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Microbiology is the study of microorganisms /microbe which is visible only with a
microscope.The diverse group of organisms includes algae, archae, bacteria,cyanobacteria,
fungi, protozoa, viruses.Most of the microorganisms are harmless.99% are good. Eg:
Cynobacteria (blue green algae)1% are bad. Eg: Pathogens MICROBIOLOGY Discovery Era
Transition Era Golden Era Modern Era
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The term microbe is used by Sedillot in 1878. In 1676, he observed and described
microorganisms such as bacteria and protozoa as “Animalcules”. Father of Bacteriology and
protozoology. Descriptions of Protozoa, basic types of bacteria, yeasts and algae.3.
DISCOVERY ERA: “Spontaneous generation”Aristotle (384-322) and others believed that living
organisms could develop from non-living materials.In 13th century, Rogen Bacon described that
the disease caused by a minute “seed” or “germ”.Antony Van Leeuwenhoek (1632 – 1723)
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He showed that boiled broth would not give rise to microscopic forms of life. He
demonstrated that air carried germs to the culture medium. He covered the flasks with cork as
done by Redi, Still the microbes appeared on mutton broth.Lazzaro spallanzai (1729 – 1799) He
proposed that tiny organism(animalcules) arose spontaneously on the mutton gravy. Supporter
of the spontaneous generation theory. He showed that maggots would not arise from decaying
meat, when it is covered.John Needham (1713 – 1781)4. TRANSITION ERA:Francesco Redi
(1626 - 1697)
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Pasteur in 1897 suggested that mild heating at 62.8°C (145°F) for 30 minutes rather
than boiling was enough to destroy the undesirable organisms without ruining the taste of the
product, the process was called Pasteurization. He pointed that no growth took place in swan
neck shaped tubes because dust and germs had been trapped on the walls of the curved necks
but if the necks were broken off so that dust fell directly down into the flask, microbial growth
commenced immediately. He is the father of Medical Microbiology.5. GOLDEN ERA:Louis
Pasteur
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He developed live attenuated vaccine for the disease. He demonstrated that anthrax
was caused by bacteria and also produced the vaccine for the disease. He disproved the theory
of spontaneous germination. He coined the term “microbiology”, aerobic, anaerobic. Pasteur
demonstrated diseases of silkworm was due to a protozoan parasite.Contributions of Loius
2. pasteur: He invented the processes of pasteurization, fermentation and the development of
effective vaccines ( rabies and anthrax).6.
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He also devised a method to destroy microorganisms in the operation theatre by
spraying a fine mist of carbolic acid into the air. Lister concluded that wound infections too were
due to microorganisms. He is the father of antiseptic surgery. Prolonged boiling or intermittent
heating was necessary to kill these spores, to make the infusion completely sterilized, a process
known as Tyndallisation.Lord Joseph Lister (1827-1912) He discovered highly resistant
bacterial structure, later known as endospore.7. John Tyndall (1820 - 1893)
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Robert Koch used gelatin to prepare solid media but it was not an ideal because (i) Since
gelatin is a protein, it is digested by many bacteria capable of producing a proteolytic exoenzyme
gelatinase that hydrolyses the protein to amino acids. (ii) It melts when the temperature rises
above 25°C. He perfected the technique of isolating bacteria in pure culture. He
demonstrated the role of bacteria in causing disease.8. Robert Koch (1893-1910)
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9. Kochs postulates
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He developed the Petri dish (plate), a container used for solid culture media. Agar is
better than gelatin because of its higher melting pointing (96°c) and solidifying (40 –
45°c)points.Richard Petri (1887) It was not attacked by most bacteria. One of Kochs assistant
first proposed the use of agar in culture media.10. Fanne Eilshemius Hesse (1850 - 1934)
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He Studied toxins and antitoxins in quantitative terms He discovered the treatment of
syphilis by using arsenic He discovered the penicillin from penicillium notatum that destroy
several pathogenic bacteria.Paul Erlich (1920) He discovered the technique of
vaccination.Alexander Flemming First to prevent small pox.11. Edward Jenner (1749-1823) &
laid foundation of biological standardization.
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Edward Jenner - Vaccination for Smallpox. Twort and d’Herelle - Bacteriophages.
Charles Chamberland, one of Pasteur’s associates constructed a porcelain bacterial filter.
GoodPasteur - Cultivation of viruses on chick embryos. Pasteur developed Rabies vaccine.
Beijerinck (1898) - Coined the term Virus for filterable infectious agents. Roux and Yersin –
Diphtheria toxinViruses: Loeffler (1884) – Diphtheria bacillus Ogston (1881) –
3. Staphylococcus Neisser (1879) – Gonococcus Hansen (1874) – Leprosy bacllus12.
IMPORTANT DISCOVERIES: Bacteria:
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13. MODERN ERA:Nobel Laureates Years Nobel laureates Contribution 1901 Von
behring Dipth antitox 1902 Ronald Ross Malaria 1905 Robert koch Tb 1908 Metchnikoff
Phagocytosis 1945 Flemming Penicillin 1962 Watson,Crick Structur DNA 1968 Holley,Khorana
Genetic code 1997 Pruisner Prions 2002 Brenner, Hervitz Genetic regulation of organ
development &cell deathIngredients Grams/Litre
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EMB AGAR COMPOSITION:
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Peptone 10.0
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Lactose 10.0
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Dipotassiummonohydrogenphosphate 2.0
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Methylene Blue 0.065
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Eosine Y 0.4
Agar 15.0
4. Capsule staining:
Capsules may be visualized by methods including electron microscopy, phase-contrast microscopy,
and various staining techniques. Two staining techniques originally developed in the early 1900s will
be described in this protocol section: Anthony‘s capsule stain was developed by E. E. Anthony in
1931 (1) and Maneval’s staining method.
Purpose
The purpose of the capsule stain is to reveal the presence of the bacterial capsule. The water-soluble
capsule of some bacterial cells is often difficult to see by standard simple staining procedures or after the
Gram stain. Capsule staining methods were developed to visualize capsules and yield consistent and
reliable results.
PROTOCOL FOR ANTHONY'S CAPSULE STAIN
A. General materials
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Staining tray
· Staining rack
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Slide holder
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Disposable gloves
B. Staining reagents
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Crystal violet 1% solution (primary stain)
Copper sulfate 20% (decolorizer agent)
C. Procedure
1. Prepare a smear from a 12- to 18-hour culture grown in milk broth or litmus milk. (Serum protein may
be used to prepare the smear if the organism was not grown in milk broth or litmus milk.) This is to
provide a proteinaceous background for contrast.
2. Allow the smear to air dry. DO NOT HEAT FIX (to avoid destroying or distorting the capsule or causing
shrinkage).
3. Cover the slide with 1% crystal violet for 2 minutes.
4. Rinse gently with a 20% solution of copper sulfate.
5. Air dry the slide. DO NOT BLOT. (Blotting will remove the un-heat-fixed bacteria from the slide and/or
cause disruption of the capsule.)
6. Examine the slide under an oil immersion lens. Bacterial cells and the proteinaceous background will
appear purplish while the capsules will appear transparent.
5. HANGINING DROP METHOD:
Motility is an inheritable phenotype and is a useful criterion for identification and classification of bacteria.
Microscopic examination of live bacteria in wet mounts reveals whether the bacteria are motile or
nonmotile. Because unstained transparent cells are examined, more examination time is usually needed to
visualize and locate the cells than for stained preparations. This is particularly true because 400X rather
than 1,000X magnification is used to see bacterial cells in this type of preparation, and therefore
examination is critical. Due to these limitations, special techniques are used in order to prevent the wet
mount from drying during the time required for microscopic examination. The hanging drop technique is a
method in which a drop of bacterial suspension, preferably in mid-logarithmic phase, is enclosed in an airtight chamber prepared in a special depression slide (having a concave depression in the center) or
assembled from modelling clay (plasticine) which is a soft, malleable, and nonhardening material available
at toy or hobby stores.
Upon inoculating a batch of culture medium, bacterial cells go through successive phases starting with a
"lag" adaptation phase the length of which varies with type of organism and nature of the environment.
During this phase cells may experience shock due to the change in environment. In addition, if cells were
transferred from an old culture or from the refrigerator, their flagella may have deteriorated; therefore at
this stage of growth examination for bacterial motility is not recommended. Bacteria then start dividing at
the maximum rate, entering a "log' phase in which numbers increase logarithmically (exponentially).
Young actively dividing cells demonstrate the best motility at the log phase. Further incubation, over 8
hours, leads bacteria to enter a "stationary" phase of growth during which the number of bacterial
cells that die is almost equivalent to the number of newly formed cells. Also, at this stage it is not
recommended to examine cells as motility may be weak or considerably lost. Extended incubation in batch
cultures leads eventually to the death and possibly lysis of bacterial cells, a phase usually described as the
"decline" phase.
This original Flash animation (made with Macromedia Flash 5) illustrates two techniques used to carry out
microscopic motility testing in wet mounts and gives details of the two procedures and hints about some
observations made during microscopy.
This animation could serve as a useful tutorial aid in the microbiology laboratory for
undergraduates studying microbiology as well as for high school students. Page 1 of 1
Preparation of a Hanging Drop Slide
A hanging drop slide allows you to view live bacteria under microscope. This method allows organisms to
maintain their natural shape and makes it possible to observe their behavior, but keeps the organisms
well
contained.
If an organism is motile, its activity is clearly apparent, and directional, place-to-place locomotion will be
observed. Non-motile organisms can exhibit Brownian Movement, a vibration or oscillation caused by the
6. action of water molecules striking the microorganism; this must be distinguished from true locomotion.
Materials:
1. Depression slide and glass coverslip
2. Vaseline and toothpicks
3. Inoculating loop
4. Bunsen burner
5. Broth culture
7. Compound microscope
8. Tray of disinfectant
Procedure:
1. Place a coverslip on the desk and with a toothpick place a small dot of Vaseline in each corner of the
coverslip.
3. Sterilize an inoculating loop and obtain a loopful of the broth culture being used. Transfer a loopful of
culture to the center of the coverslip. Resterilize the inoculating loop.
4. Obtain a depression slide, invert it and gently press it against the coverslip so that the depression is
over
the drop of culture.
5. Invert the slide so that the drop may hang free from the coverslip into the space of the depression.
6. Microscopically examine the preparation.
NOTE: Focusing on live organisms can be tricky. Many microbes are nearly colorless, and will have little
contrast with their surroundings. Be sure to use low-intensity light, so that you do not dry out the drop.
Reducing light intensity is best accomplished in this case by closing down the iris diaphragm nearly all the
way; doing so will greatly increase the contrast within the preparation (simply turning down the light dial
7. isn’t as effective). Also focusing will be easier if you first locate the edge of the drop, which you should be
able to focus on. Then determine which side of the drop edge should have organisms on it, and attempt to
focus in that area when you go to higher power.
9. Once you have completed your observation, place the entire slide into the tray of disinfectant provided
on
the front table. Because these slides contain living organisms they must be autoclaved (steam and
pressure sterilized) before being cleaned.
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