
dark. This is interference.

The basic construction of the phase contrast microscope is like a bright field microscope
except for two special attachment – 1. Special type condenser and 2. phase – plate.

The condenser has a special diaphragm consisting of an annular stop – It allows only a
hollow cone of light to pass through a condenser and bright the object on the slide.

The phase plate is a special optical disc located in the focal plane of the objective.

It has a special phase ring coated with the material that can either advance or retards the
direct rays depending on its construction.

The rays of light (direct rays) that emerge from the object as solid lines converge on the
phase ring within the objective.

Here depending on the coated material, the desired phase shift is produced.

The diffracted rays that pass through the object on the slide miss the phase ring & are not
affected by phase plate.

The resultant image will depend on whether the diffracted and direct rays are allowed to
converge or interfere.

If they converge, it will result in a bright image against a dark background (Bright phase
microscope) or if they interfere, it will result in dark image against a bright background
(Dark phase microscope).

If any specimen or cell, there will be difference in thickness between the structures or
components.

When the light rays pass through these, there will be variable refraction of the rays and
these phase changes are converted into visible difference of light intensity.

Even some structures which are difficult to stain & observe under light microscope
becomes visible in a phase contrast microscope because small phase changes results in
interference of light waves resulting in high contrast images.
EXPLAIN ABOUT ELECTRON MICROSCOPE
INTRODUCTION:
27
(Section-C)
In electron microscope , the illumination source is a beam of electrons emiited by a heated
tungsten filament.
The lens system consists of series electromagnets.
The electron beams is focused either on a fluorescent screen of zinc sulphide for direct
visualization of image or on a photographic film.
ELECTRON MICROSCOPE IS OF 3 BASIC DESIGNS:
1. Scanning electron microscope.
2. Transmission electron microscope
3. Scanning tunneling electron microscope
TRANSMISSION ELECTRON MICROSCOPE:
 It was the first type of electron microscope.
 It was developed by max knoll and Ernst Ruska in Germany inm 1931.
 TEM works much like a slide projector.
 Specimen for tem is prepared by shadow casting technique .this will give three
dimension structure.
 Structures appears after types of microscopy are called artifacts.
THE VACUUM SYSTEM:
 Because electrons cannot travel very far in air, a strong vacuum must be maintained along
the entire path of electron beam.
 A standard rotary pump and oil diffusion pump are used.
 Function of rotary pump is to achieve low vacuum when the instrument is first started up.
 An oil diffusion pump achieves high vacuum for operation.
 It is oil filled reservoir in which oil is vaporized by heating.
 The oil pump rises it traps air molecules and than condensed by vanes which are cooled
by circulating cold water.
 Cold finger, a metal inserts in the column of the microscope attracts the gases and random
contaminating molecules, which then solidify the cold metal surface.
 The vacuum3 is maintained at about 1x10-4 mmhg.
ELECTRON GUN
 The electron beam in TEM is generated by electron gun.
 The cathode a tungsten filament similar to light bulb, it emits electron when
heated.
 The cathode tip is near a circular opening in a metal housing called as wehnelt
cylinder.
28
 A negative voltage on the cylinder helps to control electron emission and shape
the beam.
 At one end of the cylinder is the anode, the anode is kept at 0 v, while cathode is
maintained at 50-100kv.
 The difference in voltage is called as accelerating voltage; it causes the electrons
to accelerate as they pass through the cylinder.
ELECTROMAGENTIC LENSES AND IMAGE FORMATION
 Image formation depends on both the wave like and particle like properties of the
electrons.
 Trajectory of electron can be controlled by using electromagnets.
 As electrons are charged, they are subject to magnetic forces when they move.
 The principle is used for the change in direction of electron beam.
 After passing through eh condenser lens system, electron beam enters the lenses made of
electromagnets.
 The lens itself is simply a space influenced by an electromagnetic field.
 The focal length of each les can be increased or decreased by varying the current applied
to its energizing coils. The use of condenser lens system with 2 lenses to achieve better
focus of electron beam.
 Specimen is positions on the specimen stage within the objective lens.
 The objective lens is concern with intermediate lens and projector lens, produce final
image on a viewing screen of ZnS that fluoresces when struck by electron beam.
PHOTOGRAPHIC SYSTEM
Using fluorescent screen can see the images and the image can be recorded by using electron
micrograph, which then becomes a permanent photographic record of the specimen. Most TEM
have camera chamber placed below the viewing screen.
SCANNING ELECTRON MICROSCOPY
INTRODUCTION
SEM generates an image by scanning the specimen with a beam of electrons.
PARTS OF SEM
 The vacuum system and electron are similar to that of TEM, but the accelerating voltage
is much lower (about 5-30kv).
 In SEM, the magnetic lens system focuses the beam of electrons in to a intense spot on
the surface of the specimens spot is moved back and for the across the specimen by
charged plates called beam deflectors located between the condenser lens and specimen.
29
 The beam reflector attracts or repels the beam according to the signals sent tot hem by the
deflector circuiting. When the electron beam hits the specimen, the molecule sin the
specimen gets excited to high energy level and emit se3condary electrons.
 These secondary electrons are used to form the final image of the specimen surface.
 The essential part of the detector is a scintillator, which emits photons of light when
excited by electrons incident upon it.
 The photons are used to generate an electronic signal to a video screen.
 The image then develop point by point, line by line on the screen as primary electron
beam sweeps over the specimen.
 A polarize camera can be used directly by photographing the video screen.
APPLICATIONS:
a. Viewing of ultra structure of the cell organelles is made possible.
b. Structural details of large biomolecules such as proteins can be studied.
Minute details of the topographic architecture is possible which is of systematic value as in the
case of pollen gains
LIMITATIONS:
Even though electron microscope is a great advancement over the light microscope in
terms of higher magnification and better resolution, there are certain limitations and light
microscopes have their own useful role to play in the microbiological laboratory. The limitations
imposed by electron microscope are:
1. Hydrated specimens are not to be used, hence living specimens cannot be observed.
2. Dehydration even if carefully done will cause some distortion.
3. Artifacts may appear.
4. As electrons have a low penetration power extremely thin sections are necessary.
SCANNING TUNNELING ELECTRON MICROSCOPE (STEM)
 Contains elements of both TEM & SEM.
 Like SEM, STEM uses an electron beam that sweeps over the specimen.
 But the electrons transmitted through the specimen as with the TEM then form the
image.
 STEM is technically sophisticated and electronically complex.
USES:
30
STEM is capable of distinguishing specific characteristic a of the electrons that are transmitted
by the specimens, thus deriving information about the specimen not obtainable with a
conventional TEM.
DISADVANTAGE:
 Technically sophisticated.
 Requires very high vacuum.
 Much more electronically complex than SEM or TEM.
DIFFERENCE BETWEEN LIGHT AND ELECTRON MICROSCOPES
CHARACTER
LIGHT MICROSCOPE
ELECTRON
MICROSCOPE
Highest practical
About 1000 – 15000
Over 100,000
magnification
Best resolution
0.2m
0.5m
Radiation source
Visible light
Electron beam
Medium of travel
Air
High vacuum
Types of lens
Glass
Electromagnet
Source of contrast
Differential light adsorption
Scattering of electron
Focusing mechanism
Adjust lens portion
Adjust current to the
mechanically
magnetic lens
Switch the objective lens
Adjust current to the
Method of changing
magnification
Specimen mount
magnetic lens
Glass slide
31
Metal grid usually copper
DARKFIELD MICROSCOPE
OIL IMMERSION LENS
32
PHASE CONTRAST MICROSCOPE
33
FLOURESCENT MICROSCOPE
34
FLUORESCENT MICROSCOPE
35
ELECTRON MICROSCOPE
36
STAIN AND STAINING METHODS.
INTRODUCTION:
 The colouring agents impart a colour to the colorless microorganisms. Due to this
coloration the microorganisms become visible, so as to observe the cell shape and
structures.
 The dyes which are used for biological purpose are called “stains”. These stains are
composed of a positive and negative ion, one of which is coloured is known as
chromophore.
 Dyes are divided into three classes based on their charged group.
Basic dyes – Methylene blue, basic fuchsin, crystal violet, safranine, malachite green are cationic
or positively charged groups. So, basic dyes bind to negatively charged molecules like nucleic
acids and many proteins. Because the surfaces of bacterial cells are also negatively charged, basic
dyes are most often used.
Acidic dyes – Eosin, acid fuchsin – anionic or negatively charged cell structures such as capsule
etc.
37
Neutral dyes- are formed by mixing together aqueous solute of concentratic acidic and basic
dyes. The coloring matter in neutral strains is present in both negatively and positive under the
microscope.
AIMS OF STAINING:
 Bacteria are semitransparent hence it is difficult to see them in unstained state under
microscope except they are stained.
Stains are used:
 To render the microscopic and semi-transparent bacteria visible by producing colour
contrast.
 To reveal size and shape of bacteria – Morphology of bacteria.
 To study various internal & external structures of bacteria which help in classification and
identification of bacteria.
STEPS IN STAINING PROCESS:
Any staining procedure can be divided into three steps:
 Preparation of the smear.
 Fixation of smear
 Staining of the smear with one or more staining solutions.
PREPARATION OF THE SMEAR:

Smear is made on a clean, grease-free glass slide. Smear is a thin uniformly
spreaded film of the fluid material present on the specified area.

Alternatively, it can be prepared by placing a loopful of sterile distilled water on
the slide. Then the organism is emulsified and spreaded uniformly to make a thin
layer film. Allow the smear to air dry.
FIXATION OF SMEAR:

The physical agent heat is applied for the smear fixation. It is done by passing the slide
slowly through the flame, so that the slide becomes hot.

Physical or chemical agents which immobilize the organisms and their structures are
called fixatives and the process is “fixation”.

Fixation brings about the denaturation of proteins. Chemical agents such as acids,
alcohols, chromic acid, glacial acetic acid, chromic acid, picric acid, mercuric chloride
are employed as fixatives.

Fixation is an essential step before staining because it :
38

Kills the bacteria – safety against pathogens.

Increases the cell permeability to dyes.

Prevents the autolysis of cell.

Avoids the shrinkage of the cell.

Protects the structures that might be soluble in staining reagents.
STAINING OF THE SMEAR:
After fixation, the smear is stained according to the aim of the experiment.
Mordants are chemical agents which have the power of making dyes to stain materials which are
unstainable.
Decolouriser – if the cells are over stained, excessive amounts are removed by the use of
decolourisers. Alcohol or acid are the decolourisers used.
SIMPLE STAINING (MONOCHROME STAINING OR POSITIVE STAINING)
(Section – B)
INTRODUCTION:

The coloration of bacteria by applying a single staining solution to a fixed smear is
“simple staining”. It is called as “monochrome staining” because only one dye is used.

Basic dyes like methylene blue, crystal violet, safranine, malachite green are used to
determine the size, shape and arrangement of bacteria.
PROCEDURE:

Prepare the smear and heats fix it.

Flood the smear with methylene blue stain and allow it to react for 1 minute.

Wash the slide with distilled water, blot and air dry.

Examine the slide under microscope.
RESULT:
Organisms are stained blue in colour.
ADVANTAGES:

Simple staining provides morphological characteristics under examination.

The shape, size and cell arrangements of various bacteria can be studied.

Bacteria are difficult to be seen under microscope unless they are stained.

When organisms are stained with a particular stain they take up the stain and can be seen
more clearly when examined under microscope.

Bacteria are seen blue, violet or red depending upon the stain used against the colorless
background.
39
NEGATIVE STAINING (RELIEF STAINING) (Section – B)
INTRODUCTION:

It is a technique by which bacterial cells are not stained but
the background is stained with an acidic dye, leaving the cells
colorless.

Negative stains include Indian ink, Congo red, Eosin, etc.
This technique is used for those cells or structures that are
difficult to stain directly.

Acidic dye is negative charge, So, it does not combine with
the negatively charged bacterial cell surface.

As a result, it forms a deposit around the cell, resulting in
appearance of bacterial cell colorless against a dark
background.
PROCEDURE:

Place a drop of 5% nigrosine or Indian ink on a slide.

Mix it into a drop of 24 hours old bacterial cultural
suspension.

Spread the drop with the help of another slide and air
dried.

Examine under oil immersion objective lens.
RESULT:
Bacteria are seen as clear transparent – colorless objects on a dark-black background.
(Section – B)
DIFFERENTIAL STAINING:
INTRODUCTION:
Gram staining is a differential staining technique by which bacteria are classified as “Gram
positive or Gram negative” depending upon whether they retain or lose the primary stain –
crystal violet, when subjected to treatment with a decolorizing agent such as alcohol.
Christian Gram in 1884 discovered this technique.
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PRINCIPLE:
 The difference between Gram positive & Gram negative bacteria is based on the cell wall
composition.
 The cell wall of Gram – negative bacteria possess higher percentage of lipids (10 – 20%)
to that in the cell wall of G+ve bacteria (1 – 2 %).
 The peptidoglycan layer in cell wall of G+ve is thick than that of G-ve bacteria. The
smear is subjected to Crystal violet – Primary stain, Grams iodine – Mordant, Alcohol –
Decolourising agent and Safranine – Counter stain.
 The primary stain (CV) forms a complex with the mordant Gram’s iodine (CV-I
complex) in the cell.
 When the bacterial cells are treated with decolorizing agent – alcohol, lipids from the cell
wall of G-ve bacteria are extracted, this results in increased porosity of the cell wall.
 Therefore CV-I complex escapes the cell and thus G-ve organisms are decolorized.
 To render the colorless G-ve bacteria visible, counter stain – safranine is applied where
the cells take up the colour of safranine, hence appear red in colour.
 In contrast, the cell wall of G+ve bacteria because of their low lipid content becomes
dehydrated during treatment with alcohol.
 The cells shrinked in size results in decreased pore size and the CV-I complex cannot be
extracted but it is retained in the cell. Therefore, G+ve cells appear purple violet in
colour.
PROCEDURE:
 Prepare a smear from the given bacterial culture on a clean sterile glass slide & allow it to
air dry & fix it by heat treatment.
 Flood the smear with crystal violet for 2 minutes.
 Pour off the stain & wash the slide in distilled water.
 Flood the smear with Gram’s iodine solution for 30 – 60 seconds.
 Rinse the slide in distilled water.
 Apply 95% ethanol in a drop-wise manner until excess crystal violet comes out (During
depolarization, the slide is held in inclined position).
 Wash the slide in distilled water.
 Apply safranine & allow it to react for 2 mins.
 Rinse the slide in distilled water, drain, blot and air dry.
 Examine the smear under microscope.
RESULT:
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Gram-positive bacteria are stained violet in colour while Gram-negative bacteria are stained pink
in colour.
ACID – FAST STAINING (ZIEHL-NEELSEN METHOD):
(Section – B)
INTRODUCTION:
 Acid fast staining is widely used differential staining procedure for Mycobacteria. It
distinguishes acid-fast from non-acid fast bacteria.
 Acid-fast means certain bacteria retain the initial stain – carbol fuchsin & difficult to
decolorize with acid-alcohol.
PRINCIPLE:
 Acid-fastness is due to the presence of high lipid content (60%) in the cell wall which
binds with the fuchsin dye, so that it is not destained with acid-alcohol.
 The major lipids are mycolic acids, glycolipids and mycosides. The dye basic fuchsin is
more soluble in phenol than in water.
 Phenol and heat act as intensifiers. Once basic fuchsin has penetrated with the aid of heat
and phenol, acid – fast cells are not easily decolorized by an acid – alcohol wash & hence
remain red.
 Non-acid fast bacteria are decolorized by acid-alcohol & thus are stained blue bu
methylene blue, counter stain.
 This method is used to identify Mycobacterium tuberculosis and Mycobacterium leprae,
the pathogens responsible for tuberculosis & leprosy.
PROCEDURE:
 Prepare the smear on a clean glass slide & heat fix it.
 Flood the smear with carbol fuchsin & heat the stain-covered slide till steam rises for
about 5 minutes.
 Care should be taken to avoid evaporation of dyes.
 Cool the slide, rinse with water.
 Decolorize with 20% sulphuric acid, until no more stain appears.
 Wash the smear with water.
 Counter stain the smear with methylene blue for 1-2 minutes.
 Wash the smear with water and air dry.
 Examine under microscope.
RESULT:
Acid fast organisms are stained red, while the non-acid fast organisms are stained blue.
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(Section – B)
CYTOLOGICAL STAINING:
ENDOSPORE STAINING (SCHAEFFER-FULTON METHOD)
INTRODUCTION:

Endospores are thick walled spores formed in the bacterial cell. They are resistant to
physical and chemical agents.

Bacillus and Clostridium have the ability to form endospores.
PRINCIPLE:
 Spores are resistant to unfavorable conditions like excessive heat, freezing, radiation,
desiccations and chemical agents.
 Due to unfavorable conditions, spores are formed particularly with the exhaustion of
nutrients. Primary stain, malachite green is allowed to stain the spore under the
influence of physical intensifier like heat and chemical intensifier like phenol.
 Due to heat treatment, pores of spores become wider and stain can penetrate the spore.
So, vegetative cells take up the counter stain, safranine.
PROCEDURE:
 Smear was made on a clean glass slide & heat fixes it.
 Flood the smear with malachite green & heat the stain-covered slide till steam rises for
about 2-3 mins.
 Care should be taken to avoid evaporation of the stain.
 Cool the slide & wash the smear with water.
 Then the smear was flooded with the counter stain, safranine for 30 secs.
 The slide is washed with water & air dried & examined under the microscope.
RESULT:
Spores retain the primary stain & appear as green in colour whereas vegetative cell appear as red.
(Section – B)
CAPSULE STAINING:
INTRODUCTION:
 Capsule is an envelope or slime layer surrounding the cell wall of certain
microorganisms. It is a slimy, gummy or mucilaginous layer made up of
polysaccharides.
 Capsules appear to be amorphous gelatinous surrounding the cell.
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 A capsule is non-ionic & does not take up the dye.
PROCEDURE:
 Place a drop of 5% nigrosine or Indian ink on a slide.
 Mix it into a drop of 24 hours old bacterial cultural suspension.
 Spread the drop with the help of another slide and air dried.
 Examine under oil immersion objective lens.
RESULT:
Bacteria are stained red in colour. Capsules appear colourless against dark background.
CELL WALL STAINING (DYAR’S METHOD):
(Section – B)
INTRODUCTION:
1. Cell wall is a rigid structure which protects the cell against severe physical conditions.
Gram positive cell wall contains teichoic acid and teichuronic acid. Gram negative cell
wall contains lipoproteins & lipopolysaccharides.
2. Generally cell wall in bacteria is made up of peptidoglycan.
PRINCIPLE:
1. Cetyl pyridinium chloride (CPC) is used. As the cell wall is negatively charged, CPC
makes it positively charged surface.
2. Subsequent treatment with Congo red stains the cell wall. Cytoplasm is stained with
methylene blue.
PROCEDURE:
 Smear was prepared, dried & heat fixed.
 Flood the smear with CPC for one minute.
 Then cover the smear with Congo red & allow for 30 – 60 secs after the removal of CPC
solution.
 Wash in tap water & counter stain with methylene blue for 30 secs.Wash with water, blot
dry and examine under microscope.
RESULT: Cell wall appears red in colour while cytoplasm is blue in colour.
FLAGELLAR STAINING (LEIFSON’S METHOD)
(Section – B)
INTRODUCTION:

Bacterial flagella are thin, filamentous appendages on cells, responsible for swimming
motility (0.01 – 0.02 m in diameter).
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
The flagellum consists of three parts: a basal body – associated with cytoplasmic
membrane & cell wall, a short hook & a helical filament, the hook & filament are
composed of “flagellin” protein.
PRINCIPLE:
1. Leifson’s stain forms deposit on the surface of flagella thereby increases the
diameter of flagella which makes it visible.
2. The counter stain, methylene blue will stain the cell.
PROCEDURE:
 Clean the slide in alcohol, then with chromic acid solution & wash with distilled water.
 Before making smear, to the culture slant add sterile distilled water upto half inch above
the slant surface.
 Rotate the slant slowly & incubate at 37 degree C for 15 – 20 mins.
 After incubation, add one drop of the suspension to the slide. Tilt the slide & allow the
drop to run to the other end & remove the excess with the blotting paper.
 Allow the slide to air dry & do not heat fix it. Flood the smear with leifson’s stain & allow
it to act at room temperature for 5 – 15 mins.
 Rinse with tap water & counter stain with 1% methylene blue for about 1 minute. Wash in
tap water, allow to air dry & examine under microscope.
RESULT: Flagella are stained red & the vegetative cell appears blue in colour.
NUCLEAR MATERIAL STAINING (FUELGEN STAINING METHOD) (Section – B)
INTRODUCTION:
Nuclear material consists of a single, circular DNA molecule & RNA. It can be made visible
under the microscope by fuelgen’s staining which is specific for DNA.
PRINCIPLE:
 In fuelgen reaction, the hydrolysis with the acid releases purine from DNA.
 The freeing of sugars from purines cause a shift from furanose to aldehyde.
 This aldehyde then forms addition compound with leuco para rosaniline of the schiff’s
reagent.
PROCEDURE:
 Prepare a thick smear & fix it with bouin’s fixative for 15 mins. Wash with distilled
water.
 Place the slide in 1N HCL at 60 degree C. The slide is removed at an interval of 1 min
after 6 mins till 12 mins.
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 Rinse it with distilled water. Stain with Schiff’s reagent for 30 – 45 mins. Drain off the
stain & treat with potassium metabisulphite solution for 2 – 6 mins.
 Rinse in distilled water, dry & examine.
RESULT: Nuclear material appear as pinkish purple while cytoplasm is colorless.
METACHROMATIC GRANULE STAINING (ALBERT’S METHOD): (Section – B)
INTRODUCTION:
1. Metachromatic granules are intracellular granules of polyphosphates found in certain
organisms. These are also known as volutin granules or babes – Ernst granules.
2. Metachromatism means the change of colour. Granules appear to be of colour different
from that of the applied dye.
3. It is due to change in concentration of dye resulting into shift in absorption maxima.
PRINCIPLE:
1. Albert’s stain contains toludine blue – O & malachite green. Dyes have good affinity for
the substrate with lower pH than the pH of the staining solution.
2. pH of Albert’s stain is 2.8 which are basic to volutin granules. Toludine blue – O stains
volutin granules while malachite green stains the cytoplasm.
PROCEDURE:
1. Prepare a thin smear & heat fixes it. Flood the film with Albert’s stain for 5 mins. Drain
the slide & dry.
2. Apply Lugol’s iodine for 1 min. Rinse the slide with water, drain, blot, air dry & examine
under microscope.
RESULT :Granules appear bluish black & Cytoplasm appear green in colour.
INTRACELLULAR LIPID STAINING (BURDON’S METHOD): (Section – B)
Introduction:
 Lipid is an important constituent of the cell found to be present in cell wall & cell
membrane. They are made up of PHB (Poly -  - hydroxy butyric acid).
 PHB is a polymer of fatty acid. Fat globules are known as sudanophilic granules & are
found mainly in old cultures.
 Bacillus cereus produces large sudanophilic bodies & they are the reserve source of
carbon.
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Principle: Fat globules are not stained by water- soluble dye but by fat-soluble dye. Stain moves
into lipid material of cell & retained there.
Procedure:
 Prepare a smear & heat fixes it. Flood the slide with Sudan black – B stain for 10 – 15
mins. Do not wash with water.
 Rinse with xylene & blot dry. Counter stain with safranine for 10 secs. Rinse with water,
blot dry & examine under microscope.
Result: Lipid granules appear deep blue against light pink cytoplasm.
POLYSACCHARIDE STAINING (HOTCHKISS METHOD):
(Section – B)
Introduction:
Polysaccharide granules are present in the cytoplasm & they act as reserve source of food.
Principle:

Polysaccharide is oxidized with periodate to form polyaldehyde which reacts with schiff’s
fuchsin sulphite to give rise to red colour.

Protein & nucleic acids remain colourless.
Procedure:
 Prepare a smear & heat fixes it. Flood with periodate solution for 5 mins. Rinse with 70%
ethyl alcohol.
 Cover the smear with reducing rinse solution for 5 mins. Rinse with 70% ethyl alcohol.
 Stain with fuchsin-sulphite solution for 15-45 mins.
 Wash with sulphite wash solution & then with water.
 Counter stain with malachite green for few seconds.
 Wash, dry & observe under microscope.
Result: Polysaccharides stains red colour, while cell is in green colour.
SPIROCHAETE STAINING (FONTANA’S SILVER IMPREGNATION METHOD)
(Section – B)
Introduction:
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 Spirochaetes are helical & flexible bacteria. They exhibit swimming motility due to
periplasmic flagella & they are responsible for motility.
 On solid surfaces, Spirochaetes exhibit creeping or crawling motility. Spirochaetes are so
thin that they cannot be easily seen by light microscopy even when gram stained.
Principle:
Fontana’s fixative & mordant’s are used before stains were used. On the application of
ammonical silver nitrate & heating, silver oxide is formed which form precipitate on the
organism, thereby the apparent diameter of organism is increased & organisms become visible.
Procedure:

Prepare the smear & air dry but do not heat fix it. Fix the smear using Fontana’s fixative.

Rinse the smear with ethanol to wash off the fixative. Flood the smear with Fontana’s
mordant & heat the slide until steam rises & allow it to act for 30 secs.

Wash well in distilled water & dry the slide. Cover the smear with Fontana’s stain & heat
the slide until steam rises & the smear becomes brown in colour.

Wash well in distilled water, air dry & examine under the microscope.
Result: Spirochaetes are stained brownish – black on a brownish yellow background.
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