List of top seven microbiologists of all times:- 1. Leeuwenhoek 2. Louis Pasteur 3. Robert Koch 4. Joseph Lister 5. Dmitri Iwanowski 6. Sergius Winogradsky and Martinus Willem Beijerinck 7. Paul Ehrlich.
Microbiologist # 1. Leeuwenhoek: Father of Microbiology:
Leeuwenhoek lived in a town, called Delft in Holland. By profession, he was a dress-maker and had hardly any training in science. But he developed a remarkable aptitude in making powerful biconvex lenses by grinding good quality glass.
He used the lenses to inspect cloth fibres. He used these lenses to construct very simple-looking microscopes which had hardly any similarities with the present-day microscopes. But his instruments were powerful enough to magnify small objects by 200 times or even more. His microscopes consisted of a single biconvex lens which was fitted between two metal plates.
The object to be magnified had to be placed on the tip of a needle which could be brought into focus by means of several Screws fitted to a bracket. Using proper illumination the observer could see the magnified image of the object from the other side of the lens.
A diagrammatic sketch of one of his microscopes is shown in Fig. 1.2.:
It astounds one to believe what Leeuwenhoek achieved with this improvised microscope. He was the first to describe the spermatozoa, the red blood corpuscles, free-living as well as parasitic protozoa and, above all, the bacteria which he called animalcules (small animals). He was specially fascinated with these tiny organisms which he observed in many samples collected by him.
They were first detected by him in the scrapings of his own teeth (1683). He described not only the different morphological types, like spheres, rods and spirals, but also accurately observed the characteristic motility of some of these types. His descriptions supported by drawings were so precise that it is possible even to identify some of these bacteria.
He communicated his observations in the form of a series of letters to the Royal Society of London. His achievements were given due recognition during his lifetime and he was honoured by a Fellowship of the Royal Society. He continued his scientific pursuits till his death at the age of 91.
Leeuwenhoek’s discovery of the animalcules and other microbes revealed the presence of a hitherto unknown world—the microbial world. However, definite knowledge about the activities of microbes began to be clear only after the lapse of one and a half century, primarily through experimental microbiology initiated by the French chemist, Louis Pasteur (1822-1895).
Microbiologist # 2. Louis Pasteur: The Germ Theory:
One of the fields that attracted Pasteur’s attention first was fermentation. Although alcoholic fermentations for production of beer and wine as well as of vinegar were practiced for many years, the involvement of microbes in them was not unequivocally proved. Before Pasteur, Cagniard-Latour (a French physicist), Schwann (a German physiologist) and Kiitzing (a German botanist) independently claimed, during 1836-37, that alcoholic fermentations were due to the vital activity of living yeasts.
But leading chemists of that time—like Leibig, Berzelius and Wohler, who dominated the scientific world— rejected the ‘vital’ theory. They claimed that fermentation was a purely chemical process and that yeasts found in fermentation liquors were merely a by-product produced by various non-crystalline substances and by no means the cause of fermentation.
In 1854 when Pasteur was appointed as the Head of the Science Department in the University of Lille, his attention was drawn to the problem of ‘souring’ of wine. It was noticed by wine merchants that sometimes the fermentation went wrong and the wine produced went sour making it unsuitable for market.
Pasteur was able to demonstrate that souring was due to the growth of an organism which were uniformly rod-shaped and smaller than the larger elliptical yeast cells, and that they produced lactic acid which turned wine sour.
Eventually, Pasteur formulated the general principle that for each type of fermentation there was a specific living ferment which resulted in formation of specific products, like ethyl alcohol, acetic acid, lactic acid and butyric acid. He showed that butyric acid fermentation occurred in complete absence of oxygen or air, thus giving rise to the concept that life could also exist in complete absence of air (anaerobic).
Pasteur also showed that in presence of sufficient air yeast did not produce alcohol and he concluded that air inhibited alcoholic fermentation (Pasteur Effect). Pasteur’s observations on fermentations laid a solid foundation for the development of the ‘germ theory’.
Just as a ‘bad’ fermentation (e.g. souring of wine) is caused by the invasion of a ‘wrong’ organism (e.g. lactic acid bacteria), so many diseases could be caused by invasion of germs. This idea was developed into the ‘germ theory of disease’ by Pasteur and another great contemporary scientist, Robert Koch (1843-1910).
Microbiologist # 3. Robert Koch: Father of Medical Microbiology:
Jacob Henle (1840) formulated for the first time the conditions to be fulfilled for proving the causal relation of a disease with a specific microbe or germ. Some 36 years later, his student Robert Koch, a German physician, demonstrated experimentally that a bacterial disease called anthrax was caused by a spore-forming rod-shaped bacterium, Bacillus anthracis.
Pasteur and Koch working independently proved this. The conditions originally laid down by Henle were tested and fulfilled in the investigation on anthrax and Koch restated them in clear terms (1876). They have come to be known as Koch’s Postulates.
In recognition of his work on anthrax, Koch was appointed at the Imperial Office of Health in Berlin in 1880. There he started his work on tuberculosis which earned him great fame and a Nobel Prize (1905). He produced scientific evidence to prove that tuberculosis, the most dreadful disease of that time, was caused by ‘tubercle bacilli’ (T.B.).
Koch was able to cultivate the bacteria in artificial culture in the laboratory after overcoming many initial difficulties. He had to develop specialized staining method for observing the bacteria under the microscope. He was able to show the presence of characteristic bacteria in the tubercles of animals which were artificially infected. Thus Koch’s Postulates were fulfilled to prove the causal relation of Mycobacterium tuberculosis and tuberculosis. The bacteria are also popularly known as Koch’s bacillus.
Like Pasteur, Koch was a versatile experimenter. Not only his associates discovered the causal organisms for many important diseases, but many new microbiological techniques were developed in his laboratory. The isolation of culture of typhoid bacillus (Salmonella typhi) by Gaffky, the diphtheria bacilli (Corynebacterium diphtheriae) by Loeffler, discovery of diphtheria antitoxin by von Behring, culture of tetanus bacillus (Clostridium tetani) by Kitasato were all carried out in Koch’s laboratory.
At the same time, important discoveries of medical microbiology were being made in Pasteur’s laboratory. Roux and Yersin demonstrated that an extracellular toxin produced by diphtheria bacilli was responsible for the symptoms of the disease. Metchnikoff discovered (1889-90) phagocytosis of bacteria by the white blood corpuscles in Pasteur’s laboratory. Pasteur’s fame reached a climax with the discovery of anti-rabies vaccine (1885).
These pioneering works laid the foundation of medical microbiology which made it possible to discover the causal agents of most of the infectious diseases caused by bacteria. To Koch’s laboratory goes the credit of using agar as an agent for solidification of bacteriological culture media for the first time.
Agar replaced gelatin which melts at a temperature at which pathogenic bacteria can grow best. Also in Koch’s laboratory, the Petri dish was first designed (by Petri) and used. Both of these helped a lot in isolation and culturing of pathogenic bacteria.
A Historical Controversy: Does Life Arise Spontaneously?
It was a general belief which persisted through ages that living creatures, particularly small ones like maggots, flies or even toads and rats could arise spontaneously from various decaying organic matters. This was known as spontaneous generation.
Although the development of maggots from rotten meat or of mice from decaying rags etc. was no longer believed in the 19th century, the spontaneous generation of microbes in meat broth and infusions remained a controversial issue, and time and again this issue came to the forefront.
Some scientists, like Needham (1749) and Buffon, a famous 18th century naturalist, even produced some experimental proof to show that different kinds of infusions, boiled or un-boiled, invariably gave rise to microorganisms.
Spallanzani (1767), an Italian naturalist, came forward to disprove Needham’s findings and showed that hermetic sealing of the flask containing broth followed by boiling for sufficiently long time would keep the broth free of microorganisms for an indefinite period and that microbial growth would start only if fresh air was let in. Taking clue from Spallanzani’s experiment, a French wine-maker developed a method for preservation of food materials, thus initiating the canning industry.
Spallanzani’s experiments should have rested the controversy for good. But they did not, because with the discovery of oxygen in 1774, the supporters of spontaneous generation raised the question whether exclusion of air from the flasks containing broth was the cause for absence of microorganisms. They contended that microbial growth failed to appear because there was no air which contained oxygen essential for life and growth appeared when fresh air was allowed to enter. This argument was countered by Schwann (1837) by showing that a boiled infusion would remain free of microbial growth if air was admitted through a strongly heated coiled tube.
Pasteur became actively interested in the problem, because he firmly believed that life could arise only from life. He wanted to prove that this was not only true for higher organisms, but also for microorganisms. In 1861, he published his findings and he demonstrated in it that microorganisms were always present in air, though their number in a given volume of air varied depending on the location and altitude.
He proved that when a few microorganisms from air entered into boiled infusions, they rapidly multiplied in number. His famous “swan-neck” flask experiments showed beyond doubt that a boiled infusion remained microbe-free, even if free access to air was allowed, provided the microbes were prevented entry.
The long drawn out neck of the flasks trapped the microbes present in air (Fig. 1.3). Pasteur’s experiment finally silenced the critics who argued that exclusion of air was the cause of absence of microbial growth in the infusions and the long-drawn “war of infusions” came to an end.
Microbiologist # 4. Joseph Lister: Antiseptic Surgery:
One of the serious problems facing surgeons was post-operational sepsis of surgical wounds resulting in high percentage of mortality. Joseph Lister (1827-1912) came to know that Pasteur demonstrated the presence of microorganisms in air. He reasoned that he could prevent or reduce sepsis of surgical wounds by preventing the access of microorganisms from air to the wounds.
He, therefore, resorted to careful sterilization of surgical instruments, dressings etc. and carried out the operation of the patient under a spray of diluted carbolic acid which was known to be a strong disinfectant. These measures proved very successful in reducing mortality due to sepsis. Lister’s antiseptic surgery (1867) together with the discovery of chloroform as an anaesthetic agent greatly improved surgery.
Another important contribution of Lister is of basic significance in microbiology. He was the first person to isolate a pure culture of bacteria by a method based on serial dilution of a suspension containing a mixed population of microorganisms.
He used a special kind of syringe capable of transferring a small measured quantity of fluid. Starting with a lactic acid fermentation liquor he continued transferring small quantities to sterile water until the transferred fluid contained a single bacterium.
The gradual diminution in the number of organisms was checked microscopically. By this method he was able to make a pure culture of lactic acid bacteria (1878). The principle of this method was adopted and refined by Koch using a solidified medium.
He developed the dilution-streak and the dilution pour plate methods using solidified medium in Petri dishes. The individual bacteria were immobilized in gelatin or agar gel and multiplied in situ to form colonies. These methods are still being used routinely in microbiological laboratories for isolation of microorganisms.
Microbiologist # 5. Dmitri Iwanowski: Discovery of Virus:
Chamberland, an associate of Pasteur, constructed a porcelain filter in 1864, the pores of which were so small that bacteria could not pass through them i.e. the filtrate was bacteria-free. In 1892, a Russian botanist named Dmitri Iwanowski was surprised to find that an extract of tobacco leaves infected with mosaic disease which was passed through such a porcelain filter could still infect healthy tobacco plants.
This was a very significant observation, because the bacteria were believed to be the smallest infective agents known at that time. Iwanowski’s observation was soon confirmed by others and within a short time several other plant and animal diseases were reported to be caused by such filterable agents. Since microscopic examination of the filtrates revealed no cellular structures, the agents were believed to be poisonous infective fluids.
About 30 years later Beijerinck, a Dutch microbiologist, showed that the infective agent of tobacco mosaic disease could be precipitated by treating the filtrate with alcohol. He concluded that the agent was not a living organism, but a “fluid infectious principle”, a poison or virus. Stanley (1935), an American scientist, made the dramatic discovery that the infectious principle of tobacco mosaic disease could be crystallized. The crystals were at first thought to be made up entirely of protein.
But later on closer scrutiny it was revealed that they also contained a small amount of ribonucleic acid (RNA). Thus the agent came to be known as tobacco mosaic virus or TMV. That the viruses have also a particulate structure which was different in size and shape for different viruses became gradually clear only after the discovery and refinement of electron microscopy.
Microbiologist # 6. Sergius Winogradsky and Martinus Willem Beijerinck: Microorganisms as Geo-Biochemical Agents:
The importance of the roles played by microorganisms living in soil and water for maintenance of the lives of plants and animals has been realized slowly. The beginning of such realization started with the works of two great microbiologists, Sergius Winogradsky (1856-1953), who worked in the Pasteur Institute, Paris, and Martinus Willem Beijerinck (1851-1931) who worked in Delft, Holland.
Through their work it became evident that there existed different groups of microorganisms in natural habitats, each group carrying out a distinctive type of function and having characteristic nutritional requirements. Each group of organisms took active part in specific geo-biochemical cycles of nature which are of profound significance in maintaining the lives of plants and animals.
Winogradsky’s major contribution concerned the understanding of the role of a specific group of microorganisms involved in conversion of ammonia to nitrate, a process known as nitrification. Ammonia, a product of protein decomposition, is oxidized by a group of bacteria, called nitrifying bacteria, through nitrite to nitrate, which is then taken up by most plants as a source of nitrogen for synthesis of their own proteins.
Nitrification is an important part of the nitrogen-cycle. Winogradsky was able to isolate the bacteria in pure culture which carry out the two-step oxidation process which itself was and still is a difficult task. But what was far more important was that by studying their physiology, he concluded that these bacteria were autotrophic and were able to grow in purely inorganic media supplied with an ammonium salt, carbon dioxide and some other inorganic salts.
Not only they did not require any organic compound like most other bacteria, but they failed to grow in presence of any organic compound. He advanced conclusive evidence to prove that the nitrifying bacteria were able to utilize CO2 as the sole carbon-source like photosynthetic organisms and utilized chemical oxidation energy instead of light. He designated this process as chemosynthesis (1891).
Beijerinck devoted himself to another group of organisms which were also involved in the nitrogen cycle of nature viz. the nitrogen fixers. Molecular nitrogen (N2) is present in abundance in the atmosphere (nearly 80% by volume in air), but nitrogenous compounds which sustain growth of plants in soil and water are generally in short supply.
This is so because very few organisms (entirely prokaryotes) have the ability to convert the inert atmospheric molecular nitrogen to nitrogenous compounds. The process is known as nitrogen fixation or, more appropriately, di-nitrogen (N2) fixation. Beijerinck succeeded in isolating a number of such bacteria—both aerobic and anaerobic—from natural habitats.
During their studies of the nitrifying and nitrogen-fixing bacteria, Winogradsky and Beijerinck independently developed the enrichment culture technique which provided the principle for designing methods for isolation of many other important groups of microorganisms and mutants.
The technique of enrichment makes use of the knowledge about the physiological capability of a specific group of microorganisms to create environmental conditions which would selectively support the growth of that group only. This would result in increase in number of one particular desired group (enrichment) in preference to others present in a mixed population. A step-wise enrichment may eventually eliminate totally or most of the undesirable organisms, making it easy to isolate the desired organism in pure culture by the serial dilution method.
Microbiologist # 7. Paul Ehrlich: Chemotherapy:
Artificial immunization provided a powerful tool to fight against communicable diseases caused by different agents. It is a prophylactic measure which prevents pathogens to establish themselves in the body by increasing the defense (immunity). A second front of attack on pathogens is to kill them by drugs.
So, use of drugs as medicines is a curative measure, because they act on pathogens after they have secured a foothold in the body making it sick. Various types of drugs have been used in different medicinal systems for very long time. An ideal drug would be one that would selectively destroy the pathogen in vivo without causing much harm to the patient.
Paul Ehrlich (1909) was the first to use the term chemotherapy to describe the application of such drugs for curing diseases. He systematically searched for a drug against syphilis which would selectively kill the pathogen of syphilis without adversely affecting the patient. After many attempts he was able to synthesize an organic arsenic compound with desired properties. It was named as ‘Salvarsan’. Salvarsan and Ehrlich won the 1908 Nobel Prize for Physiology or Medicine with Metchnikoff.
A breakthrough in chemotherapy research came after quite some time. In 1935 Domagk discovered that a dye called ‘Prontosil’ was effective in controlling bacterial infections. Prontosil contained a dye conjugated with a sulfanilamide molecule. Domagk showed that the antibacterial activity of Prontosil was actually due to the sulfanilamide part and not to the dye itself.
However, though sulfanilamide had antibacterial activity, it was found to be too toxic for internal use in humans. Later, chemical modifications of this simple compound gave rise to a series of very effective and much less toxic derivatives which came to be known as ‘sulfa-drugs’. They have been extensively used to control various bacterial infections before the antibiotics were discovered. Structures of sulfanilamide, the parent molecule and some of its derivatives (sulfa-drugs) are shown above (Fig. 1.4.)
The golden age of chemotherapy was ushered in with the discovery of antibiotics. An antibiotic is a chemical substance of biological origin which can inhibit the growth or kill microorganisms at a very low concentration. Hundreds of antibiotics have been discovered by systematic search, but relatively few have proved good enough for human application.
The history of antibiotics started with the observation of Alexander Fleming (1929) that a bluish-green mold growing in a petridish containing staphylococci (bacteria) inhibited their growth. The fungus, identified as Penicillium notatum, was found to secrete a substance in the growth medium which inhibited growth of bacteria. The substance was named penicillin.
In 1939, the work on isolation and purification of penicillin was begun by H.W. Florey and E.B. Chain. During the Second World War (1939-45), large-scale production of this antibiotic was completed and penicillin proved to be a ‘wonder-drug’. Encouraged by the success of penicillin, systematic search for antibiotics began all over the world.
The second clinically usable antibiotic was discovered by S.A. Waksman and his coworkers in 1944. It was streptomycin which proved highly effective against the tubercle bacilli (Mycobacterium tuberculosis). Streptomycin is produced by a kind of bacteria Streptomyces griseus belonging to the actinomycetes. Several other clinical antibiotics, like Chloromycetin, aureomycin, terramycin, neomycin, erythromycin, kanamycin, rifamycin etc. have been isolated from different species of the genus Streptomyces.
The antibiotics brought a revolution in the medical world. Many bacterial diseases which claimed thousands of valuable human lives can now be controlled with relative ease by application of antibiotics. Human longevity throughout the world has been extended significantly as a result of their use. Large- scale production of antibiotics also revolutionized the industry based on microbes.