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PART-A
1
DEFINITION AND HISTORY
OF MICROBIOLOGY
INTRODUCTION
Microbiology is the study of small organisms called microorganisms that are very small
to be seen by naked human eye. An object less than 0.1 mm (100 µm; 1 mm = 1000 µm)
diameter cannot be seen by the naked eye, whereas in the object less than 1 mm very little
detail can be observed. Thus, organisms with a diameter 1 mm or less are considered as
microorganisms and systematic study of these microorganisms comes under the branch of
microbiology and must be examined with a microscope.
Human society benefits from microorganisms in many number of ways. They are necessary
for various products that are required in daily human life. Microorganisms are considered to
be indispensable components of our entire ecosystem. They are considered to be the first
living organisms evolved on this earth which is 4.5 billion years old (Fig. 1.1). This is evident
from the stromatolites available in various fossil materials.
Even though, microorganisms are ubiquitous in its distribution but their identity was
found only during 17th century by using primitive microscopes. However, as branch of science,
microbiology has greater impact in 19th century by the work of meticulous scientists such as
Louis Pasteur and Robert Koch.
Today microorganisms are used extensively as research tools in divercified field from
medicine to waste management.
DEFINITION OF MICROBIOLOGY
Microbiology is thus concerned primarily with organisms and agents such as viruses,
bacteria, cyanobacteria, fungi, many algae, helminths and protozoa. The word microbiology
is derived from micros means small; bios means life; logos means study.
Microbiology often has been defined as a branch of science which deals with the living
organisms that are too small to be seen by unaided eye. These small organisms are called
microorganisms or microbes.
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The word ‘microbe’ was coined by Se’dillot, a French army retired surgeon in 1878. The
existence of these microbes was not known until the discovery of microscopes. Microscopes
are the optical instruments used to magnify objects that are too small to be clearly observed
by the naked eye.
Microscopes invented at the beginning of the seventeenth century opened the biological
world of the very small to the systematic scientific exploration. The early microscopes were
of two types. The first types were simple microscopes with a single lens of very short focal
length which was similar to magnifying lenses. The second types were compound microscopes
with two lenses made up of ocular and objective lenses. These compound microscopes were
able to magnify objects several thousand times and eventually replaced the conventional
simple microscopes. But, majority of the original discoveries were done by using the simple
microscopes by several microbiologists.
B illion yea rs
0
P h an erozoic
e ra
B illion yea rs
0
C a en ozoic era
0 .06
M eso zo ic e ra
0 .24
P a le ozoic era
0 .57
–1
la te
m etazoa
–2
P rotero zo ic e ra
0 .9
S ing le celle d
e ukaryo tes
m id dle
1 .6
A e rob ic re spiration
la te
–3
O xyg en ic
2 .9 p ho tosynthe sis
m id dle
A rcha e ic era
S ta b le O 2 -co ntaining
a tm osph ere (0 .0 1% )
e arly
2 .5
–4
In crea se o f O 2 co nte nt
to a bo ut 20 %
A n oxyge nic
3 .3
p ho tosynthe sis
e arly
No
a ccum u la tio n
o f ro cks
A n oxyge nic au to trop h y
3 .9 (m etha no ge ns a nd su lph u r
re sp iring o rga nism s)
B a nd ed iron -ston e
(fro m 2 .9 to 1.6 b illio n yea rs)
S ta rt of ph otosynth etic
O 2 p rod u ction
S ta rt of
C o rg a ccu m ulation
Stromatolites
4 .5
Fig. 1.1 Chronological history of the earth. (1 billion = 1000 million/10 9).
Earlier, living organisms were classified as kingdom Animalia (animals) and Plantae
(plants) by their differences in form and constitution (Fig. 1.2). The difference in the modes
of nutrition forms the key factor. The organisms which have the capacity to synthesize their
DEFINITION AND HISTORY OF MICROBIOLOGY
5
own food by using simple inorganic materials and sunlight as a source of energy are known
as phototrophs. The plants are best example for the phototrophs and are also known as
autotrophs. The organisms which feed on complex organic substances are called heterotrophs.
The animals are best example for the heterotrophs. But with the discovery of large number
of microorganisms, difficulty was faced with placing them either in plants or animals. Thus
a third kingdom Protista was proposed by Haeckel in 1866.
The kingdom Protista contains those organisms that are differentiated from plants and
animals by their absence of morphological specialization and most of them are unicellular in
nature. These Protista are further divided into higher protists and lower protists. Higher
protists are eukaryotes having resemblance with the animal and plants and include microalgae,
fungi and protozoa. Lower protists are prokaryotes and include bacteria and cyanobacteria
and their cellular structure is different from that of other living organisms. Microorganisms
are included in all these above groups and also consist of acellular particles such as viruses,
prions and viroids.
P lan ts
A n im a ls
E u ka ryotes
(E u cytes)
E u glen a
Fu ng i
M icro alga e
P ro to zo a
M icro org an ism s
E u ba cteria
A rch ae ba cteria
P ro karyotes
(P ro to cyte s)
Fig. 1.2 Different types of microorganisms.
The term “microorganisms” does not have the precise taxonomic importance because it
is not restricted to a particular living organism’s taxonomic group such as vertebrate,
invertebrate or angiosperm. Each of these groups defines a restricted group, all the members
of which share many common structural and functional properties. But, in the case of
microorganisms it is not the same. Any organism of microscopic dimensions, by the definition,
comes under the microorganisms category irrespective of its taxonomic position.
Microorganisms are present in all the four spheres of the environment viz., biosphere,
hydrosphere, geosphere, and atmosphere.
HISTORY OF MICROBIOLOGY
Even before the discovery of microorganisms certain microbial processes caused by their
living activities, such as fermentation of wine, milk, yeast and others were known to mankind.
In ancient times at the beginning of the human civilization, man by using these microbial
processes learned to prepare various food products such as sour milk, curd, cheese and other
products.
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The attempts made to learn the causes of infectious diseases led to the proposal of living
nature (contagium vivum) of contagious diseases. Even before microorganisms were seen,
some scientists proposed their existence and responsibility for disease. Roman philosopher
Lucretius (about 98–55 B.C.), Roger Bacon (1220–1292) and physician Girolamo Fracastoro
(1478–1553) suggested that disease was caused by invisible living creatures.
During the 17th century due to the success attained in the field of optics, the previously
unknown invisible mysterious world of minute organisms was discovered.
Glass polishers Hans and Zaccharius Jansen in 1590 constructed first a device with
magnifying glasses which allowed them to see minute objects. This started the era of microscopes.
In 1609–1610 Galileo Galilei (1564-1642) made the first simple microscope. In 1617–1619 the
first compound microscope with a convex objective and ocular lenses appeared. The inventor of
this microscope was thought to be the physicist C. Drebbel. This type of microscope was later
used to study the cells of plant and animal tissues and also minute organisms.
The remarkable and pioneer discovery was made by the Dutch cloth merchant Antony
van Leeuwenhoek (1632-1723). He himself made simple lenses which magnified 160 to 300
fold. In 1678, Antony van Leeuwenhoek published his work on ‘animalicula viva’, live
animalcules, which he observed in water, various infusions, faeces and teeth scrapings along
with the meticulous drawings.
The pioneer discovery made by Antony van Leeuwenhoek stimulated a live interest
among scientists. This also became the starting point for the systematic study of the microbial
world and formed the foundation stone for the science of new biological science called
microbiology. The optical device which Antony van Leeuwenhoek made formed the forerunner
for the modern world microscope. The animalicula viva (living animals), which Antony van
Leeuwenhoek observed, are now considered as bacteria and protozoa.
The development of microbiology as a branch of science is considered in the following
three era. The summary of some of the major events with respect to microbiology is represented
in Table 1.1.
Table 1.1 A few important events in the development of microbiology
Year
Microbiological Events
1546
1590
1676
1838
1857
1885
1892
1910
1915
1923
1929
1944
1953
1970
Fracastoro suggests that invisible organisms cause disease.
Jansen develops first useful compound microscope.
Leeuwenhoek discovers “animalcules”.
Schwann and Schleiden proposed cell theory.
Pasteur showed lactic acid fermentation by microbes.
Pasteur develops rabies vaccine.
Ivanovsky proved viruses causation of tobacco mosaic disease.
Ehrlich develops chemotherapy for syphilis.
D’Ilerelle and Twort discovered bacteriophages.
First edition of Bargey’s manual.
Fleming discovers penicillin.
Waksman discovers streptomycin.
Watson and Crick discovered DNA structure.
Arber and Smith discovered restriction endonucleases.
DEFINITION AND HISTORY OF MICROBIOLOGY
1983
1986
1995
1996
1997
1998
2000
2001
2002
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Gallo and Montagnier isolated and identified HIV.
First genetically engineered Hepatitis vaccine approved for human use.
Haemophilus influenzae genome sequenced.
Methanococcus jannaschii genome sequenced.
Escherichia coli genome sequenced.
Mycobacterium tuberculosis genome sequenced.
Vibrio cholerae genome sequenced.
Mycobacterium leprae genome sequenced.
Plasmodium falciparum genome sequenced.
ERA OF MICROBIAL DISCOVERY
Antony van Leeuwenhoek (1632–1723)
Antony van Leeuwenhoek (Fig. 1.3) was born to Dutch couple in 24th October, 1632
in Delft, Netherlands (earlier known as Holland). He had a formal primary education in
school in the Warmond town. During 1654 his family shifted back to Delft and he spent entire
life in the same city. He started his own business and started different hobbies. He was a
draper (a cloth merchant) and he sold clothes to the wealthy ladies of the city.
Antony van Leeuwenhoek was also a qualified surveyor, official wine taster of the city
and a minor city official. During 1666, he started grinding lenses as his hobby and constructed
his own simple microscopes. He used such simple microscopes for observing the weaving
pattern of various types of cloths. For the curiosity, he started observing the things surrounding
him. He was also inspired by the work of Robert Hooke and his illustrated book—Micrographia.
Later, he took up lense grinding work completely and started making more perfect lenses of
various types.
Fig. 1.3 Antony van Leeuwenhoek (1632-1723).
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During his entire life time Antony van Leeuwenhoek made nearly 500 microscopes of
different magnifications. He was so involved in grinding the lenses that he entirely gave up
the cloth business and took the lense making work as full time job. He designed the microscope
in such a way that all were simple but had highly powerful magnifying optical devices. When
compared to the present day microscopes they were very simple and around 4 inches in
length. It consisted of one lense (a), which was mounted in a small hole in the brass plate
that formed the body of the device (See Chapter 5 Fig. 5.3A). The specimen was placed at
the tip of blunt pin (b) attached to back plate. The pin tip was brought into focus by adjusting
the two screws (c) and (d). This device was held very close to eye during the observation. The
main disadvantage of his microscopes was that the magnifying power of each microscope
could not be increased.
Antony van Leeuwenhoek had great curiosity and by using his own microscopes he
observed variety of materials. He observed seeds, embryo, leaves, plant tissues, hair, insects’
eye, crystals, different types of fluids (rainwater, pond water, blood). He also used scrapings
from his teeth, tail fins of fishes for his observations.
Even though Antony van Leeuwenhoek did not have formal university education, he had
kept all his observations recorded. Each recorded observation was supported with the accurate
drawings. He made observation on blood and found tiny rounded objects which are considered
as red blood cells. Leeuwenhoek also observed yeasts and said that they were made of small
round particles.
Antony van Leeuwenhoek’s fame in the field of microbiology came from his discovery
and explanation of microbes which he called ‘animalcules’ (tiny animals). He observed them
in different sources such as pond water, stagnant water, rainwater, fruits and seeds infusion,
scrapings from his teeth and others. He was very much excited to see them living, showing
movement in the microscopic field. The different types of main unicellular microorganisms
that we know today—algae, bacteria, protozoa and yeasts, were first described by Antony van
Leeuwenhoek with great accuracy in the early years of 1676.
Antony van Leeuwenhoek started communicating his observations to the Royal Society
of England, which was established for the communication and publication of the scientific
work. In the first letter of 7th September 1674, he gave explanation to the tiny animals—
animalcules. These animalcules are now considered as protozoa. In his eighth letter written
on 9th October 1676, he gave a detailed description of the microbial forms. All the descriptions
were appropriately supported by the accurate drawings. In 1680, he was elected as Fellow
of Royal Society.
Antony van Leeuwenhoek, during explaining the microorganisms, said about these
organisms as, in his own writings—
“ ... in the year 1675, I discovered living creatures in rainwater which had stood for a
few days in a new earthen pot, glazed blue within. This invited me to view this water with
great attention, especially those little animals appearing to me ten thousand times less than
those ... which may be perceived in the water with naked eye.”
He was very meticulous and accurate in his descriptions and it is now clear that what
he had observed must have been different types of algae, bacteria, fungi, protozoa and others.
On 16th June 1675, during the observation of well water into which he had added a pepper
grain, he said—
DEFINITION AND HISTORY OF MICROBIOLOGY
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“I discovered in a tiny drip of water, incredibly very little animalcules and these were
of diverse sorts and sizes. They moved with bending, as an eel always swims with its head
in front and never, tail first; yet these animalcules swam as well backwards as forwards,
though their motion was very slow.”
Obviously what Leeuwenhoek saw were the motile bacteria. In 1683, he made observations
and explained different forms of animalcules such as rods, spheres and spiral shapes which
were nothing but the different morphological forms of bacteria. This was the first recorded
information of bacteria and even today this morphological classification of animalcules is
known to be perfect for bacteria.
Antony van Leeuwenhoek had not proceeded further beyond describing the microbes. He
never tried to associate them with their surroundings as causative agents. It is also said that
Antony van Leeuwenhoek was a secretive person and invited none to work with him, nor did
he reveal the secret of his technique of grinding lenses. During the time of Leeuwenhoek and
even later, microscopic observation was regarded as an idle man’s job with no practical
relevance.
He also made observation on spermatozoa, small invertebrate animals and red blood cells
(thus he was also considered as founder of histology) and he also described capillary circulation
of blood.
ERA OF TRANSITION
Leeuwenhoek’s discovery of microbial world took long period of time to get its importance
in the science. This was due to optical defect, which limits the observation of microorganisms
by using compound microscopes. Leeuwenhoek gave a clear indication of microbial diversity
along with its incredible abundance in the nature. This made scientists to think about their
origin.
There were two schools of thought. One group believed that animalcules were formed
spontaneously from the non-living materials and this is known as spontaneous generation
or abiogenesis. But, others including Leeuwenhoek believed that they were formed from
the ‘germs’ or ‘seeds’ of these animalcules, which were always present in the air. This was
referred as biogenesis. This raised the controversy over abiogenesis and biogenesis and
abiogenesis has had a long acceptance. Scientists at that time believed this, because meat
kept open for longer period of time produced maggots and mushrooms suddenly appeared on
rotten wood.
Later, due to increased knowledge of living organisms, abiogenesis gradually lost its
acceptance. Doubtfulness about the abiogenesis started by meticulously performed experiment
by an Italian physician Francesco Redi in 1665.
Francesco Redi (1626–1697)
Theory of abiogenesis was first challenged by Francesco Redi with experimental proof.
He placed fresh meat in three glass containers. One was kept open, the second was covered
with paper, and the third was covered with a fine gauze that would not allow flies to enter
the container. After some time flies laid eggs on the meat that was not covered. In the other
two containers maggots were not produced on the meat. But, flies moved towards gauze
covered container and laid their eggs on the gauze itself (as they could not enter the container).
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Later these eggs produced maggots. This clearly indicated that the meat did not spontaneously
produce maggots, but they were produced from the eggs that were laid by the flies on the
meat. This was not considered true for the microorganisms and they said microorganisms
arose spontaneously. To support this John Needham performed the experiment.
John Needham (1713–1781)
John Needham, an English priest, in 1748 did experiments to support the views of
abiogenesis. He boiled the mutton broth and then covered the mouth of the flasks tightly.
Later these flasks became turbid and consisted of microorganisms. He thus proposed that
organic matter contains vital force that converts a non-living material into living ones. A few
years later this was again questioned by the experiments of another priest, Lazzaro
Spallanzani.
Lazzaro Spallanzani (1729–1799)
Lazzaro Spallanzani was an Italian priest and naturalist and by his experiment strongly
argued that microorganisms were not spontaneously generated. He kept seeds and water
in glass flasks. Then it was sealed and boiled for ¾ of an hour. In these flasks no growth
of microorganisms was noticed until the seals were broken. From this he proposed that air
carried microbes to the broth medium and the said air is required for the growth of
microorganisms. But supporters of abiogenesis claimed that heating the air in sealed
container completely destroyed the ability of air to support the life forms, because during
this period Priestley and Lavoisier clearly indicated that oxygen (air) is essential to life
forms.
Many workers later tried to give a proper answer to these arguments in support of
biogenesis. The main scientists were Theodore Schwann, Friedrich Schroder, von Dusch,
Pasteur, Tyndall and others.
Theodore Schwann (1810–1882)
Theodore Schwann by passing air through red-hot tube into sterile culture medium showed
that even after providing the air microbes did not appear spontaneously in the broth medium. This
work was further improved by George Friedrich Schroder and Theodore von Dusch. They passed
the air through sterile cotton wool into sterile culture medium and allowed for growth of
microorganisms. No growth was noticed even though the air had not been heated. This paved the
way for practice of plugging the culture vessels with cotton plugs in all culture works.
But French naturalist Felix Pouchet still argued that microbial growth can occur
without air contamination. This argument attracted French scientist Louis Pasteur who
decided to settle the matter towards biogenesis.
Edward Jenner (1749–1823)
Edward Jenner (Fig. 1.4) was an English physician, who, during his treatment practice,
discovered the method of safe and proper immunity protocol for smallpox disease. He
successfully developed efficient method of vaccination for the first time against viral disease
even though its pathogenic agent was not isolated in pure form. The vaccination (Latin vacca
means cow) is a term later adopted and coined by the Pasteur in the recognition and honor
of the work contributed by Edward Jenner. Vaccination is the process of protection of an
individual by the preparation of microbes or its contents.
DEFINITION AND HISTORY OF MICROBIOLOGY
11
Fig. 1.4 Edward Jenner (1749-1823).
During Jenner’s period cow pox was a common disease and was caused by cow pox virus
(vaccinia virus). This disease is known to cause small pustules on the teats in the udder.
Sometimes similar pustules were also observed on the milkmen’s hands who generally handled
infected cows for milking. Such cow pox lesions were very much similar in character to
smallpox. But, they were restricted, localized and known to cause mild symptoms without
complications. Cow pox was also not able to disperse from one person to another person (noncommunicable). It were also observed that people who were infected with cow pox showed
complete resistance against the smallpox. The cow pox pustules by some phenomena protected
the individuals from the infection by smallpox.
These facts made Edward Jenner to take up serious observation because he was a
medical student. Later, as a physician, he made detailed investigation and recorded all his
methods of practice. By the repeated experiment for many years he considered that cow pox
lesions gave protection against smallpox disease. During his studies Jenner collected the pus
from a cow pox lesion from the hand of a dairy maid (called Sarah Nelms) in 1796. Then he
rubbed it on the skin surface of a healthy boy (called James phillip). Later the boy developed
a typical scar on the skin. After a few weeks the boy was inoculated with the pus taken from
the smallpox pustules of a diseased person. But the boy could not show any disease of
smallpox. Hence, boy showed the complete immunity to the disease. From experiments
performed later and scientific accumulation of data of his studies, he concluded that inoculation
of a person with cow pox pus could offer immunity from the smallpox disease. Later, in June
1798, he published his findings in An Inquiry into the Cause and Effect of Variole Vaccinae.
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This achievement is considered as one of the greatest achievements ever made by man in his
quest for conquering disease and is also heralded as a significant milestone in the history of
microbiology. Though vaccination was met with greater opposition in the beginning, it has
stood the test of time and proved its advantage in medical field in later dates. Through this
process of vaccination the smallpox virus and the disease have been eradicated from the
world population and now vaccination against this disease is considered as not that necessary.
GOLDEN ERA OF MICROBIOLOGY
The golden era of microbiology began with brilliantly planned experiments of Louis
Pasteur, Robert Koch, John Tyndall, Elie Metchnikoff and others. They provided the sound
scientific principles without debatable answers to the abiogenesis phenomena. This was
greatly accepted by the scientific community of that age. The majority of the microbial
discoveries, their scientific principles and advancement were made during this period of time.
These paved the way for development of microbiology as a very important branch of science.
Louis Pasteur (1822–1895)
Louis Pasteur (Fig. 1.5) was a French scientist, chemist and microbiologist and is
linked with most important discoveries in the field of microbiology.
Louis Pasteur was born on 27th December 1822, in the countryside of Dole, France. He
was first involved in the study of chemistry and obtained the doctorate in Chemistry from
the Norman School in Paris and became Professor of Chemistry at Paris.
Fig. 1.5 Louis Pasteur (1822-1895).
DEFINITION AND HISTORY OF MICROBIOLOGY
13
Death blow to the theory of abiogenesis
Louis Pasteur performed many experiments in 1860 to give a death blow to the theory
of spontaneous generation of life (abiogenesis). Pasteur first filtered air through sterile cotton
and then the sterile cotton was placed in a sterile medium. This showed the growth of
microorganisms and indicated that air contained these microorganisms. Later, he poured
sterile nutrient medium in specially designed flasks called swan-necked flasks. These are
round-bottom flasks having curved necks of various types with open ends. He then boiled the
medium to free the contamination and allowed to cool. In this, growth was not observed even
though the curved necks had opening to surrounding atmosphere.
Pasteur thus opined that no growth was seen because dust and germs had been trapped
by the walls of the curved necks. But, growth would be noticed if the curved necks were
broken because the air could enter directly into the flasks with depositing the germs and dust
on the neck walls. However, Pasteur not only conclusively disproved the theory of spontaneous
generation or abiogenesis in 1861, but also showed how to maintain any solution in sterile
condition for longer period. Later, this was also supported by the work of John Tyndall,
which even could destroy the bacterial endospore (Tyndallization—the process of repeated
heating and cooling of the medium that can completely destroy all living germs in the
solution) that are highly resistant to regular boiling.
Pasteur with his knowledge in chemistry came into the limelight while working on
variety of biological fields including medicine because he had great insight into the problems
which he came across. Thus, he was called for the majority of the unsolved problems in the
different fields. The major contribution of Pasteur to the field of microbiology in particular
and to the science in general is listed below.
Tartaric acid isomers
Tartaric acid is an organic compound which has the capacity to get crystallized. When
working with tartaric acid crystals he found out two types of crystals based on their capacity
to refract light in different directions called polarization. One type of crystals refracted light
(polarized) towards right and others towards left. These were microscopically separated into
two different types of crystals and were called isomers.
Microbial fermentation
The process of conversion of carbohydrates (sugar) into alcohol is known as fermentation.
Large numbers of people in France are involved in wine production. In the meantime Theodore
Schwann (1837) showed that yeast cells are responsible for the process of fermentation. But
German chemist Justin Liebeg proposed that fermentation was a chemical process. During this
period distillers of France faced a problem of their alcohol turned into sour taste. This totally
hampered the entire industry in France and they approached Pasteur to solve this problem.
Pasteur took up this challenge and started observation on wine preparation in greater
detail. He could observe yeast cells and bacteria under microscopic field. Pasteur considered
that yeast cells were responsible for the fermentation process. By setting up a number of
conclusive experiments he demonstrated that yeast cells turned grape juice (sugar) into
alcohol. By the application of heat Pasteur destroyed the yeast cells in the grape juice
mixture. In this case no alcohol was formed and alcohol formation could be regained when
the fresh yeast cells were added into the grape juice.
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He also showed that bacteria were responsible for sour taste of the wine. This sour
taste is formed due to the growth of the specific type of bacteria and their fermentation
in the mixture. The basic discovery of microbial fermentation was remarkable in
microbiology, because up to that time microbes were considered as the things of curiosity
and none could link them with any biochemical activity of significance. Pasteur published
a research paper on fermentation that formed the basis of emergence of microbiology as
a branch of Biology. This could also gave a solution to the problem which occurred in the
French distilleries.
Aerobes and anaerobes discovery
Priestley showed that organisms required air (oxygen) for the growth and development.
Hence, scientists during 19th century believed that microorganisms also needed oxygen for the
growth and development. Microorganisms that require oxygen for the growth are referred as
aerobes or aerobic microorganisms (E.g.: Bacillus, Pseudomonas). Pasteur during his studies
on butyric acid fermentation showed that some of the microorganisms did not require oxygen.
Observations of the fermented mixture under the microscope indicated that large numbers of
microbes were actively showing the motility. These microbes could not show the motility when
exposed to the air. He also showed that fermentation of the mixture could be stopped by
aeration of the broth. Pasteur named those groups of microorganisms that did not require
oxygen for their growth as anaerobes or anaerobic microorganisms (E.g.: Clostridium).
Process of pasteurization
The process of heating liquids to destroy microbes which are harmful in nature is known
as pasteurization. This was first adopted for the wine and beer industry by Pasteur to kill
the undesirable microbes present in the grape juice. These microorganisms produced lactic
acid and acetic acid in the fermentation mixture. By this process Pasteur showed that spoilage
of taste of wine (due to the presence of acid) can be prevented by exposing fermentation
mixture to low heating at 60°C for a brief period instead of complete boiling. This temperature
was more than enough for the killing of undesirable bacteria which acted as contamination
in the juices.
This pasteurization process is now employed for a number of liquids such as beer, milk,
milk products and other beverages. This process has become one of the necessary processes
in the food industry and others. Present day pasteurization temperatures are heating of
liquid at 62.8°C for 30 minutes (slow pasteurization) or 72°C for 15 seconds (flash
pasteurization or high-temperature short-term [HTST] pasteurization).
Work on pebrine disease
The southern parts of France was the major area for the silk industry that produced the
large economy to the France. During 1865 this silk producing area suffered a disastrous
economic loss for the country. This was due to the pebrine disease affecting the silk worms and
resulted in large scale spread of the disease in majority of the silk worm rearing areas. Then
Ministry of Agriculture appointed Pasteur as a major person to study and find the proper
solution for the disease.
Pasteur after detailed investigation in the affected area for three years was able to find
out the causative agent of the disease as a protozoa member (Nosema bombysis). He also
showed the farmers to get rid off the disease by using the disease-free eggs for rearing and
DEFINITION AND HISTORY OF MICROBIOLOGY
15
destroying the affected eggs and older leaves that were used as silk worms feed. Thus by using
hygienic source material the disease could be completely eliminated. The silk industry of
France was thus saved from the major economic loss. This path-breaking discovery led him to
become a great patriot apart from being an eminent scientist and scholar.
Work on anthrax
In France anthrax was a fatal disease and affected the sheep industry. The same type
of disease was also observed in the other animals that became a major problem for the
Government economy. In the meantime, work done by the German physician Robert Koch
revealed that the anthrax bacterium (Bacillus anthracis) was associated with the disease but
could not establish the relationship with the disease causing ability of the bacterium. It was
Pasteur’s idea which proved that the bacteria present in the culture medium was causing the
disease in healthy animals.
Pasteur also got a clue on the process of vaccination or protective inoculation of healthy
host to get resistance against disease. He successfully demonstrated that inoculation of
weakened microbes to animals could give them immunity and ward-off from the disease.
Through this, Pasteur discovered the effective vaccine for the anthrax. This created the
curiosity and interest in many of the contemporary scientists towards vaccination. Pasteur
developed anthrax vaccine along with his associate Charles Chamberland (1851–1908).
Charles Chamberland also developed the bacteriological filters.
The most famous and remarkable discovery in the field of medicine came from the
research work of Pasteur through the work on rabies. Rabies or hydrophobia is a viral
(Rhabdovirus group) disease that comes to humans through mad dog bite. Even though
Pasteur was not able to observe or isolate the causative agent of the disease under microscope,
he successfully developed the treatment or protection against the disease.
Pasteur first started his work on the animals on a trial basis. By observing the observation
of Edward Jenner, he planned to place an avirulent (non-virulent; made by growing virus in
an abnormal host, the rabbit) mixture of dried, grinded spinal cord under the dog skin
surface. This process continued for a few days with increased virulent strain preparation
from spinal cord extract. In the end of the experiment, the animal successfully got resistance
against the disease. The rabid dog bite and application of most potent extracts to brain of
healthy animal failed to induce rabies. With this success in the case of animals, he also
explained the need for further research work in the application of this process directly to
human beings.
Before the further research work could occur the situation had aroused which led Pasteur
to practice it even on humans. During the course of his experiments, Joseph Meister, a nine
year old boy who had been bitten by a rabid dog, was brought to the observation of Pasteur.
As boy’s death was certain in the absence of treatment, Pasteur considered for trying
vaccination. The boy was vaccinated 13 times over the next 10 days with increasingly virulent
preparations of the attenuated strain of virus. To the surprise the boy survived with the
vaccination and later became the faculty in Pasteur Institute.
In the gratitude for Pasteur’s development of vaccination process, people from different
parts of the world immensely contributed to initiation of research work in a separate institute
called Pasteur Institute in Paris, France. He died in Paris, on 28th September, 1895. His
tomb was constructed in the basement of the Pasteur Institute, Paris.
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Joseph Lister (1827–1912)
Joseph Lister (Fig. 1.6), was an English surgeon born in Upton, England on 5th
April, 1827. In 1852 he earned his degree in medicine. Lister initiated the study on the
coagulation of blood and inflammation that occurred after injuries and surgical wounds. He
became Professor of Surgery at Glasgow and Edinburgh. Lister started concentrating to
reduce the gangrene and bacterial infections. During his time gangrene was considered to
be caused by the bad quality air. Even after maintaining the cleanliness in surgical materials
and rooms, the death rate was very high (50%).
Lister made several contributions for the improvement of the microbiology as a whole
and medicine in particular.
Fig. 1.6 Joseph Lister (1827–1912).
Antiseptic technique
Lister had contributed to surgery in many ways, but his major contribution has been in
preventing wound infection after surgery by following antiseptic methods. As a great observer
and surgeon, Lister was attracted towards post-operative complications in patients due to
wound infections. He made detailed study the cause of such wound infections. He observed a
similarity between the wound inflammation, fermentation and putrefaction described by
Pasteur, which was caused due to microorganisms. This made him to believe that the microbes
entered the wound through air, hands, surgical instruments or bandage cloths and thus
resulted in sepsis. When he protected the wounds with dressing process that killed microbes,
inflammation never resulted. Thus later this formed the basis for antiseptic technique.
DEFINITION AND HISTORY OF MICROBIOLOGY
17
Lister by his observations came to the practice of applying carbolic acid as a microbicidal
chemical agent. He covered all the wounds with the carbolic acid-soaked bandage cloths. This
resulted in quick healing of the wounds which generally caused the fatal inflammations.
Later he made similar applications to many deadly wounds and observed the same results.
This made him to continue it as a routine for all the operation procedures and that came to
be known as antiseptic system of operation or antiseptic surgery.
Pure culture technique
Microorganisms are omnipresent in nature. They live in extremely large populations
made up of millions of cells from different species. For the study of a particular species
characteristics it is necessary to separate it from all other species present in the mixed
population. Lister in 1878, by using serial dilution technique for the first time, obtained
pure culture of bacteria in liquid media. By using a specially designed syringe, he diluted
curdled milk containing a mixture of bacteria until a single bacterium was delivered into a
container of sterile milk. After incubation he observed the bacteria in that container. They
were all of a single kind and rod shaped bacteria. Lister coined them as Bacterium lactic.
Robert Koch (1843–1910)
Robert Koch (Fig. 1.7) was born on 11th December, 1843 in Germany. In 1866 he
obtained his medical degree from the University of Gottingen and started as medical
practitioner. Later Robert Koch switched over to microscopic studies that he engaged in his
full life time. Then he became Professor of Hygiene and headed as Director in the Institute
of Infective Diseases at Berlin, Germany. Robert Koch is considered as one of the main
founders of modern bacteriology.
Robert Koch’s major contributions to the development of microbiology are as follow:
Fig. 1.7 Robert Koch (1843-1910).
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1. Germ theory of diseases
Robert Koch by his experiments proved that microbes are responsible for the diseases
in animals. During the study of anthrax disease in cattle, Koch had noticed rod shaped
microorganisms in the blood of diseased animals. For testing if these microorganisms were
the causative agents of anthrax disease, he injected a healthy mice with the blood from a
diseased animal. He found that disease was transmitted to the mice which resulted in death.
Later Robert Koch took the blood of the dead mice and injected it into another healthy mouse
and again the mouse died due to disease. Koch repeated this experiment for 20 times and
each time the healthy mice died after inoculation.
In the laboratory Robert Koch cultured these bacteria in the aqueous humour from the
eyes of a cow. He was able to reproduce the disease every time through inoculating these
microorganisms into healthy animals. With his experimental procedures he laid down certain
basic criteria for considering microorganisms as causative agents of the disease. These criteria
are referred as ‘Koch’s postulates’ and are:
(a)
The causative agent of the disease must be present in every diseased individual.
(b)
The causative agent must be isolated from the diseased individual and grown in
pure culture.
(c)
The isolated infectious microorganism must reproduce the disease when inoculated
into a healthy individual, and
(d) The same infectious agent must be reisolated from the experimentally induced
diseased individual.
These postulates of Koch continue to be useful even today with some modification.
2. Discovery of Mycobacterium tuberculosis
After the anthrax work Koch concentrated towards widespread tuberculosis disease.
With the intensive research work on tuberculosis, Robert Koch discovered and proved in 1882
that all forms of infectious tuberculosis were caused by the bacillus forms called Mycobacterium
tuberculosis. He showed method of culturing these bacilli in pure form and made detailed
study on the Mycobacterium tuberculosis.
3. Preparation of smears
As microorganisms occurred in groups of millions of cells, it was very difficult to
study the bacteria under microscope. Hence, Robert Koch showed for the first time the
technique of making smears of bacteria on glass slides and staining them with aniline
dyes to observe them more clearly under the microscope. Making of smears on the slides
distributed the microorganisms in thin layer without overlapping and gave the access for
the proper staining.
4. Plating method for isolation of pure culture
In his laboratory Robert Koch accidentally observed that a slice of potato had many
colonies of bacteria growing distinctly from one another. However, the potato had certain
disadvantages such as the moist surface of potato allowed motile bacteria to spread on entire
surface. As the surface of potato is opaque it was very difficult to observe bacterial colonies
and it is not a good nutrient medium for large number of bacterial strains.
Fi
DEFINITION AND HISTORY OF MICROBIOLOGY
19
Owing to above mentioned disadvantages, Koch planned that if a liquid nutrient medium
could be solidified by the addition of a solidifying agent such as gelatin, then it could be used
for the isolation of bacteria in the laboratory. He poured the molten gelatin medium on glass
plates and allowed it to solidify. After the gel had solidified, the surface was streaked with
a platinum wire dipped into a bacterial suspension. These plates were then covered and
incubated. After incubation period the growth was found all along the streaking line and
single colonies were found at the end of the streak. These single colonies contained only one
type of bacteria and this formed the pure cultures of bacteria. Thus, the use of gelatin for
the preparation of a solid medium and the streak plate method for the isolation of pure
bacterial cultures came into existence.
Koch’s another associate, Walter Hesse gave a clue of using agar as solidifying agent for
its advantage over gelatin. Richard Petri, a student of Robert Koch in 1887, discovered usage
of the petri dishes in the laboratory for the culturing purpose. This enables the microbiologists
to handle large number of microbes in the laboratory without the problem of contamination.
The discovery of acellular particles, viruses and their role in disease development was
made possible when Charles Chamberland (Pasteur’s colleague) developed porcelain bacterial
filters in 1884. This made the microbiologists to use such filters and study the viruses in
greater detail.
During this period development was also taking place in determining how animals
resisted disease and in understanding immunity. After the discovery of diphtheria toxin, Emil
von Berhring (1854–1917) and Shibasaburo Kitasato (1852–1931) inoculated inactivated toxin
into rabbits. Later a substance known as antitoxin (antibody against diphtheria toxin) was
produced in the blood and this protected against the disease by inactivating the toxin.
Fig. 1.8 Elie Metchnikoff (1845-1916).
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BIOTECHNOLOGY-II
The antitoxin work provided evidence that immunity could result from soluble substances
present in the blood. This is now well known that these substances are antibodies that are
responsible for the humoral immunity (antibody-induced immunity). Later, Elie Metchnikoff
(1845–1916) (Fig. 1.8) discovered that blood leukocytes (WBC’s) were also responsible for the
immunity. He referred these cells as phagocytes and the process as phagocytosis. Thus, was
opened a new branch of immunity named cellular immunity.
Alexander Fleming (1881–1955)
Alexander Fleming (Fig. 1.9), a British scientist, was born on 6th August, 1881, at
Lochfield in Scotland. He studied medicine and obtained his degree from St. Mary’s Medical
School, London University. Later, he joined the military service and worked as captain in
Army Medical Corps throughout First World War.
During 1921, working with blood and its action on bacteria he discovered the bacteriolytic
properties in the body tissues and secretions. For these substances he coined the term Lysozyme.
In 1928, Alexander Fleming, a student of Almroth Wright (discoverer of opsonins) and
an excellent bacteriologist working at St. Mary’s Hospital, in London, discovered Penicillin
the first wonder drug.
Fig. 1.9 Alexander Fleming (1881–1955).
When he was working in the laboratory, one of the petri plates containing the bacterial
culture (Staphylococcus aureus) attracted his attention. In his laboratory, Fleming was
performing experiments in search of novel bactericidal agents, particularly against wound
infections. During the course of experiments, he observed that a culture plate of Staphylococcus
aureus was contaminated by a blue-green coloured fungal growth. The area surrounding the
edges of the fungal colony was clear. The growth of Staphylococcus aureus near the fungal
colony was completely inhibited or was killed by the fungus. Fleming by careful examination
DEFINITION AND HISTORY OF MICROBIOLOGY
21
opined that something that is excreted by the fungal colony proved to be fatal to Staphylococcus
cells. The contaminated fungal colony was then isolated and identified as a common bluegreen fungal member Penicillium notatum.
Later, Fleming cultured this fungal colony on broth medium in a separate flask. Then
from the broth medium he obtained the exudates content. In order to test his hypothesis that
the fungal secretions could destroy Staphylococcus he inoculated this extraction into laboratory
mice infected with Staphylococcus, Streptococcus and Pneumococcus. The mysterious compound
present in this extraction killed all the microorganisms in the body of the mice as it had done
similar thing on Staphylococcus aureus in the petri plate. Then, Fleming named this mysterious
compound as Penicillin. Fleming, in 1929, published a research article in the British Journal
of Experimental Pathology.
This work later drew the attention of Sir Howard W. Florey and Ernst B. Chain at
Oxford University. In due course Howard Florey and Ernst Chain successfully purified the
penicillin from the culture filtrate of Penicillium notatum.
This meant that the penicillin could be used for the large scale purpose. During 1941
industrial production of penicillin had started and proved efficient against bacterial infection
during First World War. In Medicine category Fleming shared the Nobel Prize along with
H.W. Florey (Australia) and E.B. Chain (Great Britain) in 1945.
FURTHER DEVELOPMENT OF MICROBIOLOGY
Ecological role of microorganisms was studied by a few of the early microbiologists.
Particularly they studied by taking soil and aquatic habitats as the environment and
worked in detail microbial involvement in the carbon, nitrogen and sulfur cycles. Sergei N.
Winogradsky (1856–1953) and Martinus W. Beijerinck (1851–1931) are the two pioneers in
this category.
Fig. 1.10 Sergei N. Winogradsky (1856-1953).
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BIOTECHNOLOGY-II
Sergei N. Winogradsky (Fig. 1.10), a Russian microbiologist, made several important
contributions to the soil microbiology. Winogradsky discovered that soil bacteria are involved
in oxidization of iron, sulfur and ammonia to get their energy requirement. He also discovered
that photosynthetic bacteria, similar to plants, can also use CO2 to produce organic materials.
Another new groups of microorganisms were discovered by Winogradsky including anaerobic
nitrogen-fixing microorganisms. He isolated thems from the soil habitat and also studied in
detail the decomposition of cellulose in the soil.
Martinus W. Beijerinck (Fig. 1.11), another Russian microbiologist also made many basic
contributions in the development of microbial ecology and other branch of microbiology.
Beijerinck isolated the Azotobacter, an aerobic nitrogen-fixing bacterium (soil bacterium);
Rhizobium, a root nodule forming symbiotic bacterium capable of fixing nitrogen and many
sulfate-reducing bacteria from soil.
Fig. 1.11 Martinus W. Beijerinck (1851-1931).
Later Beijerinck and Winogradsky together developed the enrichment technique of
culturing of microbes and the use of selective media in the growth and isolation of pure
culture. This became a great importance in the microbiological field.
Heinrich Anton de Bary (1831–1888), a German doctor, made large number of contributions
to the fungi and mycology and established Phytophthora infestans as cause of the late blight
of potato. Anton de Bary (Fig. 1.12) laid clear foundations of mycology and hence he is
commonly known as founder of modern mycology or father of plant pathology.
Dmitry Ivanovsky (1864–1920) (Fig. 1.13) found a new field of microbiology called virology
by discovery of tobacco mosaic disease, causative agent, a virus (later named as Tobacco
mosaic virus). This gave way for observing the organisms that are not easily studied by using
conventional microscopes and started virology as a major field in microbiology.
DEFINITION AND HISTORY OF MICROBIOLOGY
23
Fig. 1.12 Heinrich Anton de Bary (1831-1888).
Fig. 1.13 Dmitry Ivanovsky (1864-1920).
Selman Waksman (1888–1973) working at Rutgers University in US isolated a soil
actinomycetes bacterium Streptomyces and discovered streptomycin, a major broad spectrum
antibiotic. Selman Waksman (Fig. 1.14) was the person who coined the word ‘antibiotic’ and
won the Nobel Prize in 1952 for the same.
24
BIOTECHNOLOGY-II
Fig. 1.14 Selman Waksman (1888-1973).
David Hendricks Bergey (1860–1937) was the person who headed the manual preparation
team for the entire bacterial organisms identifications called Bergey’s Manual. This is most
important for Bary proper identification and placing of newly discovered bacterium. David
Hendricks Bergey’s (Fig. 1.15) name is still used in the recent edition of the manual for his
great work done in bacteriology.
Fig. 1.15 David Hendricks Bergey (1860-1937).
In the early part of twentieth century, microbiology developed independently of other
biological disciplines due to difference in focus. During this period microbiologists were more
DEFINITION AND HISTORY OF MICROBIOLOGY
25
concerned with the agents of infectious disease, the immune response, the search of new
chemotherapeutic compounds and bacterial metabolism.
Branch of microbiology made a closer relationship with other branches of biology during
1940s. This was due to its closer association with genetics, physiology and biochemistry.
Microorganisms are found to be extremely used as subjects in the experiments because they
are relatively simple, grow rapidly and can be grown in large quantities in small area. The
mutants of bread mold Neurospora were used by George W. Beadle and Edward L. Tatum
in 1941 for studying the relationship between genes and enzymes. In 1943, Salvadore Luria
and Max Delbruck studied spontaneous gene mutation by using bacterial mutants. Later in
1944, Oswald T. Avery, Colin M. MacLeod and Maclyn McCarty proved conclusively that DNA
was the genetic material and carried genetic information during the gene transformation.
The intimate interactions between microbiology, genetics and biochemistry soon helped in the
development of modern molecular oriented genetics. J.D. Watson and F.H.C. Crick discovered
the double helix structure of DNA molecules in 1953 and got Nobel Prize in 1962.
In recent times microbiology has become one of the major contributors to the rise of
molecular biology (the branch of biology which deals with the physical and chemical aspects
of living matter and their function) into greater heights. Microbiologists have been deeply
involved in detailed studies on the mechanisms of DNA, RNA, genetic code and protein
synthesis. Microorganisms were more often employed in many of the early studies on the
gene regulation and enzyme regulation. Discoveries made during 1970s contributed to the
emergence of recombinant DNA technology and genetic engineering.
This is clearly evident when we observe the list of Nobel Prizes awarded in Physiology or
Medicine category. About 1/3 of the prizes have been awarded to the scientists working on
microbiological or related problems. Thus, it shows the importance of microbiology in the
branch of science.
Microbiology is one of the most rewarding of professions. This is because it gives its
practitioners the opportunity to be in contact with all the other natural sciences and thus to
contribute in many different forms and ways to the improvement of science in general and
human life in particular.
SUMMARY
Microbiology is the branch of biology which deals with the study of organisms that are
usually too small to be seen by naked eye. It includes viruses, viroids, prions, bacteria, many
algae, fungi and protozoa. Microbiology is not a mere study of structure and classification of
microbes. The various aspects of microbiology, both basic and applied aspects can be studied.
Discovery of microorganisms made by Antony van Leeuwenhoek in 1675 was considered
to be a starting point for microbiology. The history of microbiology can be divided into era of
discoveries, the era of transition and the golden the era of microbiology.
The era of discoveries involved the first scientific study of microbes and its structure.
Antony van Leeuwenhoek’s discovery and explanation of microbes which he called
‘animalcules’ (tiny animals) is most significant scientific observation in the era of discoveries.
Along with the microbes he also made observation on spermatozoa, small invertebrate animals
and red blood cells (thus considered as founder of histology) and also described capillary
circulation of blood. The era of transition considered to be the scientific method of accepting
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BIOTECHNOLOGY-II
the life forms as produced only from pre-existing living forms and not from non-living organisms
or materials. Microbiologists such as Francesco Redi, John Needham, Lazzaro Spallanzani,
Theodore Schwann and Edward Jenner were the major contributors to this era. In the golden
era of microbiology contributions made by large number of microbiologists helped in the
establishment of a new branch of science, microbiology. Microbiologists namely Louis Pasteur,
Joseph Lister, Robert Koch, Winogradsky, Beijerinck and Alexander Fleming were the very
important scientists and their large number of contributions helped in better understanding
of microbes and their applications to the human society. Hence, it is rightly called the golden
era of microbiology.
The major contributions of Louis Pasteur include proving the biogenesis, discovery of
microbial fermentations, identification of aerobes and anaerobes, causative agent of pebrine
disease, pasteurization, isolation and identification of isoforms of tartaric acid and vaccination.
Pasteur began his scientific career as chemist but soon became a pioneer microbiologist. For
these large number of contributions he is commonly known as the father of microbiology.
Joseph Lister advocated the aseptic surgery method that has tremendous impact on the
wound healing in the post-operative therapy. Robert Koch found germ theory of diseases,
smear preparations, pure culture methods and Mycobacterium tuberculosis bacterium.
Alexander Fleming discovered the antibiotic penicillin that opened up a new era of
chemotherapy of various infectious diseases. Soil microbiology was developed because of the
pioneer contributions made by Winogradsky and Beijerinck. They isolated and identified
Azotobacter, Rhizobium and other soil microorganisms and their role in soil fertility and
biogeochemical cycles.
Later both technological developments and basic scientific developments in variety of
fields contributed to the development of microbiology as a complete field of science.
EXERCISE
1.
What do you mean by microbiology?
2.
Describe the importance of microbiology as a branch of modern science.
3.
Explain historical events of microbiology.
4.
Define Koch’s postulates and their importance.
5.
What is meant by pure culture technique and who proposed this technique?
6.
What do you mean by vaccination and who started this process?
7.
Write the contributions of the following microbiologists in the field of microbiology:
(a)
Louis Pasteur
(b)
Robert Koch (April, 1998)
(c)
Joseph Lister
(d) Alexander Fleming
(e)
Winogradsky
(f)
Beijerinck
(g)
Edward Jenner
(h) Tatum
(i)
Metchnikoff
(j)
John Tyndall
DEFINITION AND HISTORY OF MICROBIOLOGY
27
8.
Explain how Antony van Leeuwenhoek contributed to initiating microbiology.
9.
What are the contributions of Robert Koch, Alexander Flemming and Joseph Lister to the
field of microbiology? (April/May 2000, April/May 2001).
10.
11.
Discuss contributions of Louis Pasteur (Nov./Dec., 1999).
Write short notes on:
(a) Penicillin
(b) Cotton plug
(c) Abiogenesis
(d) Pebrine disease
(e) Pasteurization
(f) Tartaric acid
(g) Bacterial smears
(h) Anthrax
(i) Aerobes and anaerobes
(j) Fermentation
12.
Who coined the word microbes?
13.
Name other scientists who got the Nobel Prize along with A. Fleming for penicillin related
discovery and work.
14.
Antiseptic agents.
15.
Which microbiologist discovered the bacterium Mycobacterium tuberculosis?
16.
Differentiate between biogenesis and abiogenesis.
17.
Azotobacter and its usefulness.
18.
Soil microbiology and its applications.
19.
What is the major contribution of Watson and Crick?
20.
Charles Chamberland and his contributions.