Microbiology: Study of organisms and agents that can’t be seen from naked eyes.

It includes
viruses, bacteria, certain algae and fungi & protozoa.

Large bacteria (visible without microscope): Thiomargarita and Epulopiscium

Discovery of Microbial World

Lucretius (98-55 BC) & Girolamo Fracastoro (1478-1533): Disease is caused by invisible living
creature.

Francesco Stelluti (1625-1630): Microscopic observation on bees and weevils.

Antony Van Leeuwenhoek (1632-1723): Observing and describing microorganisms. He

constructed a simple microscope consisting double convex lens held between two silver plates
with a magnification between 50-300 times. In 1673, detailed letter was sent to Royal Society of
London. He saw bacteria and protozoa.
Landmark/Discoveries in the Field of Microbiology

This Table is about the various landmark discoveries which took place in the field of
microbiology.

Year Person behind discovery Landmark discoveries

1546 Fracastoro Invisible organisms are responsible for disease (suggestion)
1590-1608 Jansen Development of first compound microscope
1676 Leeuwenhoek Animalcules
1688 Redi Work on spontaneous generation of maggots was
published
1765-1776 Spallanzani Attacked on theory of spontaneous generation
1786 Muller First classification of bacteria
1798 Jenner Cowpox vaccination for small pox
1838-1839 Schwann and Schleiden The cell theory
1835-1844 Bassi Silkworm disease is caused by a fungus.
Proposed that many diseases are microbial in origin.
1847-1850 Semmelweis Childbed fever is transmitted by physicians.
The use of antiseptics to prevent the disease.
1849 Snow The epidemiology of a cholera epidemic in London.
1857 Pasteur Lactic acid fermentation is due to microorganism.
1858 Virchow All cells come from cells
1861 Pasteur Microorganisms do not arise by spontaneous generation.
1867 Lister Antiseptic surgery work published
1869 Miescher Discovery of nucleic acids
1876-1877 Koch Anthrax is caused by Bacillus anthracis
1880 Laveran Discovery of Plasmodium (causes malaria)
1881 Koch Cultures bacteria on gelatin
Pasteur Developed anthrax vaccine
1882 Koch Discovered tubercle bacillus, Mycobacterium tuberculosis
1884 Koch Koch’s postulates were first published
Metchnikoff Phagocytosis was described
- Autoclave was developed
- Gram staining was developed
1885 Pasteur Develops vaccine for rabies
Escherich Discovered Escherichia coli (causes diarrhea)
1886 Fraenkel Discovers Streptococcus pneumonia (causes pneumonia)
1887 Richard Petri Developed petri dish (plate)
1887-1890 Winogradsky Study of sulfur and nitrifying bacteria
1889 Beijerinck Isolation of root nodule bacteria
1890 Von Behring Prepared antitoxins for diphtheria and tetanus
1892 Ivanowsky Provided evidence for virus causation of tobacco mosaic
disease
1894 Kitasato & Yersin Discovers Yersinia pestis (causesplague)
1895 Bordet Discovers complement
1896 Van Ermengem Discovers Clostridium botulinum (causes botulism)
1897 Ross Malaria parasite is carried by mosquito
Buchner Prepared extract from yeast that ferments
1899 Beijerinck Proved that virus particle causes the tobacco mosaic disease
1900 Reed Proved that yellow fever is transmitted by mosquito
1902 Landsteiner Discovery of blood groups
1903 Wright and others Discovered antibodies in the blood of immunized animals
1905 Schaudinn & Hoffmann Treponema pallidum causes syphilis
1906 Wassermann Developed complement fixation test for syphilis
1909 Ricketts Rocky mountain spotted fever is transmitted by ticks and
caused by Rickettsia rickettsii
1910 Ehrlich Developed chemotherapeutic agent for syphilis
1911 Rous Discovered virus that causes cancer in chickens
1915-1917 D’Herelle & Twort Discovered bacterial viruses.
1921 Fleming Discovered lysozyme
1923 - First edition of Bergey’s manual
1928 Griffith Discovered bacterial transformation
1929 Fleming Discovered penicillin
1931 Van Niel Photosynthetic bacteria use reduced compounds as electron
donors without producing oxygen
1933 Ruska Developed first transmission electron microscope
1935 Stanley Crystallized the tobacco mosaic virus
1937 Chatton Division of living organisms into prokaryotes and eukaryotes
1941 Beadle & Tatum One –gene-one-enzyme hypothesis was proposed
1944 Avery DNA carries information during transformation
Waksmann Discovered streptomycin
1946 Lederberg & Tatum Bacterial conjugation
1949 Enders, Weller & Robbins Growing poliovirus in human tissue cultures
1950 Lwoff Induced lysogenic bacteriophages
1952 Hershey and Chase Bacteriophages inject DNA into host cells
Zinder & Lederberg Discovered generalized transduction
1953 - Phase-contrast microscope was developed
Medawar Discovered immune tolerance
1955 Jacob & Wollman Discovered that the F factor is a plasmid
Jerne & Burnet Proposed the clonal selection theory
1959 Yalow Developed the radioimmunoassay technique
1961 Jacob & Monod Proposed the operon model of gene regulation
1961-1966 Nirenberg, Khorana & Described the genetic code
others
1962 Porter Proposed the basic structure for immunoglobulin G
- First quinolone antimicrobial (nalidixic acid) was synthesized
1970 Arber & Smith Discovery of restriction endonucleases
Temin & Baltimore Discovery of reverse transcriptase in retroviruses
1973 Ames Developed bacterial assay for the detection of mutagens
Cohen, Boyer, Chang & Used plasmid vectors to clone genes in bacteria
Helling
1975 Kohler & Milstein Developed technique for the production of monoclonal
antibodies
- Lyme disease was discovered
1977 Woese & Fox Recognition of archaea as a distinct microbial group
Gilbert & Sanger Developed technique for DNA sequencing
1979 - Insulin synthesized using recombinant DNA techniques
- Small declared officially eliminated
1980 - Development of the scanning tunneling microscope
1982 - Recombinant hepatitis B vaccine developed
1982-1983 Cech & Altman Discovery of catalytic RNA
1983-1984 Gallo & Montagnier HIV was identified and isolated
Mullis Polymerase chain reaction was developed
1986 - First vaccine (hepatitis B vaccine) produced by genetic
engineering approved for human use
1990 - First human gene-therapy testing begun
1992 - First human trials of antisense therapy
1995 - Chickenpox vaccine approved for US use
- Haemophilus influenzae genome sequenced
1996 - Methanococcus jannaschii genome sequenced
- Yeast genome sequenced
1997 - Discovery of Thiomargarita namibiensis (largest known
bacterium)
- Escherichia coli genome sequenced
2000 - Discovery that Vibrio cholera has two separate chromosomes
 Controversy over spontaneous generation
Theory of spontaneous generation: Living organisms could develop from non-
living matter
Aristotle (384-322BC): Thought some simpler invertebrates could arise by spontaneous
generation.

Francesco Redi (1626-1697):

 Challenged Aristotle’s view and theory of SG.

 Carried out experiments on decaying meat and its ability to produce maggots
spontaneously. Placed meat in 3 containers.

1st container (open): Flies laid their eggs on uncovered meat and maggots developed.

2nd container (covered with paper): Maggots not produced spontaneously on decaying meat.

3rd container (covered with fine gauze): Maggots not produced spontaneously. Flies laid eggs on
the gauze, these eggs produced maggots.

“The generation of maggots by decaying meat resulted from the presence of fly eggs, and meat
did not spontaneously generate maggots as previously believed”

John Needham (1713-1781):

 In 1748, boiled mutton broth and then tightly stoppered the flasks.
 Many of the flasks became cloudy and contained microorganisms.
 Organic matter contained a vital force that could confer the properties of life on nonliving
matter.

Lazzaro Spallanzani (1729-1799):

 Improved Needham’s experiment design by first sealing glass flasks that contained water
and seeds.
 Sealed flasks were kept in boiling water for ¾ hour. No growth occurred until the flasks
were sealed.
 Proposed that “air carried germs to culture medium but external air might be required for
growth of animals already in the medium”.

The supporters of spontaneous generation maintained that heating the air in sealed flasks
destroyed its ability to support life.
Theodore Schwann (1810-1882):

 Air passed through a red-hot tube to enter a flask containing sterile nutrient solution.
 The flask remained sterile.

Georg Friedrich Schroder and Theodor von Dusch:

 Air was passed through sterile cotton wool to enter a flask of heat-sterilized medium.
 No growth was seen in medium even the air was not heated.

Felix Pouchet claimed in 1859 to have carried out experiments conclusively proving that
microbial growth could occur without air contamination.

Louis Pasteur (1822-1895):

 First, he filtered air with cotton and found that objects resembling plant spores had been
trapped. If the cotton was placed in sterile medium, microbial growth occurred.
 Then he placed nutrient solutions in flasks, heated their
necks in flame to make variety of curves while keeping
the neck open.
 Boiled solution and allowed to cool.
 No growth even the content is exposed to air
 No growth occurred because dust and germs had been
trapped on the walls of curved necks.
 If necks were broken, growth occurred immediately.
 Resolved controversy by 1861 and explained how to
keep medium sterile.
 This setup is also referred as swan neck flask experiment.

John Tyndall (1820-1893):

 Dust carried germs and if it was absent, broth remained sterile even after direct exposure.
 He provided evidence for the existence of exceptionally heat-resistant forms of bacteria.
 Role of Microorganisms in the Causation of disease
It took years to establish connection between m/o and illness. Once it was clear, microbiologist
explored the ways in which hosts defended themselves against m/o and how illness can be
prevented “IMMUNOLOGY was born”.

Recognition of relationship between microorganisms and disease

Agostino Bassi showed that microorganisms could cause disease. In 1835, he demonstrated that
silkworm disease was due to fungal infection. He also suggested many diseases were due to
microbial infection.

M. J. Berkeley in 1845 proved that the great Potato Blight of Ireland was caused by fungus.

Pasteur investigated perbrine disease of silkworms and showed that it was due to protozoan
parasite. Disease was controlled by raising caterpillars from eggs produced by healthy moths.

Joseph Lister in 1867 developed a system for antiseptic surgery to prevent microbial entry in
wounds. Instruments were heat sterilized, and phenol was used on surgical dressing and sprayed
over surgical area. It also provided strong indirect evidence for the role of microorganisms in
disease because phenol, which killed bacteria, also prevented wound infections.

Robert Koch (First direct demonstration of the role of bacteria in disease):

Study of anthrax and Bacillus anthracis (1876).
Injected healthy mice with material from diseased animals → Mice become ill.
Transferred anthrax by inoculating through a series of 20 mice, he incubated spleen containing
anthrax bacillus in beef serum. The bacilli grew, reproduced and produced spores. When bacilli
or spores injected in mice → anthrax developed.
Koch’s postulates
1. The microorganism must be present in every case of the disease but absent from healthy
organisms.
2. The suspected microorganism must be isolated and grown in a pure culture.
3. The same disease must result when the isolated microorganism is inoculated into a
healthy host.
4. The same microorganism must be isolated again from the diseased host.

Koch’s proof that Bacillus anthracis caused anthrax was independently confirmed by Pasteur
and his coworkers. They discovered that after burial of dead animals, anthrax spores survived
and were brought to the surface by earthworms. Healthy animals then ingested the spores and
became ill.

Molecular Koch’s postulates:

It was published in 1884 on the cause of tuberculosis.

It was not always possible to apply Koch’s Postulates in studying human diseases. Ex. Some
pathogens can’t grow outside host and hence these require human studies. The identification,
isolation and cloning of genes responsible for pathogenic virulence have made possible a new
Molecular form of Koch’s postulates that resolve some of these difficulties. The focus is on
virulence gene irrespective to agent. The molecular postulates can be described as:

1. The virulence trait under study should be associated much more with pathogenic strains
of the species than with nonpathogenic strains.
2. Inactivation of the gene or genes associated with the suspected virulence trait should
substantially decrease pathogenicity.
3. Replacement of the mutated gene with the normal wild-type gene should fully restore
pathogenicity.
4. The gene should be expressed at some point during the infection and disease process.
5. Antibodies or immune system cells directed against the gene products should protect the
host.

Development of techniques for studying microbial Pathogens

Koch first isolated pathogenic bacteria on sterile surface of cut, boiled potatoes →
Unsatisfactory (Bacteria would not grow well)

Then Koch solidified regular liquid media with gelatin → Separate bacterial colonies developed
after streaking with bacteria sample (gelatin not ideal → bacteria digest gelatin and it melts
above 28°C)

Fannie Eilshemius Hesse (wif of Walther Hesse, Koch’s assistant) provided new alternative →
Agar as solidifying agent (she made jellies). Not attacked by bacteria and didn’t melts at 100°C.

Richard Petri → Petri dish (plate)

Koch developed → Nutrient broth, nutrient agar and media (for growing bacteria isolated from
body)

Koch isolated bacillus that cause tuberculosis (1882).

Charles Chamberland, Pasteur’s associate developed porcelain bacteria filter in 1884. The
discovery of virus and role in disease was made possible

Tobacco mosaic disease virus → First pathogenic virus study

Immunological studies
Determining how animals resisted diseases and developing techniques for protecting humans and
livestock against pathogens.

Pasteur and Roux (during chicken cholera disease) discovered that incubating their cultures for
long intervals between transfers would attenuate bacteria i.e., bacteria lost the ability to cause
disease. If chickens were injected with attenuated cultures they didn’t fall ill and developed
resistance to disease. He called attenuated culture “VACCINE”.

Pasteur and Roux developed attenuated anthax vaccine in two ways:

1. By treating cultures with potassium bichromate and

2. By incubating bacteria at 42-43°C.

Pasteur prepared rabies vaccine by different approach. The pathogen was attenuated by growing
it in rabbit. Infected rabbits died, their brain and spinal cord was removed and dried. Pasteur
injected the boy (rabid dog bite) 13 times over 10 days with increasingly virulent preparations of
the attenuated virus. Boy survived.

Diptheria bacillus produces a toxin. Emil von Behring and Shibasaburo Kitasato injected toxin in
rabbits, inducing them to produce antitoxin, a substance in blood that inactivate the toxin and
protect from disease. Tetanus antitoxin was prepared; both antitoxins were used in treatment.

The antitoxin work provided evidence that immunity could result from soluble substances in the
blood, now known to be antibodies (humoral immunity). It became clear that blood cells were
also important in immunity (cellular immunity) when Elie Metchnikoff discovered that some
blood leukocytes could engulf disease-causing bacteria. He called these cells phagocytes and the
process phagocytosis [Greek phagein, eating].

Role of Microorganisms in Transformation of organic matter

Theodore Schwann and others (1837): Proposed that yeast cells were responsible for
conversion of sugars to alcohol (alcoholic fermentation).

Some chemists argued that the microorganisms are not involved. Fermentation was due to
chemical instability.

Louis Pasteur: Fermentation is carried by living organisms and produced asymmetric products
such as amyl alcohol that had optical activity.
  In 1856, M. Bigo business produced ethanol from fermentation of beet sugars required
Pasteur’s assistance.
 Reduced yield of alcohol with sour taste.
 Discovered that microorganisms producing alcohol was replaced with those producing
lactic acid.
 All fermentations were due to specific yeasts and bacteria. Published several papers
between 1857 and 1860. This led to study wine diseases and development of
pasteurization for preservation of wine during storage.
 Some fermentative microorganisms were anaerobic and grow only in absence of oxygen.

Microbes are involved in carbon, nitrogen and sulfur cycle which takes place in soil and aquatic
habitats.

Sergei N. Winogradsky, Russian (Soil microbiology):

 Soil bacteria could oxidize Fe, S and NH3 to obtain energy

 Many bacteria could incorporate CO2 into organic matter much like photosynthetic
organisms do.
 Isolated anaerobic nitrogen fixing soil bacteria.
 Studied decomposition of cellulose.

Martinus W. Beijerinck (Microbial ecology):

 Isolated Azetobacter (aerobic nitrogen fixing bacteria)

 Isolated root nodule bacterium capable of fixing nitrogen (Rhizobium)
 Isolated sulfate reducing bacteria

Winogradsky & Beijerinck developed enrichment culture technique and use of selective media.