Microbiology in the New Millennium

The major thrust in the specialized domain of ‘microbiology’ got a tremendous boost in speed
and momentum during the twentieth century towards the development of judicious control and management
of infectious human diseases ; elaborated studies in immunity profile ; as exceptionally attractive
models for investigating fundamental life processes viz., activities related to metabolizing, growing,
reproducing, aging, and dying ; and microbes’ broad spectrum physiological and biochemical potentialities
than all other organisms combined. In addition, the science of microorganisms have propogated
other allied disciplines, for instance : biochemistry, genetics, genetic engineering, molecular biology,
and the like.
Historic revelation of DNA (deoxyribonucleic acid), which being the key to life and genetics,
was duly discovered by two world famous biologists Watson and Crick. DNA forms the basic funda

* A special type of virus-bacterial cell interaction maintained by a complex cellular regulatory mechanism.
Bacterial strains freshly isolated from their natural environment may contain a low concentration of
bacteriophage. This phage will lyse other related bacteria. Cultures that contain these substances are said to
be lysogenic.
** A rod-shaped, Gram-negative, non pathogenic soil and water bacteria that fix atmospheric nitrogen ; the
single genus of the family Azotobacteraceae

mental structure of each and every chromosome in the precise shape of a ‘double-helix’.* In fact,
microorganisms helped extensively and intensively in the better understanding of the exact mechanism
whereby the most critical and valuable information meticulously stored in the ‘genetic material’ is
ultimately transcribed and subsequently translated into proteins. Later on, Escherichia coli i.e., a colon
bacterium, served as a via-media or a common tool for the geneticists, microbiologists, and biochemists
to decepher the intricacies of various cellular processes. The concerted research inputs made by
Nirenberg, Khorana, Holley, Jacob, Monod, and a plethora of others substantiated copious informations
to the present day knowledge of the living systems, of course, making use of the microorganisms. It is,
however, pertinent to mention at this juncture that microbes are being skilfully and gainfully utilized to
grasp the meaning with respect to the control mechanisms directly involved in cell division as well as
reproduction.
As to date ‘microbiology’ has marked with a dent an altogether separate identity and distinct
branch of biology having an established close relationship with biochemistry and genetics. It has progressively
and aggressively emerged into an intriguing subject over the years because each and every
specific area in microbiology has virtually expanded into a large specialized subject in itself, namely :
dairy microbiology, environmental microbiology, food microbiology, industrial microbiology, medical
microbiology, sanitary microbiology, and soil microbiology. Importantly, newer techniques exploring
and exploiting microorganisms for gainful and economically viable products of interest have always
been the focus of attention across the globe. In the same vein, the absolute control and management of
certain non-productive and troublesome species have always remained another virile and fertile area of
interest in ‘microbiology’, which ultimately yielding definitely not only a purer product but also augmented
the end-product to a considerable extent.

There are ample evidences cited in the scientific literatures with respect to enormous utilization
of the microorganisms to understand both biology and the prevailing intricacies of various biological
processes towards the last two decades of the twentieth century and the early part of the New Millennium.
Besides, microbes have been adequately exploited particularly as ‘cloning vehicles’. In this context
one may always bear in mind that E. coli and other microorganisms have been used extensively in
order to carry out the spectacular piece of most innovative inventions of the century, for instance : (a)
cloning specific segments of DNA ; (b) large-scale production of vital chemicals hitherto synthesized by
tedious high-cost chemical routes, e.g., acetic acid, ethanol, citric acid, a variety of antibiotics, and
steroids.
The microbiological transformations have beneficially led to the production of a good number
of steroid variants from progesterone as illustrated under

The New Millennium shall witness the remarkable innovations and paramount advancements in
the latest recombinant DNA (rDNA) technology that has virtually revolutionized the bright futuristic
growth and prospects of manupulating the exceptionally unique ‘genetic combine’ of a microorganism,
plant, animal, and human being to fit into the appropriate requirements for the upliftment of humanity in
particular and remove the sufferings of the mankind in general. In true sense, the recombinant DNA is
considered to be a wonderful novel piece of artistic creation so as to accomplish a controlled recombination
which essentially gives rise to such techniques whereby either genes or other segments of relatively
large chromsomes may be segregated, replicated, and studied exhaustively by suitable nucleic acid
sequencing, and electron microscopy. Thus, biotechnology has really undergone a see change by
means of two vital and important technological advancements viz., rDNA, and genetic engineering in
order to expand enormously the inherent potentials of microorganisms, fungi, viruses, and yeast cells
ultimately turning into highly sophisticated and specialized miniature biochemical units.
FURTHER READING REFERENCES
1. American Society for Microbiology : Celebrating a century of leadership in microbiology,
ASM News : 65 (5), 1999.
2. Beck RW : A Chronology of Microbiology in Historical Context, ASM Press, Washing

ton DC, 2000.
3. Benacerraf B et. al. : A History of Bacteriology and Immunology, William Heinemann,
London, 1980.
4. Brock T (ed). : Milestones in Microbiology, Prentice-Hall, Eaglewood Cliffs., NJ., 1961.
5. Collard P. : The Development of Microbiology, University Press, Cambridge, 1976.
6. Dowling HF : Fighting Infection, Conquests of the Twentieth Century, Harvard, Cambridge,
Mass., 1977.
7. Hellemans A and Bunck B : The Timetables of Science, Simon and Schuster, New York,
1988.
8. Lechevalier H. and Solotorovsky M. : Three Centuries of Microbiology, McGraw Hill,
New York, 1965.
9. Parisch HJ : Victory with Vaccines — The Story of Immunization, Livingstone, London,
1968.
10. Summers WC : History of Microbiology. In : Encylopedia of Microbiology, Vol. 2. J.
Ledenberg, Ed., Academic Press, San Diego, 677–97, 2000.
11. Van Iterson et. al. : Martinus Bijerinck’s, His Life and Work, Science Tech, Madison,
Wis., 1984.
12. Waterson AP and Wilkinson L. : An Introduction to the History of Virology, Cambridge
University Press, London, 1978.

Microorganisms as Geochemical Agents

The mid of the nineteenth century witnessed an ever growing interest in the pivotal role of
microorganisms in carrying out not only the various processes related to fermentations but also tackling
some of the human diseases. Nevertheless, Pasteur’s articulated contributions on fermentation evidently
proved and established that microorganisms in particular may cater as highly specific entities in performing
a host of chemical transformations.
Winogradsky and Beijerinck legitimately shared the overall merit and credibility for establishing
the precise role of microbes in the critical transformations of N and S.
Windogradsky (1856-1953) : He critically examined and observed that there exist a plethora of
distinct and discrete categories of microorganisms each of which is invariably characterized by its inherent
capability to make use of a specific inorganic energy source.
Examples :
(a) Sulphur Microbes : They oxidize inorganic sulphur containing entities exclusively.
(b) Nitrogen Microbes : They oxidize inorganic nitrogen containing compounds solely.
Interestingly, Winogradsky caused to be seen that there are certain microorganisms which either
in association with free living or higher plants may exclusively make use of gaseous nitrogen for the
synthesis of the specific cell components.
Hellriegel and Wilfarth (1888) : They showed explicitely that a predominantly mutual and
immensely useful symbiosis does exist between bacteria and the leguminous plants particularly.
Beijerinck (1901) : He meticulously observed, described, and even enumerated the usefulness
of the very presence of the ‘free-living nitrogen fixing’ organism Azotobacter** in maintaining the
fertility of the soil.

Emergence of Virology

Virology essentially refers to — ‘the study of viruses and viral diseases’.
Preamble : Towards the later part of the nineteenth century Pasteur and his co-workers were
vigorously attempting to unfold the precise and exact mechanism of the phenomenon of disease development
by examining meticulously a good number of infectious fluids (drawn from patients) for the
possible presence of specific disease producing agent(s) by allowing them to pass through filters with a
view to retain the bacterial cells. An affirmative conclusion could be reached easily in the event when
the filtrates (obtained above) failed to produce any infection, and the presence of the disease producing
bacterial agent in the original (infectious) fluid.
The following researchers determined the presence of ‘virus’ in pathological fluids in the following
chronological order :
Chamberland (1884) : First and foremost developed the specially designed ‘porcelain filters’
that exclusively permitted the passage of fluid but not the microorganisms ; and, therefore, could be
used gainfully for the sterilization of liquids. Besides, the application of such devices may also suggest
and ascertain if at all ‘infective agents’ smaller in dimensions than the bacteria could exit actually.
Iwanowski (1892) : Repeated the similar sort of test but employed an extract meticulously
obtained from the infected tobacco plants, with ‘mosaic* disease’. Iwanowski observed that the clear
filtrate was found to be extremely infectious to the healthy tobacco plants.
Beijerinck (1898) : He confirmed Iwanowski’s findings and baptised the contents of the clear
filtrate as ‘virus’ (i.e., infectious poisonous agent). He further affirmed that the virus could be propogated
strategically within the living host.
Loeffler and Frosch (1998) : They first and foremost demonstrated that the clear filtrate happened
to be the main culprit, virus, which had the capability of being transmitted from one infected
animal to another. Later on they amply proved that the lymph** obtained from infected animals suffering
from ‘foot and mouth disease’, whether it was either filtered or unfiltered, both caused infection in
healthy animals almost to the same extent. From the above critical studies one may infer that since
animals infected with the filtered lymph served as a source of inoculum*** for the infection of healthy
animals thereby suggesting overwhelmingly that the infective filterable agent never was a toxin****,
but an agent capable of undergoing multiplication.

FW Twort (1915) : Twort inoculated nutrient agar with smallpox vaccine fluid with a possible
expectation that a virulent variant of vaccinia virus could grow up eventually into colonies. In fact, the
only colonies which actually showed up on the agar plates were nothing but bacteria that proved to be
contaminants in the vaccine lymph. However, these bacterial colonies had undergone a transformation
that turned into a ‘glassy watery transparent substance’, which could not be subcultured anymore.
Salient Features of ‘Glassy-Watery Transparent Substance : The various salient features of
the glassy-watery transparent substance are as given under

* Genetic mutation wherein the tissues of an organism are of different genetic kinds even though
they were derived from the same cell.
** An alkaline fluid found in the lymphatic vessels and the cisterna chyli.
*** A substance introduced by innoculation.
**** A poisonous substance of animal or plant origin

(1) When a ‘normal bacterial colony’ was contacted even with a trace of the ‘glassy-watery
transparent substance’, the normal colony would in turn be transformed right from the
point of contact.
(2) Even when the ‘glassy-watery transparent substance’ subjected to a million-fold dilution
it affords transformation as well as gets across the porcelain bacteria-proof filters.
(3) By successive passages from glossy to normal colonies it could be feasible to transmit the
disease for an indefinite number of times ; however, the specific agent of the disease would
neither grow of its own on any medium, nor would it cause the glassy transformation of heat
killed microorganisms.
(4) The specific agent may also be stored for more than 6 months at a stretch without any loss in
activity whatsoever ; however, it would certainly be deprived of its activity when heated to
60°C for 1 hour.
Twort, in 1915, put forward three logical and possible explanations based on his original discoveries,
namely :
(1) The bacterial disease may represent a stage of life-cycle of the bacterium, wherein the bacterial
cells would be small enough to pass via the porcelain bacteria proof filters, and are
also unable to grow on media which actually support the growth of normal microorganisms.
(2) The causative organism (agent) could be a bacterial enzyme that invariably leads to its own
production and destruction, and
(3) The organism (agent) could be a virus that ultimately grows and infects the microorganisms.
It is, however, pertinent to state here that the later two probabilities (i.e., ‘1’ and ‘2’ above)
gained tremendous recognition and turned out to be the hottest topic of various vigorous investigations
inspite of the brief forceful and unavoidable interruptions caused by the World War 1.

F. d’Herelle (1917) : For almost two years the splendid research and observations of Twort
remained unnoticed until the investigations of d’ Herelle-an entomologist who incidentally encountered
during that period a particular transmissible disease of bacteria while investigating the organisms causing
diarrohea in locust. While experimenting with the coccobacilli* d’Herelle observed that the cell-free filtrates
could give rise to ‘glassy’ transformation. Besides, he watched carefully that in the absence of
cocobacilli the agent i.e., ‘glassy-watery transparent substance’ failed to grow in any culture media.
Interestingly, d’Herelle carried out his research absolutely in an independent manner without the least
knowledge about Twort’s findings. His work prominently and emphatically attracted immense and widespread
attention which ultimately paved the way towards the dawn of a relatively more clear picture of
bacterial viruses.
In addition, d’Herelle helped in the discovery of certain earlier preliminary methodologies for
the assay** of bacteriophages.*** It has been duly observed that the lysates displayed practically little
effect upon the inactivated organisms (bacteria), which fact was further looked into and adequately
established that the bacteriophages are nothing but definitive self-producing viruses that are essentially
parasitic on microorganisms.

* Bacilli that are short, thick, and somewhat ovoid.
** The analysis of a substance or mixture to determine its constituents, and the relative proportion of each.
*** A virus that infects bacteria

A. Lwoff (1921) : Lwoff further ascertained and proved the fact that bacteria invariably carry
bacteriophages without undergoing ‘any sort of clearance’, and it was termed as ‘lysogeny

Emergence of Molecular Biology

Molecular Biology refers to that specific branch of biology dealing with analysis of the structure
and development of biological systems vis-a-vis the chemistry and physics of their molecular
constituents. Now, with the advent of latest laboratory methodologies and modern experimental techniques
the prevailing skill, wisdom, and knowledge pertaining to the characteristic features of microorganisms
accumulated with a tremendous momentum and speed. Based upon the intensive and extensive
information(s) with respect to the in-depth biochemical activities of various microorganisms virtually
became an ‘open-secret’.
Importantly, a careful and critical examination of the copious volume of accumulated data evidently
revealed and suggested that there existed quite a lot of similarities amongst the different microorganisms,
whereas the points of dissimilarities revolved essentially around the variations on a major
central biochemical pathway. Interestingly, at that point in time there prevailed a distinct world-wide
emergent growing recognition between the ensuing unity of the biochemical life processes in microorganisms
and the higher forms of life (including the humans). As a result, it more or less turned out to
be definitely much beneficial and advantageous to employ the microorganisms as a befitting tool for

deciphering and exploring the basic life phenomena. In order to accomplish the aforesaid aims and
objectives the microorganisms do offer invariably a plethora of advantages for this type of research
activities, namely :
􀂳 they reproduce (i.e., cultivate) extremely fast,
􀂳 they may be cultured (grown) either in small or large quantum easily, conveniently, and
quickly,
􀂳 their growth may be manipulated and monitored in a not-so-difficult manner by means of
chemical and physical methods, and
􀂳 their cells may be cleaved and torn apart, and the contents segregated into different fractions
of varying particle sizes.
Conclusively, the above cited characteristic features together with certain other vital factors help
to render the ‘microorganisms’ an extremely vulnerable and a very convenient research-role-model
in pin-pointing and establishing precisely the modus operandi of various life processes that essentially
occur with respect to certain particular chemical reactions, besides the specific structural features
involved intimately.
In the light of the above statement of facts showing the enormous strengths of microorganisms in
the revelation of the intricacies of life processes various scientists and researchers of all disciplines viz.,
physicists, chemists, geneticists, biologists, and microbiologists not only joined their hands together but
also put their intellectual resources and wisdom in a concerted manner to evolve an altogether new
discipline christened as molecular biology. According to Professor Luria* molecular biology may be
defined as — ‘the programme of interpreting the specific structures and functions of organisms in terms
of molecular structure’.
The outcome of the results obtained from the brilliant studies accomplished in the field of molecular􀁏 Elucidation of enzyme structure and mode of action,
􀁏 Cellular regulatory mechanisms,
􀁏 Energy metabolism mechanisms,
􀁏 Protein synthesis,
􀁏 Structure of viruses,
􀁏 Functionality of membranes, and
􀁏 Structure and function of nucleic acids.
Significance of Discoveries : The major significance of discoveries with regard to molecular
biology may be ascertained by virtue of the following breakthroughs :
􀁑 Fundamental information(s) regarding DNA and genetic processes at the molecular level via
bacteria and bacteriophages**, and
􀁑 Many Nobel Prizes bagged due to researches carried out in molecular biology related to
various arms of biology

* Professor Salvador E Luria — at the Massachusetts Institute of Technology (MIT) as a Professor of Biology
was awarded the Nobel Prize in 1969 for his splendid research in the field of molecular biology.
** Viruses that infect bacteria

Industrial Microbiology

An exponential growth in the ever expanding domain of industrial microbiology commenced
logically from the mid of the nineteenth century to the end of the said century. The various vital and
important ‘milestones’ in the field of industrial microbiology may be summarized as stated under :
Emil Christian Hansen (1842-1909) : a Dane*, who actually showed up the brilliant and fertile
way to the extremely investigative field of industrial fermentations. He meticulously examined and
methodically developed the pure culture study of microorganisms and yeasts exclusively utilized in
the large-scale manufacture of ‘fermented vinegar’. This simultaneously encouraged as well as promulgated
the application of pure cultures termed as ‘starters’ associated with the elaborated study of
various fermentation processes.
L. Adametz (1889) : an Austrian, augmented the commercial production of cheese by making
use of pure cultures (i.e., starters).
* A native of Denmark.

HW Conn (in Connecticut, USA) and H Weigmann (in Germany) (1890–1897) : developed
miraculously a host of pure culture starters for the commercial production of butter.
Alcohol Fermentations : Pure culture of yeasts were used to produce alcohol (ethanol) from a
variety of fermentable carbohydrates such as : corn, molasses, potatoes, sugar beets, grapes etc., employed
throughout the world.
In addition to the above mentioned widely consumed and need based products there are several
other highly in-demand industrial products derived exclusively from molds that are being used largel
across the globe as detailed under

:

Pharmaceutical Microbiology

The remarkable and spectacular breakthroughs accomplished by Pasteur, Koch, Jenner, and a

host of others more or less paved the way towards several miraculous discoveries in curing fatal and

dreadful human ailments thereby minimising their immense sufferings. Many meaningful and wonderful

researches also led to the discovery of a good number of causative agents of diseases and altogether

newer techniques for diagnosis, which ultimately rendered the diagnosis of these ailments rather rapid

and precise.

Examples : (a) Widal Test* — for typhoid fever, and

(b) Wasserman Test** — for syphilis.

Importantly, a plethora of dreadful diseases were duly identified and characterized by the presence

of their specific causative microorganisms, such as : Hensen (1874) leprosy (Mycobacterium

leprae) ; Neisser (1879) gonorrhea (Neisseria gonorrhoeae) ; Ogston (1881) wound infections

(Staphylococcus aureus) ; Nicolaier (1885) tetanus (Clostridium titani) ; Kitasato and Yersin (1894)

plague (Yersinia pestis) ; Shiga (1898) dysentry (Shigella dysenteriae) ; Schaudin and Hoffmann (1905)

syphilis (Treponema pallidum) ; Bordet and Gengou (1906) whooping cough (Bordetella pertussis) ;

Ricketts (1909) rocky mountain spotted fever (Rickettsia ricketsii) ;

Some of the important events that mark the history of pharmaceutical microbiology are enumerated

below in a chronological arrangement

Antibiotics : Antibiotic refers to a natural or synthetic substance that destroys microorganisms

or inhibits their growth. Antibiotics are employed extensively to treat infectious, diseases in humans,

animals, and plants. In fact, the terminology ‘antibiotic’ etymologically evidently signifies anything

against life. Obviously, in the event when the microorganisms are critically present in a natural

medium two situations may arise invariably viz., (a) favouring the growth of bacteria usually termed as

‘symbiosis’ ;* and (b) antagonizing the growth of bacteria normally called as ‘antibiosis’.**

Charles Robert Darwin (1809–1882), a British naturalist) aptly commenced scientific and methodical

investigative explorations into the fundamental problems of natural selection and struggle amongst

the interspecies ; and later on came up with his famous doctrine — ‘Survival of the fittest’. Louis

Pasteur (1822–1895) observed for the first time the characteristic antagonistic interrelations prevailing

between the microorganisms of different species.

Joubert and Pasteur first observed the critical destruction of cultures of Bacillus anthracis by

means of certain air-borne microbes. A follow up by Sirotinin (1888) emphatically proved the antagonistic

action of Bacillus anthracis upon the enteric fever, and Blagoveshchensky (1890) carefully ascertained

the antagonistic effect of the blue-pus organism on the Bacillus anthracis. It was ultimately the

miraculous discovery of Lashchenkov (1909) and Alexander Fleming (1922) who meticulously isolated

the enzyme lysozyme***, that was chiefly capable of inhibiting a relatively larger segment of microorganisms.

Chain, Florey, and co-workers (1929) made the epoch making historical development in the

emerging field of antibiotics with the remarkable discovery of wonderful therapeutic and interesting

pharmacological properties of the extracts obtained from the cultures of the mold Penicillium notatum

that eventually gave rise to the formation of the wonder drug ‘penicillin’.

Specifically the antibiotics are extremely useful in the control, management and treatment of a

good number of human infectious diseases but their diversified applications are found to be equally

useful in the meticulous curing and controlling of plant and animal diseases as well. Penicillin has been

effectively employed in the management and control of pests. Antibiotics, in general, are invariably

employed in animal husbandry as ‘feed additive’ to cause enhancement in the fattening of food animals.

Food handling and processing industries extensively make use of antibiotics to critically minimise inevitable

spoilage of fish, vegetables, and poultry products. Present day modern scientific researches

being conducted across the globe do make use of antibiotics as useful and indispensable tools for the

elaborated study of biochemical cellular mechanisms.

Since the discovery of penicillin many more antibiotics came into being as stated under :

Waksman (1944) : Streptomycin — [Streptomyces griseus] — a soil microbe ;

— (1945) : Bacitracin — [Bacillus subtilis] ;

— (1947) : Chloramphenicol (Chloromycetin) — [Streptomyces venezuelae] ;

— (1947) : Polymixin — [Bacillus polymixa] — and various designated polymixins

A, B, C, D, and E.

— (1948) : Chlorotetracycline — [Streptomyces aureofaciens] — a broad-spectrum

antibiotic.

— (1948) : Neomycin — [a species of Streptomyces] — isolated from soil.

— (1950) : Oxytetracycline — [a strain of Streptomyces].

— (1952) : Erythromycin — [Streptomyces erythreus].

It is, however, pertinent to state here that the ‘antibiotics’ may be broadly classified into nine

categories as given below :

[Kar, Ashutosh : Pharmacognosy and Pharmacobiotechnology, New Age International (P)

Ltd., Publishers, New Delhi, 2003].

Important Points : The various important points with respect to the development of antibiotics

are summarized below :

􀁏 in all approximately 5000 antibiotics have been prepared, characterized, and evaluated for

their therapeutic efficacy till date.

􀁏 nearly 1000 antibiotics belonging to only six genera of filamentous fungi i.e., including

Cephelosporium and Penicillium have been reported successfully.

􀁏 about 50 antibiotics have been synthesized from two genera and belonging to the class of

non-filamentous bacteria.

􀁏 nearly 3000 antibiotics have been prepared from a group of filamentous bacteria i.e., including

streptomyces.

􀁏 approximately 50 antibiotics are at present actively used in therapeutic treatment and veterinary

medicine around the world.

Importantly, the most common bacteria that invariably attack the humans specifically, and the

diseases they cause or organs of the body they attack, are listed as under :

Medical Microbiology

Interestingly, the ‘germ theory’ of disease was very much in existence for a long duration ;
however, the direct implication and involvement of germs in causing disease was not well established,
and hence recognized and widely accepted.
The magnificent and remarkable success of Louis Pasteur and Robert Koch not only earned
them befitting honours and accolades from their beloved countrymen, but also rewarded them by
bestowing their gratitude in establishing the famous and prestigious Pasteur Institute in Paris (1888),
and Professor of Hygiene and Director of the Institute for Infective Diseases in the University of
Berlin respectively.
At this point in time altogether newer microorganisms (bacteria) were being discovered with an
ever-increasing speed and momentum, and their disease-producing capabilities were adequately established
and proved by Koch’s four cardinal postulates as stated earlier (see section 1.2.4).
In this manner, the domain of ‘medical microbiology’ gradually received a progressive advancement
through the meaningful researches conducted by several scientists and scholars as enumerated
below :
Edwin Klebs (1883) and Frederick Loeffler (1884) : discovered the diphtheria bacillus,
corynebacterium diphtheriae ; and showed that it produced its toxins (poisons) in a laboratory flask.
Emil von Behring and Shibasaburo Kitasato : devised an unique technique of producing immunity
to infections caused by C. diphtheriae by injecting the toxins into healthy animals so that an
antitoxin**** gets developed.
Shibasaburo Kitasato and Emil von Behring : cultivated (grown) the microorganism responsible
for causing tetanus (lockjaw), Chlostridium titani ; and Behring prepared the corresponding antitoxin
for the control, prevention, treatment, and management of this fatal diseas

Interestingly, the ‘germ theory’ of disease was very much in existence for a long duration ;
however, the direct implication and involvement of germs in causing disease was not well established,
and hence recognized and widely accepted.
The magnificent and remarkable success of Louis Pasteur and Robert Koch not only earned
them befitting honours and accolades from their beloved countrymen, but also rewarded them by
bestowing their gratitude in establishing the famous and prestigious Pasteur Institute in Paris (1888),
and Professor of Hygiene and Director of the Institute for Infective Diseases in the University of
Berlin respectively.
At this point in time altogether newer microorganisms (bacteria) were being discovered with an
ever-increasing speed and momentum, and their disease-producing capabilities were adequately established
and proved by Koch’s four cardinal postulates as stated earlier (see section 1.2.4).
In this manner, the domain of ‘medical microbiology’ gradually received a progressive advancement
through the meaningful researches conducted by several scientists and scholars as enumerated
below :
Edwin Klebs (1883) and Frederick Loeffler (1884) : discovered the diphtheria bacillus,
corynebacterium diphtheriae ; and showed that it produced its toxins (poisons) in a laboratory flask.
Emil von Behring and Shibasaburo Kitasato : devised an unique technique of producing immunity
to infections caused by C. diphtheriae by injecting the toxins into healthy animals so that an
antitoxin**** gets developed.
Shibasaburo Kitasato and Emil von Behring : cultivated (grown) the microorganism responsible
for causing tetanus (lockjaw), Chlostridium titani ; and Behring prepared the corresponding antitoxin
for the control, prevention, treatment, and management of this fatal disease

Emil von Behring bagged the Nobel Prize in 1901 in physiology or medicine.
De Salmon and Theobald Smith : proved amply that immunity to a plethora of infectious
diseases may be produced quite effectively and efficiently by proper timely innoculation with the killed
cultures of the corresponding microorganisms.
Elie Metchnikoff : described for the first time the manner certain specific leukocytes (i.e., white
blood cells) were able to ingest (eat up) the disease-producing microorganisms present in the body. He
baptized these highly specific defenders and crusaders against bacterial infections known as phagocytes
(‘eating cells’), and the phenomenon is termed as phagocytosis.
Metchnikoff’s Theory : Based of the aforesaid explanations Metchnikoff put forward a theory
that — ‘the phagocytes were the body’s first and most important line of defense against a variety of
infection’.
Paul Ehrlich : Paul Ehrlich (Robert Koch’s brilliant student) put forward two altogether newer
concepts with regard to the modus operandi whereby the body aptly destroys microorganisms (bacteria),
namely :
(a) Antibody* : The logical explanation of immunity based upon certain antibodies in the
blood, and
(b) Chemotherapy** and Antibiotics*** : Both these aspects virtually opened the flood gates
to the enormous future developments in combating the growth and destruction of pathogenic
bacteria.
Example : Arsphenamine [Salvarsan(R)] : A light yellow organo-metallic compound (powder)

containing about 30% Arsenic (As), was formerly used in the treatment of syphilis.
The two decades stretching between 1880–1900 proved to be indeed a golden era for the ‘science
of microbiology’ to step into adolescence from the stage of infancy. In fact, during this specific
period many researchers have gainfully identified the causative microorganisms duly responsible for the
eruption of a host of infectious human diseases, such as :
Anthrax, Gonorrhea, Typhoid fever, Malaria, Wound infections, Tuberculosis, Cholera, Diphtheria,
Tetanus, Meningitis, Gas gangarene, Plague, Dysentery, Syphilis, Whooping cough, and Rocky
Mountain spotted fever.

Immunity

Immunity refers to the state of being immune to or protected from a disease, especially an
infectious disease. This state is invariably induced by having been exposed to the antigenic marker on
an organism that invades the body or by having been immunized with a vaccine capable of stimulating
production of specific antibodies.*

Interestingly, Pasteur’s practical aspects and Koch’s theoretical aspects jointly established the
fact that the attenuated microorganisms* retained their capacity and capability for stimulating the
respective host to produce certain highly specific substances i.e., antibodies** which critically protect
against subsequent exposure to the virulent organisms.***
Examples :
(a) Edward Jenner’s successful cowpox vaccine (in 1798) : Jenner’s epoch-making successful
attempts in vaccinating (innoculating) patients with cowpox vaccine, that ultimately resulted
in the development of resistance to the most dreadful smallpox infection.
(b) Pasteur’s successful rabies vaccine : Pasteur’s charismatic fame and reputation became
well known throughout France when he successfully prepared rabies vaccine by innoculating
a rabbit with the saliva from a rabid dog. The healthy rabbit contracted the rabies virus and
died later on. The extract of dead rabbit’s brain and spinal cord were duly attenuated and
injected into rabies patient who eventually survived later on. Thus, the vaccine for rabies or
hydrophobia — a disease transmitted to humans through bites of dogs, cats, monkeys, and
other animals.

Classical Laboratory Methods and Pure Cultures

Microorganisms are abundantly found in nature in sufficiently large populations invariably comprised
of a plethora of different species. It is, however, pertinent to state here that to enable one to carry
out an elaborated study with regard to the characteristic features of a specific species it is absolutely
necessary to have it separated from all the other species.
Laboratory Methods : Well defined, articulated, and explicite laboratory methods have been
adequately developed which enable it to isolate a host of microorganisms representing each species,
besides to cultivate each of the species individually.
Pure Culture : Pure culture may be defined as — ‘the propogation of microorganisms or of
living tissue cells in special media that are conducive to their growth’.
In other words it may also be explained as the growth of mass of cells belonging to the same
species in a laboratory vessel (e.g., a test tube). It was indeed Joseph Lister, in 1878, who first and
foremost could lay hand on pure cultures of bacteria by the aid of ‘serial dilution technique’ in liquid
media.
Example : Lister diluted milk, comprising of a mixture of bacteria, with a specially designed
syringe until a ‘single organism’ was strategically delivered into a container of sterile milk. The container
on being subjected to incubation for a definite period gave rise to a bacteria of a single type, very
much akin to the parent cell. Lister termed it as Bacterium lactis.

Colonies : Koch meticulously devised methods for the specific study of microorganism. He
smeared bacteria on a sterile glass slide, followed by addition of certain specific dyes so as to observe
the individual cells more vividly under a microscope. Koch carefully incorporated some specific solidifying
agents, such as : gelatin, agar into the media in order to obtain characteristic isolated growths of
organisms usually called as colonies. Importantly, each colony is essentially comprised of millions of
individual bacterial cells packed tightly together.
Now, from these identified colonies one may transfer pure cultures to other sterile media. However,
the development of a liquefiable solid-culture medium proved to be of immense fundamental
importance.
Example : Koch thoroughly examined material obtained from subjects suffering from pulmonary
tuberculosis, and succeeded in the isolation of the tubercle bacillus Mycobacterium tuberculosis.
In conclusion, one may summarize the remarkable importance of ‘pure cultures’ toward the
overwhelming development in the field of microbiology, because by the help of pure-culture techniques
several intricate and complicated problems could be answered with reasonable clarification and
complete satisfaction, namely :
􀂳 Microorganisms causing a large number of infections,

􀂳 Certain specific fermentative procedures,
􀂳 Nitrogen-fixation in soil,
􀂳 High-yielding alcohol producing strains from ‘malt wort’, and ‘molasses’,
􀂳 Selected good cultures for making top-quality wines, and
􀂳 Specific cultures for manufacturing dairy products viz., cheeses, yogurt.
Futuristic Goals
The futuristic goals of ‘pure cultures’ are exclusively based upon the following two cardinal
aspects, namely :
(a) better understanding of the physiology of individual microorganisms present in the pure
culture, and
(b) ecological relationships of the entire microbial populations in a given environment.
Thus, the following new horizons in the domain of microbiology may be explored with great
zeal and gusto :
􀁏 Advancements in marine microbiology,
􀁏 Rumen microbiology,
􀁏 Microbiology of the gastro-intestinal tract (GIT), and
􀁏 Several other systems.

Germ Theory

A plethora of observant researchers had already conceptualized and opined rather vehemently
the much applauded and widely accepted ‘germ theory’ of disease even before Pasteur established
experimentally that microbes (or bacteria) happen to be the root cause of several human dreadful
diseases. Later on various other scientists supported and proved the aforesaid ‘germ theory’ in one way
or the other as stated under :
Girolamo Fracastro (1483–1553) : advocated that certain diseases might be caused by virtue of
invisible organisms transmitted from one subject to another.
Plenciz (1762) : stated that the living microbes (or agents) are the ultimate cause of disease but
at the same time aired his views that different germs were responsible for different ailments.
Oliver Wendell Holmes (1809–1894) : suggested that puerperal fever** was highly contagious
in nature ; besides, it was perhaps caused by a germ carried eventually from one mother to another
either by midwives or physicians.
Ignaz Philipp Semmelweis (1818–1865) : pioneered the usage of antiseptics specifically in the
obstetrical practices.
Joseph Lister (1890) : made known in England the importance of antisepsis, which was subsequently
fully appreciated by the medical profession all and sundry.

Robert Koch (1843–1910) : discovered the typical bacilli having squarish ends in the blood
sample of cattle that had died due to anthrax.*
Koch’s Modus Operandi — Koch adopted the following steps to isolate microbes causing anthrax
:
(1) First of all these bacteria were duly grown in cultures in the laboratory.
(2) Bacteria examined microscopically so as to ascertain only one specific type was present.
(3) Injected bacteria into other animals to observe whether they got also infected, and subsequently
developed clinical symptoms of anthrax.
(4) Isolated microbes from experimentally infected animals squarely matched with those obtained
originally from sheep that died due to infection of anthrax.
Koch’s Postulates : The series of vital observations ultimately led to the establishment of Koch’s
postulates, that essentially provided four vital guidelines to identify the particular causative agent for an
infectious disease, namely :
(a) A particular microbe (organism) may invariably be found in association with a given disease.
(b) The organism may be isolated and cultivated in pure culture in the laboratory.
(c) The pure culture shall be able to cause the disease after being duly inoculated into a susceptible
animal.
(d) It should be quite possible to recover conveniently the causative organism in its pure culture
right from the infected experimental animal.

Fermentation

France having the strategical geographical location developed the commercial manufacture of a
large variety of wines and beer as a principal industry. Pasteur played a critical and major role in the
proper standardization of various processes and techniques intimately associated with the said two
‘alcoholic beverages’ in order to obtain a consistently good product. Pasteur used his God gifted wonderful
skill and wisdom to explore and exploit the unique capabilities of microbes in the fermentation
industry exclusively using fruits and grains resulting in alcohol-based table wines, dry-wines, champagne,
whiskies, etc. Pasteur meticulously isolated, typified, and characterized ‘certain microbes’ exclusively
responsible for the ‘good batches’ predominantly in comparison to the ones found solely in
the ‘poor products’.
In fact, the overall net outcome of such extensive as well as intensive investigations helped in a
long way for the assured and successful production of consistently good and uniform ultimate product.
Pasteur vehemently argued and suggested that the unwanted/undesirable types of microbes must be
destroyed and removed by heating not enough to alter the original and authentic inherent flavour/aroma
of the fruit juice, but just sufficient to cause and afford the legitimate destruction of a relatively very high
percentage of the ‘bad microbial population’. This ‘destructive microbial phenomenon’ could be accomplished
successfully by holding the juices at a temperature of 145°F (≡ 62.8°C) for a duration of 30
minutes.
Pasteurization. Nowadays, the large-scale handling of such destructive microbial process may be
achieved by ‘pasteurization’* in commercial fermentation industries using either ‘malt wort’ (having ~
10% solids) or molasses (~ 10% solids) or even fruit-juices.

Spontaneous Generation Vs Biogenesis

The wonderful discovery of microbes both generated and spurred enough interest not only in the
fundamental origin of ‘living things’ but also augmented argument and speculation alike.
Based upon the various experimental evidences the following observations were duly made by
scientists as enumerated below :
John Needham (1713-1781) : Precisely in the year 1749, while experimenting with raw meat
being exposed to hot ashes, he observed meticulously the appearance of organisms that were not present
at the initial stages; and, therefore, inferred that the bacteria virtually originated from the raw meat itself.
Lazaro Spallanzani (1729-1799) : actually boiled ‘beef broth’ for a duration of 60 minutes,
and subsequently sealed the flasks tightly. After usual incubation for a certain length of time, practically
no microbes appeared. However, Needham never got convinced with Spallanzani’s findings, and
vehemently insisted that ‘air’ happened to be an essential component to the process of spontaneous
generation of the microbes, and that it had been adequately excluded from the flasks by sealing them
precisely by the later.
Franz Schulze (1815-1873) and Theodor Schwann (1810–1882) : these two scientists independently
fully endorsed and justified the earlier findings of Spallanzani by allowing air to pass through
strong acid solutions into the boiled infusions, and by passing air into the flasks via red-hot tubes
respectively (Fig. 1.A). In neither instance did microorganisms appear.

H. Schröder and T. von Dusch (~ 1850) : carried out a more logical and convincing experimental
design by passing air via cotton fibers so as to prevent the bacterial growth ; and thus, it ultimately
initiated and gave rise to a basic technique of ‘plugging’ bacterial culture tubes with ‘cotton plugs’
(stoppers), which technique being used still as to date (Fig. : 1.B).
Felix Archimede Pouchet (1800–1872) : revived once again the concept and ideology of spontaneous
generation via a published comprehensive and extensive research article thereby proving its
occurrence. Pasteur (1822–1895) carried out a number of experiments that virtually helped in concluding
the on-going argument once for all time. Pasteur designed a flask having a long and narrow gooseneck
outlet (Fig. : 1.C). Thus, the nutrient broths were duly heated in the above specially–designed flask,
whereby the air — untreated and unfiltered — may pass in or out but the germs settled in the ‘very
gooseneck’ ; and, therefore, practically no microbes ultimately appeared in the nutrient broth (solution).
John Tyndall (1820-1893) : conducted finally various well planned experiments in a specifically
designed box (Fig. : 1.D) to establish and prove the fact that ‘dust’ actually contained and carried
the ‘microbes’ (i.e., germs). He subsequently demonstrated beyond any reasonable doubt that in a
particular situation whereby absolutely no dust was present, the sterile nutrient broth could remain
free of any sort of microbial growth for an indefinite length of time.

The Microscope

The evolution of microscope gathered momentum in the year 1674, when a Dutch cloth merchant
Antony van Leeuwenhoek first of all had a glimpse at a drop of lake-water via a lens made of glass
that he had ground himself. Through this simple device using a magnifying lens Leeuwenhoek first and
foremost ever had an ‘amazing sight’ of the most fascinating world of the microbes.

Later on, Leeuwenhoek critically and explicitly described the finer details of a plethora of microorganisms
viz., protozoa, algae, yeast, and bacteria to the august Royal Society of London (UK) in a
series of letters. It is worthwhile to mention here that the entire description was so precise and accurate
that as to date it is now quite possible to assign them into each particular genera without any additional
description whatsoever.
The earlier observations of microorganisms were made duly by several researchers chronologically
as given below :
Roger Bacon (1220–1292) : first ever postulated that a disease is caused by invisible living
creatures.
Girolamo Fracastoro (1483–1553) and Anton von Plenciz (1762) : these two reseachers also
made similar observations, assertions, and suggestions but without any experimental concrete evidences/
proofs.
Athanasius Kircher (1601–1680) : made reference of these ‘worms’ that are practically invisible
to the naked eyes and found in decaying meat, milk, bodies, and diarrheal secretions. Kircher was,
in fact, the pioneer in pronouncing the cognizance and significance of bacteria and other microbes in
disease(s).
Antony van Leeuwenhoek (1632–1723) : initiated the herculian task of ‘microscope making’
through his inherent hobby of ‘lens making’. During his lifespan stretching over to 89 years he meticulously
designed more than 250 microscopes ; of which the most powerful one could magnify about 200-
300 times only. However, these microscopes do not have any resemblance to the present day ‘compound
light microscope’ that has the ability to even magnify from 1,000-3,000 times.

HISTORICAL DEVELOPMENT OF MICROBIOLOGY — MILESTONES

It is more or less a gospel truth that in science the ultimate credit, glory, and fame goes to the one
who actually succeeds to convince the world, and not to the one who first had conceived the original
concept and idea. Hence, in the development of microbiology the most popular and common names
are invariably of those researchers/scientists who not only convinced the world in general, but also
developed a tool or a specific technique or an idea (concept) which was virtually adopted or who expatiated
their observations/findings rather vividly or astronomically that the science grew and prospered in
particular.
Evidence from the literature reveals that Antony van Leeuwenhoek’s (1632-1723) lucid explanations
with regard to the ubiquitous (i.e., found everywhere) nature of the microbes practically enabled
Louis Pasteur (1822–1895) almost after two centuries to discover the involvement of these microorganisms
in a variety of fermentation reaction procedures that eventually permitted Robert Koch (1843-
1910), Theobald Smith, Pasteur and many others to establish and ascertain the intimate relationship of
the various types of microbes with a wide range of dreadful human diseases. In fact, Robert Koch
bagged the most prestigious Nobel prize in the year 1905 for his spectacular and wonderful discovery
for the isolation and characterization of the bacteria that cause anthrax*** and tuberculosis.****
With the passage of time the ‘mankind’ has won several gruesome battles with dreadful microorganisms
quite successfully and have adequately mustered the knack not only to make them work in an
useful and beneficial manner but also to control and prevent some of those that are rather dangerous and
harmful in nature.