PART-Anewagepublishers.com/samplechapter/001399.pdf · Antony van Leeuwenhoek (1632–1723) Antony van Leeuwenhoek (Fig. 1.3) was born to Dutch couple in 24th October, 1632 in Delft,

PART-A

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DEFINITION AND HISTORY

OF MICROBIOLOGY

3

INTRODUCTION

Microbiology is the study of small organisms called microorganisms that are very smallto 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 littledetail can be observed. Thus, organisms with a diameter 1 mm or less are considered asmicroorganisms and systematic study of these microorganisms comes under the branch ofmicrobiology and must be examined with a microscope.

Human society benefits from microorganisms in many number of ways. They are necessaryfor various products that are required in daily human life. Microorganisms are considered tobe indispensable components of our entire ecosystem. They are considered to be the firstliving organisms evolved on this earth which is 4.5 billion years old (Fig. 1.1). This is evidentfrom the stromatolites available in various fossil materials.

Even though, microorganisms are ubiquitous in its distribution but their identity wasfound 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 asLouis Pasteur and Robert Koch.

Today microorganisms are used extensively as research tools in divercified field frommedicine 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 microbiologyis 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 livingorganisms that are too small to be seen by unaided eye. These small organisms are calledmicroorganisms or microbes.

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The word ‘microbe’ was coined by Se’dillot, a French army retired surgeon in 1878. Theexistence of these microbes was not known until the discovery of microscopes. Microscopesare the optical instruments used to magnify objects that are too small to be clearly observedby the naked eye.

Microscopes invented at the beginning of the seventeenth century opened the biologicalworld of the very small to the systematic scientific exploration. The early microscopes wereof two types. The first types were simple microscopes with a single lens of very short focallength which was similar to magnifying lenses. The second types were compound microscopeswith two lenses made up of ocular and objective lenses. These compound microscopes wereable to magnify objects several thousand times and eventually replaced the conventionalsimple microscopes. But, majority of the original discoveries were done by using the simplemicroscopes by several microbiologists.

Phanerozoic e ra

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Noaccum ula tiono f rocks

Caenozoic era

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Increase o f O content to about 20%

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O2

Banded iron-stone(from 2 .9 to 1.6 b illion years)

Sta rt of photosynthetic p roductionO2

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Stromatolites

Fig. 1.1 Chronological history of the earth. (1 billion = 1000 million/109).

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 modesof 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 knownas phototrophs. The plants are best example for the phototrophs and are also known asautotrophs. 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 numberof microorganisms, difficulty was faced with placing them either in plants or animals. Thusa third kingdom Protista was proposed by Haeckel in 1866.

The kingdom Protista contains those organisms that are differentiated from plants andanimals by their absence of morphological specialization and most of them are unicellular innature. These Protista are further divided into higher protists and lower protists. Higherprotists are eukaryotes having resemblance with the animal and plants and include microalgae,fungi and protozoa. Lower protists are prokaryotes and include bacteria and cyanobacteriaand their cellular structure is different from that of other living organisms. Microorganismsare included in all these above groups and also consist of acellular particles such as viruses,prions and viroids.

Euglena

An im a lsP lan ts

Fung iM icroalgae Pro tozoa

Eubacter ia ArchaebacteriaProkaryotes(Pro tocytes)

Eukaryotes(Eucytes)

M icroorgan ism s

Fig. 1.2 Different types of microorganisms.

The term “microorganisms” does not have the precise taxonomic importance because itis 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 membersof which share many common structural and functional properties. But, in the case ofmicroorganisms 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 theirliving 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 microbialprocesses learned to prepare various food products such as sour milk, curd, cheese and otherproducts.

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The attempts made to learn the causes of infectious diseases led to the proposal of livingnature (contagium vivum) of contagious diseases. Even before microorganisms were seen,some scientists proposed their existence and responsibility for disease. Roman philosopherLucretius (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 previouslyunknown invisible mysterious world of minute organisms was discovered.

Glass polishers Hans and Zaccharius Jansen in 1590 constructed first a device withmagnifying 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 thefirst compound microscope with a convex objective and ocular lenses appeared. The inventor ofthis microscope was thought to be the physicist C. Drebbel. This type of microscope was laterused 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 Antonyvan Leeuwenhoek (1632-1723). He himself made simple lenses which magnified 160 to 300fold. In 1678, Antony van Leeuwenhoek published his work on ‘animalicula viva’, liveanimalcules, which he observed in water, various infusions, faeces and teeth scrapings alongwith the meticulous drawings.

The pioneer discovery made by Antony van Leeuwenhoek stimulated a live interestamong scientists. This also became the starting point for the systematic study of the microbialworld and formed the foundation stone for the science of new biological science calledmicrobiology. The optical device which Antony van Leeuwenhoek made formed the forerunnerfor the modern world microscope. The animalicula viva (living animals), which Antony vanLeeuwenhoek observed, are now considered as bacteria and protozoa.

The development of microbiology as a branch of science is considered in the followingthree era. The summary of some of the major events with respect to microbiology is representedin Table 1.1.

Table 1.1 A few important events in the development of microbiology

Year Microbiological Events1546 Fracastoro suggests that invisible organisms cause disease.1590 Jansen develops first useful compound microscope.1676 Leeuwenhoek discovers “animalcules”.1838 Schwann and Schleiden proposed cell theory.1857 Pasteur showed lactic acid fermentation by microbes.1885 Pasteur develops rabies vaccine.1892 Ivanovsky proved viruses causation of tobacco mosaic disease.1910 Ehrlich develops chemotherapy for syphilis.1915 D’Ilerelle and Twort discovered bacteriophages.1923 First edition of Bargey’s manual.1929 Fleming discovers penicillin.1944 Waksman discovers streptomycin.1953 Watson and Crick discovered DNA structure.1970 Arber and Smith discovered restriction endonucleases.


1983 Gallo and Montagnier isolated and identified HIV.1986 First genetically engineered Hepatitis vaccine approved for human use.1995 Haemophilus influenzae genome sequenced.1996 Methanococcus jannaschii genome sequenced.1997 Escherichia coli genome sequenced.1998 Mycobacterium tuberculosis genome sequenced.2000 Vibrio cholerae genome sequenced.2001 Mycobacterium leprae genome sequenced.2002 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 inschool in the Warmond town. During 1654 his family shifted back to Delft and he spent entirelife in the same city. He started his own business and started different hobbies. He was adraper (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 cityand a minor city official. During 1666, he started grinding lenses as his hobby and constructedhis own simple microscopes. He used such simple microscopes for observing the weavingpattern of various types of cloths. For the curiosity, he started observing the things surroundinghim. 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 ofvarious 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 ofdifferent magnifications. He was so involved in grinding the lenses that he entirely gave upthe cloth business and took the lense making work as full time job. He designed the microscopein such a way that all were simple but had highly powerful magnifying optical devices. Whencompared to the present day microscopes they were very simple and around 4 inches inlength. It consisted of one lense (a), which was mounted in a small hole in the brass platethat formed the body of the device (See Chapter 5 Fig. 5.3A). The specimen was placed atthe tip of blunt pin (b) attached to back plate. The pin tip was brought into focus by adjustingthe two screws (c) and (d). This device was held very close to eye during the observation. Themain disadvantage of his microscopes was that the magnifying power of each microscopecould not be increased.

Antony van Leeuwenhoek had great curiosity and by using his own microscopes heobserved 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 scrapingsfrom his teeth, tail fins of fishes for his observations.

Even though Antony van Leeuwenhoek did not have formal university education, he hadkept all his observations recorded. Each recorded observation was supported with the accuratedrawings. He made observation on blood and found tiny rounded objects which are consideredas red blood cells. Leeuwenhoek also observed yeasts and said that they were made of smallround particles.

Antony van Leeuwenhoek’s fame in the field of microbiology came from his discoveryand explanation of microbes which he called ‘animalcules’ (tiny animals). He observed themin 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, showingmovement in the microscopic field. The different types of main unicellular microorganismsthat we know today—algae, bacteria, protozoa and yeasts, were first described by Antony vanLeeuwenhoek with great accuracy in the early years of 1676.

Antony van Leeuwenhoek started communicating his observations to the Royal Societyof England, which was established for the communication and publication of the scientificwork. 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 writtenon 9th October 1676, he gave a detailed description of the microbial forms. All the descriptionswere appropriately supported by the accurate drawings. In 1680, he was elected as Fellowof Royal Society.

Antony van Leeuwenhoek, during explaining the microorganisms, said about theseorganisms as, in his own writings—

“ ... in the year 1675, I discovered living creatures in rainwater which had stood for afew days in a new earthen pot, glazed blue within. This invited me to view this water withgreat attention, especially those little animals appearing to me ten thousand times less thanthose ... 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 whathe 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 peppergrain, he said—


“I discovered in a tiny drip of water, incredibly very little animalcules and these wereof diverse sorts and sizes. They moved with bending, as an eel always swims with its headin 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 observationsand explained different forms of animalcules such as rods, spheres and spiral shapes whichwere nothing but the different morphological forms of bacteria. This was the first recordedinformation of bacteria and even today this morphological classification of animalcules isknown to be perfect for bacteria.

Antony van Leeuwenhoek had not proceeded further beyond describing the microbes. Henever tried to associate them with their surroundings as causative agents. It is also said thatAntony van Leeuwenhoek was a secretive person and invited none to work with him, nor didhe reveal the secret of his technique of grinding lenses. During the time of Leeuwenhoek andeven later, microscopic observation was regarded as an idle man’s job with no practicalrelevance.

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 circulationof blood.

ERA OF TRANSITION

Leeuwenhoek’s discovery of microbial world took long period of time to get its importancein the science. This was due to optical defect, which limits the observation of microorganismsby using compound microscopes. Leeuwenhoek gave a clear indication of microbial diversityalong with its incredible abundance in the nature. This made scientists to think about theirorigin.

There were two schools of thought. One group believed that animalcules were formedspontaneously from the non-living materials and this is known as spontaneous generationor abiogenesis. But, others including Leeuwenhoek believed that they were formed fromthe ‘germs’ or ‘seeds’ of these animalcules, which were always present in the air. This wasreferred as biogenesis. This raised the controversy over abiogenesis and biogenesis andabiogenesis has had a long acceptance. Scientists at that time believed this, because meatkept open for longer period of time produced maggots and mushrooms suddenly appeared onrotten wood.

Later, due to increased knowledge of living organisms, abiogenesis gradually lost itsacceptance. Doubtfulness about the abiogenesis started by meticulously performed experimentby 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 coveredwith paper, and the third was covered with a fine gauze that would not allow flies to enterthe container. After some time flies laid eggs on the meat that was not covered. In the othertwo containers maggots were not produced on the meat. But, flies moved towards gauzecovered 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 spontaneouslyproduce maggots, but they were produced from the eggs that were laid by the flies on themeat. This was not considered true for the microorganisms and they said microorganismsarose 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 thatorganic matter contains vital force that converts a non-living material into living ones. A fewyears later this was again questioned by the experiments of another priest, LazzaroSpallanzani.

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 waterin glass flasks. Then it was sealed and boiled for ¾ of an hour. In these flasks no growthof microorganisms was noticed until the seals were broken. From this he proposed that aircarried microbes to the broth medium and the said air is required for the growth ofmicroorganisms. But supporters of abiogenesis claimed that heating the air in sealedcontainer completely destroyed the ability of air to support the life forms, because duringthis period Priestley and Lavoisier clearly indicated that oxygen (air) is essential to lifeforms.

Many workers later tried to give a proper answer to these arguments in support ofbiogenesis. 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. Thiswork was further improved by George Friedrich Schroder and Theodore von Dusch. They passedthe air through sterile cotton wool into sterile culture medium and allowed for growth ofmicroorganisms. No growth was noticed even though the air had not been heated. This paved theway 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 occurwithout air contamination. This argument attracted French scientist Louis Pasteur whodecided 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. Hesuccessfully developed efficient method of vaccination for the first time against viral diseaseeven though its pathogenic agent was not isolated in pure form. The vaccination (Latin vaccameans cow) is a term later adopted and coined by the Pasteur in the recognition and honorof the work contributed by Edward Jenner. Vaccination is the process of protection of anindividual by the preparation of microbes or its contents.


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 handledinfected cows for milking. Such cow pox lesions were very much similar in character tosmallpox. But, they were restricted, localized and known to cause mild symptoms withoutcomplications. Cow pox was also not able to disperse from one person to another person (non-communicable). It were also observed that people who were infected with cow pox showedcomplete resistance against the smallpox. The cow pox pustules by some phenomena protectedthe individuals from the infection by smallpox.

These facts made Edward Jenner to take up serious observation because he was amedical student. Later, as a physician, he made detailed investigation and recorded all hismethods of practice. By the repeated experiment for many years he considered that cow poxlesions gave protection against smallpox disease. During his studies Jenner collected the pusfrom a cow pox lesion from the hand of a dairy maid (called Sarah Nelms) in 1796. Then herubbed it on the skin surface of a healthy boy (called James phillip). Later the boy developeda typical scar on the skin. After a few weeks the boy was inoculated with the pus taken fromthe smallpox pustules of a diseased person. But the boy could not show any disease ofsmallpox. Hence, boy showed the complete immunity to the disease. From experimentsperformed later and scientific accumulation of data of his studies, he concluded that inoculationof a person with cow pox pus could offer immunity from the smallpox disease. Later, in June1798, 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 hisquest for conquering disease and is also heralded as a significant milestone in the history ofmicrobiology. Though vaccination was met with greater opposition in the beginning, it hasstood the test of time and proved its advantage in medical field in later dates. Through thisprocess of vaccination the smallpox virus and the disease have been eradicated from theworld 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 LouisPasteur, Robert Koch, John Tyndall, Elie Metchnikoff and others. They provided the soundscientific principles without debatable answers to the abiogenesis phenomena. This wasgreatly accepted by the scientific community of that age. The majority of the microbialdiscoveries, 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. Hewas first involved in the study of chemistry and obtained the doctorate in Chemistry fromthe Norman School in Paris and became Professor of Chemistry at Paris.

Fig. 1.5 Louis Pasteur (1822-1895).


Death blow to the theory of abiogenesisLouis 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 cottonand then the sterile cotton was placed in a sterile medium. This showed the growth ofmicroorganisms and indicated that air contained these microorganisms. Later, he pouredsterile nutrient medium in specially designed flasks called swan-necked flasks. These areround-bottom flasks having curved necks of various types with open ends. He then boiled themedium to free the contamination and allowed to cool. In this, growth was not observed eventhough the curved necks had opening to surrounding atmosphere.

Pasteur thus opined that no growth was seen because dust and germs had been trappedby the walls of the curved necks. But, growth would be noticed if the curved necks werebroken because the air could enter directly into the flasks with depositing the germs and duston the neck walls. However, Pasteur not only conclusively disproved the theory of spontaneousgeneration or abiogenesis in 1861, but also showed how to maintain any solution in sterilecondition 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 repeatedheating and cooling of the medium that can completely destroy all living germs in thesolution) that are highly resistant to regular boiling.

Pasteur with his knowledge in chemistry came into the limelight while working onvariety of biological fields including medicine because he had great insight into the problemswhich he came across. Thus, he was called for the majority of the unsolved problems in thedifferent fields. The major contribution of Pasteur to the field of microbiology in particularand to the science in general is listed below.

Tartaric acid isomersTartaric 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 capacityto refract light in different directions called polarization. One type of crystals refracted light(polarized) towards right and others towards left. These were microscopically separated intotwo 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 TheodoreSchwann (1837) showed that yeast cells are responsible for the process of fermentation. ButGerman chemist Justin Liebeg proposed that fermentation was a chemical process. During thisperiod distillers of France faced a problem of their alcohol turned into sour taste. This totallyhampered 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 greaterdetail. He could observe yeast cells and bacteria under microscopic field. Pasteur consideredthat yeast cells were responsible for the fermentation process. By setting up a number ofconclusive experiments he demonstrated that yeast cells turned grape juice (sugar) intoalcohol. By the application of heat Pasteur destroyed the yeast cells in the grape juicemixture. In this case no alcohol was formed and alcohol formation could be regained whenthe 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 sourtaste is formed due to the growth of the specific type of bacteria and their fermentationin the mixture. The basic discovery of microbial fermentation was remarkable inmicrobiology, because up to that time microbes were considered as the things of curiosityand none could link them with any biochemical activity of significance. Pasteur publisheda research paper on fermentation that formed the basis of emergence of microbiology asa branch of Biology. This could also gave a solution to the problem which occurred in theFrench distilleries.

Aerobes and anaerobes discoveryPriestley showed that organisms required air (oxygen) for the growth and development.

Hence, scientists during 19th century believed that microorganisms also needed oxygen for thegrowth and development. Microorganisms that require oxygen for the growth are referred asaerobes or aerobic microorganisms (E.g.: Bacillus, Pseudomonas). Pasteur during his studieson 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 ofmicrobes were actively showing the motility. These microbes could not show the motility whenexposed to the air. He also showed that fermentation of the mixture could be stopped byaeration of the broth. Pasteur named those groups of microorganisms that did not requireoxygen for their growth as anaerobes or anaerobic microorganisms (E.g.: Clostridium).

Process of pasteurizationThe 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 killthe undesirable microbes present in the grape juice. These microorganisms produced lacticacid and acetic acid in the fermentation mixture. By this process Pasteur showed that spoilageof taste of wine (due to the presence of acid) can be prevented by exposing fermentationmixture to low heating at 60°C for a brief period instead of complete boiling. This temperaturewas more than enough for the killing of undesirable bacteria which acted as contaminationin 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 processesin the food industry and others. Present day pasteurization temperatures are heating ofliquid at 62.8°C for 30 minutes (slow pasteurization) or 72°C for 15 seconds (flashpasteurization or high-temperature short-term [HTST] pasteurization).

Work on pebrine diseaseThe 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 disastrouseconomic loss for the country. This was due to the pebrine disease affecting the silk worms andresulted in large scale spread of the disease in majority of the silk worm rearing areas. ThenMinistry of Agriculture appointed Pasteur as a major person to study and find the propersolution for the disease.

Pasteur after detailed investigation in the affected area for three years was able to findout the causative agent of the disease as a protozoa member (Nosema bombysis). He alsoshowed the farmers to get rid off the disease by using the disease-free eggs for rearing and


destroying the affected eggs and older leaves that were used as silk worms feed. Thus by usinghygienic source material the disease could be completely eliminated. The silk industry ofFrance was thus saved from the major economic loss. This path-breaking discovery led him tobecome a great patriot apart from being an eminent scientist and scholar.

Work on anthraxIn 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 theGovernment economy. In the meantime, work done by the German physician Robert Kochrevealed that the anthrax bacterium (Bacillus anthracis) was associated with the disease butcould not establish the relationship with the disease causing ability of the bacterium. It wasPasteur’s idea which proved that the bacteria present in the culture medium was causing thedisease in healthy animals.

Pasteur also got a clue on the process of vaccination or protective inoculation of healthyhost to get resistance against disease. He successfully demonstrated that inoculation ofweakened 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 thecuriosity and interest in many of the contemporary scientists towards vaccination. Pasteurdeveloped 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 theresearch 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 thoughPasteur 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 observationof Edward Jenner, he planned to place an avirulent (non-virulent; made by growing virus inan abnormal host, the rabbit) mixture of dried, grinded spinal cord under the dog skinsurface. This process continued for a few days with increased virulent strain preparationfrom spinal cord extract. In the end of the experiment, the animal successfully got resistanceagainst the disease. The rabid dog bite and application of most potent extracts to brain ofhealthy animal failed to induce rabies. With this success in the case of animals, he alsoexplained the need for further research work in the application of this process directly tohuman beings.

Before the further research work could occur the situation had aroused which led Pasteurto practice it even on humans. During the course of his experiments, Joseph Meister, a nineyear 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 tryingvaccination. The boy was vaccinated 13 times over the next 10 days with increasingly virulentpreparations of the attenuated strain of virus. To the surprise the boy survived with thevaccination and later became the faculty in Pasteur Institute.

In the gratitude for Pasteur’s development of vaccination process, people from differentparts of the world immensely contributed to initiation of research work in a separate institutecalled Pasteur Institute in Paris, France. He died in Paris, on 28th September, 1895. Histomb 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 thecoagulation of blood and inflammation that occurred after injuries and surgical wounds. Hebecame Professor of Surgery at Glasgow and Edinburgh. Lister started concentrating toreduce the gangrene and bacterial infections. During his time gangrene was considered tobe caused by the bad quality air. Even after maintaining the cleanliness in surgical materialsand rooms, the death rate was very high (50%).

Lister made several contributions for the improvement of the microbiology as a wholeand medicine in particular.

Fig. 1.6 Joseph Lister (1827–1912).

Antiseptic techniqueLister 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 observerand surgeon, Lister was attracted towards post-operative complications in patients due towound infections. He made detailed study the cause of such wound infections. He observed asimilarity between the wound inflammation, fermentation and putrefaction described byPasteur, which was caused due to microorganisms. This made him to believe that the microbesentered the wound through air, hands, surgical instruments or bandage cloths and thusresulted 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.


Lister by his observations came to the practice of applying carbolic acid as a microbicidalchemical agent. He covered all the wounds with the carbolic acid-soaked bandage cloths. Thisresulted 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 tobe known as antiseptic system of operation or antiseptic surgery.

Pure culture techniqueMicroorganisms 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 speciescharacteristics it is necessary to separate it from all other species present in the mixedpopulation. Lister in 1878, by using serial dilution technique for the first time, obtainedpure culture of bacteria in liquid media. By using a specially designed syringe, he dilutedcurdled milk containing a mixture of bacteria until a single bacterium was delivered into acontainer of sterile milk. After incubation he observed the bacteria in that container. Theywere 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 medicalpractitioner. Later Robert Koch switched over to microscopic studies that he engaged in hisfull life time. Then he became Professor of Hygiene and headed as Director in the Instituteof Infective Diseases at Berlin, Germany. Robert Koch is considered as one of the mainfounders 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 diseasesRobert 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 shapedmicroorganisms in the blood of diseased animals. For testing if these microorganisms werethe causative agents of anthrax disease, he injected a healthy mice with the blood from adiseased 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 mouseand again the mouse died due to disease. Koch repeated this experiment for 20 times andeach time the healthy mice died after inoculation.

In the laboratory Robert Koch cultured these bacteria in the aqueous humour from theeyes of a cow. He was able to reproduce the disease every time through inoculating thesemicroorganisms into healthy animals. With his experimental procedures he laid down certainbasic criteria for considering microorganisms as causative agents of the disease. These criteriaare 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 inpure culture.

(c) The isolated infectious microorganism must reproduce the disease when inoculatedinto a healthy individual, and

(d) The same infectious agent must be reisolated from the experimentally induceddiseased individual.

These postulates of Koch continue to be useful even today with some modification.

2. Discovery of Mycobacterium tuberculosisAfter the anthrax work Koch concentrated towards widespread tuberculosis disease.

With the intensive research work on tuberculosis, Robert Koch discovered and proved in 1882that all forms of infectious tuberculosis were caused by the bacillus forms called Mycobacteriumtuberculosis. He showed method of culturing these bacilli in pure form and made detailedstudy on the Mycobacterium tuberculosis.

3. Preparation of smearsAs 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 thetechnique of making smears of bacteria on glass slides and staining them with anilinedyes to observe them more clearly under the microscope. Making of smears on the slidesdistributed the microorganisms in thin layer without overlapping and gave the access forthe proper staining.

4. Plating method for isolation of pure cultureIn 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 certaindisadvantages such as the moist surface of potato allowed motile bacteria to spread on entiresurface. As the surface of potato is opaque it was very difficult to observe bacterial coloniesand it is not a good nutrient medium for large number of bacterial strains.


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Owing to above mentioned disadvantages, Koch planned that if a liquid nutrient mediumcould be solidified by the addition of a solidifying agent such as gelatin, then it could be usedfor the isolation of bacteria in the laboratory. He poured the molten gelatin medium on glassplates and allowed it to solidify. After the gel had solidified, the surface was streaked witha platinum wire dipped into a bacterial suspension. These plates were then covered andincubated. After incubation period the growth was found all along the streaking line andsingle colonies were found at the end of the streak. These single colonies contained only onetype of bacteria and this formed the pure cultures of bacteria. Thus, the use of gelatin forthe preparation of a solid medium and the streak plate method for the isolation of purebacterial cultures came into existence.

Koch’s another associate, Walter Hesse gave a clue of using agar as solidifying agent forits advantage over gelatin. Richard Petri, a student of Robert Koch in 1887, discovered usageof the petri dishes in the laboratory for the culturing purpose. This enables the microbiologiststo 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 wasmade possible when Charles Chamberland (Pasteur’s colleague) developed porcelain bacterialfilters in 1884. This made the microbiologists to use such filters and study the viruses ingreater detail.

During this period development was also taking place in determining how animalsresisted disease and in understanding immunity. After the discovery of diphtheria toxin, Emilvon Berhring (1854–1917) and Shibasaburo Kitasato (1852–1931) inoculated inactivated toxininto rabbits. Later a substance known as antitoxin (antibody against diphtheria toxin) wasproduced in the blood and this protected against the disease by inactivating the toxin.

Fig. 1.8 Elie Metchnikoff (1845-1916).

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The antitoxin work provided evidence that immunity could result from soluble substancespresent in the blood. This is now well known that these substances are antibodies that areresponsible 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 theimmunity. He referred these cells as phagocytes and the process as phagocytosis. Thus, wasopened 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, atLochfield in Scotland. He studied medicine and obtained his degree from St. Mary’s MedicalSchool, London University. Later, he joined the military service and worked as captain inArmy Medical Corps throughout First World War.

During 1921, working with blood and its action on bacteria he discovered the bacteriolyticproperties 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) andan excellent bacteriologist working at St. Mary’s Hospital, in London, discovered Penicillinthe first wonder drug.

Fig. 1.9 Alexander Fleming (1881–1955).

When he was working in the laboratory, one of the petri plates containing the bacterialculture (Staphylococcus aureus) attracted his attention. In his laboratory, Fleming wasperforming experiments in search of novel bactericidal agents, particularly against woundinfections. During the course of experiments, he observed that a culture plate of Staphylococcusaureus was contaminated by a blue-green coloured fungal growth. The area surrounding theedges of the fungal colony was clear. The growth of Staphylococcus aureus near the fungalcolony was completely inhibited or was killed by the fungus. Fleming by careful examination


opined that something that is excreted by the fungal colony proved to be fatal to Staphylococcuscells. The contaminated fungal colony was then isolated and identified as a common blue-green fungal member Penicillium notatum.

Later, Fleming cultured this fungal colony on broth medium in a separate flask. Thenfrom the broth medium he obtained the exudates content. In order to test his hypothesis thatthe fungal secretions could destroy Staphylococcus he inoculated this extraction into laboratorymice infected with Staphylococcus, Streptococcus and Pneumococcus. The mysterious compoundpresent in this extraction killed all the microorganisms in the body of the mice as it had donesimilar thing on Staphylococcus aureus in the petri plate. Then, Fleming named this mysteriouscompound as Penicillin. Fleming, in 1929, published a research article in the British Journalof Experimental Pathology.

This work later drew the attention of Sir Howard W. Florey and Ernst B. Chain atOxford University. In due course Howard Florey and Ernst Chain successfully purified thepenicillin from the culture filtrate of Penicillium notatum.

This meant that the penicillin could be used for the large scale purpose. During 1941industrial production of penicillin had started and proved efficient against bacterial infectionduring First World War. In Medicine category Fleming shared the Nobel Prize along withH.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 andworked 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 inthis category.

Fig. 1.10 Sergei N. Winogradsky (1856-1953).

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Sergei N. Winogradsky (Fig. 1.10), a Russian microbiologist, made several importantcontributions to the soil microbiology. Winogradsky discovered that soil bacteria are involvedin oxidization of iron, sulfur and ammonia to get their energy requirement. He also discoveredthat photosynthetic bacteria, similar to plants, can also use CO2 to produce organic materials.Another new groups of microorganisms were discovered by Winogradsky including anaerobicnitrogen-fixing microorganisms. He isolated thems from the soil habitat and also studied indetail the decomposition of cellulose in the soil.

Martinus W. Beijerinck (Fig. 1.11), another Russian microbiologist also made many basiccontributions 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 manysulfate-reducing bacteria from soil.

Fig. 1.11 Martinus W. Beijerinck (1851-1931).

Later Beijerinck and Winogradsky together developed the enrichment technique ofculturing of microbes and the use of selective media in the growth and isolation of pureculture. This became a great importance in the microbiological field.

Heinrich Anton de Bary (1831–1888), a German doctor, made large number of contributionsto the fungi and mycology and established Phytophthora infestans as cause of the late blightof potato. Anton de Bary (Fig. 1.12) laid clear foundations of mycology and hence he iscommonly 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 virologyby discovery of tobacco mosaic disease, causative agent, a virus (later named as Tobaccomosaic virus). This gave way for observing the organisms that are not easily studied by usingconventional microscopes and started virology as a major field in microbiology.


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 soilactinomycetes bacterium Streptomyces and discovered streptomycin, a major broad spectrumantibiotic. Selman Waksman (Fig. 1.14) was the person who coined the word ‘antibiotic’ andwon the Nobel Prize in 1952 for the same.

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Fig. 1.14 Selman Waksman (1888-1973).

David Hendricks Bergey (1860–1937) was the person who headed the manual preparationteam for the entire bacterial organisms identifications called Bergey’s Manual. This is mostimportant for Bary proper identification and placing of newly discovered bacterium. DavidHendricks Bergey’s (Fig. 1.15) name is still used in the recent edition of the manual for hisgreat work done in bacteriology.

Fig. 1.15 David Hendricks Bergey (1860-1937).

In the early part of twentieth century, microbiology developed independently of otherbiological disciplines due to difference in focus. During this period microbiologists were more


concerned with the agents of infectious disease, the immune response, the search of newchemotherapeutic compounds and bacterial metabolism.

Branch of microbiology made a closer relationship with other branches of biology during1940s. 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 theyare relatively simple, grow rapidly and can be grown in large quantities in small area. Themutants of bread mold Neurospora were used by George W. Beadle and Edward L. Tatumin 1941 for studying the relationship between genes and enzymes. In 1943, Salvadore Luriaand Max Delbruck studied spontaneous gene mutation by using bacterial mutants. Later in1944, Oswald T. Avery, Colin M. MacLeod and Maclyn McCarty proved conclusively that DNAwas the genetic material and carried genetic information during the gene transformation.The intimate interactions between microbiology, genetics and biochemistry soon helped in thedevelopment of modern molecular oriented genetics. J.D. Watson and F.H.C. Crick discoveredthe 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 ofmolecular biology (the branch of biology which deals with the physical and chemical aspectsof living matter and their function) into greater heights. Microbiologists have been deeplyinvolved in detailed studies on the mechanisms of DNA, RNA, genetic code and proteinsynthesis. Microorganisms were more often employed in many of the early studies on thegene regulation and enzyme regulation. Discoveries made during 1970s contributed to theemergence of recombinant DNA technology and genetic engineering.

This is clearly evident when we observe the list of Nobel Prizes awarded in Physiology orMedicine category. About 1/3 of the prizes have been awarded to the scientists working onmicrobiological or related problems. Thus, it shows the importance of microbiology in thebranch of science.

Microbiology is one of the most rewarding of professions. This is because it gives itspractitioners the opportunity to be in contact with all the other natural sciences and thus tocontribute in many different forms and ways to the improvement of science in general andhuman life in particular.

SUMMARY

Microbiology is the branch of biology which deals with the study of organisms that areusually too small to be seen by naked eye. It includes viruses, viroids, prions, bacteria, manyalgae, fungi and protozoa. Microbiology is not a mere study of structure and classification ofmicrobes. The various aspects of microbiology, both basic and applied aspects can be studied.

Discovery of microorganisms made by Antony van Leeuwenhoek in 1675 was consideredto be a starting point for microbiology. The history of microbiology can be divided into era ofdiscoveries, 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 animalsand red blood cells (thus considered as founder of histology) and also described capillarycirculation of blood. The era of transition considered to be the scientific method of accepting

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the life forms as produced only from pre-existing living forms and not from non-living organismsor materials. Microbiologists such as Francesco Redi, John Needham, Lazzaro Spallanzani,Theodore Schwann and Edward Jenner were the major contributors to this era. In the goldenera of microbiology contributions made by large number of microbiologists helped in theestablishment of a new branch of science, microbiology. Microbiologists namely Louis Pasteur,Joseph Lister, Robert Koch, Winogradsky, Beijerinck and Alexander Fleming were the veryimportant scientists and their large number of contributions helped in better understandingof microbes and their applications to the human society. Hence, it is rightly called the goldenera of microbiology.

The major contributions of Louis Pasteur include proving the biogenesis, discovery ofmicrobial fermentations, identification of aerobes and anaerobes, causative agent of pebrinedisease, pasteurization, isolation and identification of isoforms of tartaric acid and vaccination.Pasteur began his scientific career as chemist but soon became a pioneer microbiologist. Forthese 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 thewound 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 ofchemotherapy of various infectious diseases. Soil microbiology was developed because of thepioneer contributions made by Winogradsky and Beijerinck. They isolated and identifiedAzotobacter, Rhizobium and other soil microorganisms and their role in soil fertility andbiogeochemical cycles.

Later both technological developments and basic scientific developments in variety offields 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


8. Explain how Antony van Leeuwenhoek contributed to initiating microbiology.

9. What are the contributions of Robert Koch, Alexander Flemming and Joseph Lister to thefield of microbiology? (April/May 2000, April/May 2001).

10. Discuss contributions of Louis Pasteur (Nov./Dec., 1999).

11. 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 relateddiscovery 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.

Documents

PART-Anewagepublishers.com/samplechapter/001399.pdf · Antony van Leeuwenhoek (1632–1723) Antony van Leeuwenhoek (Fig. 1.3) was born to Dutch couple in 24th October, 1632 in Delft,