DEFINITION AND HISTORY OF MICROBIOLOGY

Transcription

1 PART-A

2 1 DEFINITION AND HISTORY OF MICROBIOLOGY INTRODUCTION Microbiology is the study of small organisms called microorganisms that are very small to be seen by naked human eye. An object less than 0.1 mm (100 µm; 1 mm = 1000 µm) diameter cannot be seen by the naked eye, whereas in the object less than 1 mm very little detail can be observed. Thus, organisms with a diameter 1 mm or less are considered as microorganisms and systematic study of these microorganisms comes under the branch of microbiology and must be examined with a microscope. Human society benefits from microorganisms in many number of ways. They are necessary for various products that are required in daily human life. Microorganisms are considered to be indispensable components of our entire ecosystem. They are considered to be the first living organisms evolved on this earth which is 4.5 billion years old (Fig. 1.1). This is evident from the stromatolites available in various fossil materials. Even though, microorganisms are ubiquitous in its distribution but their identity was found only during 17 th century by using primitive microscopes. However, as branch of science, microbiology has greater impact in 19th century by the work of meticulous scientists such as Louis Pasteur and Robert Koch. Today microorganisms are used extensively as research tools in divercified field from medicine to waste management. DEFINITION OF MICROBIOLOGY Microbiology is thus concerned primarily with organisms and agents such as viruses, bacteria, cyanobacteria, fungi, many algae, helminths and protozoa. The word microbiology is derived from micros means small; bios means life; logos means study. Microbiology often has been defined as a branch of science which deals with the living organisms that are too small to be seen by unaided eye. These small organisms are called microorganisms or microbes. 3

3 4 BIOTECHNOLOGY-II The word microbe was coined by Se dillot, a French army retired surgeon in The existence of these microbes was not known until the discovery of microscopes. Microscopes are the optical instruments used to magnify objects that are too small to be clearly observed by the naked eye. Microscopes invented at the beginning of the seventeenth century opened the biological world of the very small to the systematic scientific exploration. The early microscopes were of two types. The first types were simple microscopes with a single lens of very short focal length which was similar to magnifying lenses. The second types were compound microscopes with two lenses made up of ocular and objective lenses. These compound microscopes were able to magnify objects several thousand times and eventually replaced the conventional simple microscopes. But, majority of the original discoveries were done by using the simple microscopes by several microbiologists. 2 Billion years 0 1 Phanerozoic era Proterozoic era Billion years 0 Caenozoic era 0.06 Mesozoic era 0.24 Paleozoic era late middle early 0.57 metazoa 0.9 Single celled eukaryotes 1.6 Aerobic respiration Increase of O content to about 20% 2 Stable O 2 -containing atmosphere (0.01%) 3 late Oxygenic photosynthesis Banded iron-stone (from 2.9 to 1.6 billion years) Start of photosynthetic O 2 production 4 Archaeic era middle early No accumulation of rocks Anoxygenic photosynthesis Anoxygenic autotrophy (methanogens and sulphur respiring organisms) Start of C accumulation org Stromatolites 4.5 Fig. 1.1 Chronological history of the earth. (1 billion = 1000 million/10 9 ). Earlier, living organisms were classified as kingdom Animalia (animals) and Plantae (plants) by their differences in form and constitution (Fig. 1.2). The difference in the modes of nutrition forms the key factor. The organisms which have the capacity to synthesize their

4 DEFINITION AND HISTORY OF MICROBIOLOGY 5 own food by using simple inorganic materials and sunlight as a source of energy are known as phototrophs. The plants are best example for the phototrophs and are also known as autotrophs. The organisms which feed on complex organic substances are called heterotrophs. The animals are best example for the heterotrophs. But with the discovery of large number of microorganisms, difficulty was faced with placing them either in plants or animals. Thus a third kingdom Protista was proposed by Haeckel in The kingdom Protista contains those organisms that are differentiated from plants and animals by their absence of morphological specialization and most of them are unicellular in nature. These Protista are further divided into higher protists and lower protists. Higher protists are eukaryotes having resemblance with the animal and plants and include microalgae, fungi and protozoa. Lower protists are prokaryotes and include bacteria and cyanobacteria and their cellular structure is different from that of other living organisms. Microorganisms are included in all these above groups and also consist of acellular particles such as viruses, prions and viroids. Plants Animals Eukaryotes (Eucytes) Fungi Microalgae Euglena Protozoa Microorganisms Eubacteria Archaebacteria Prokaryotes (Protocytes) Fig. 1.2 Different types of microorganisms. The term microorganisms does not have the precise taxonomic importance because it is not restricted to a particular living organism s taxonomic group such as vertebrate, invertebrate or angiosperm. Each of these groups defines a restricted group, all the members of which share many common structural and functional properties. But, in the case of microorganisms it is not the same. Any organism of microscopic dimensions, by the definition, comes under the microorganisms category irrespective of its taxonomic position. Microorganisms are present in all the four spheres of the environment viz., biosphere, hydrosphere, geosphere, and atmosphere. HISTORY OF MICROBIOLOGY Even before the discovery of microorganisms certain microbial processes caused by their living activities, such as fermentation of wine, milk, yeast and others were known to mankind. In ancient times at the beginning of the human civilization, man by using these microbial processes learned to prepare various food products such as sour milk, curd, cheese and other products.

5 6 BIOTECHNOLOGY-II The attempts made to learn the causes of infectious diseases led to the proposal of living nature (contagium vivum) of contagious diseases. Even before microorganisms were seen, some scientists proposed their existence and responsibility for disease. Roman philosopher Lucretius (about B.C.), Roger Bacon ( ) and physician Girolamo Fracastoro ( ) suggested that disease was caused by invisible living creatures. During the 17th century due to the success attained in the field of optics, the previously unknown invisible mysterious world of minute organisms was discovered. Glass polishers Hans and Zaccharius Jansen in 1590 constructed first a device with magnifying glasses which allowed them to see minute objects. This started the era of microscopes. In Galileo Galilei ( ) made the first simple microscope. In the first compound microscope with a convex objective and ocular lenses appeared. The inventor of this microscope was thought to be the physicist C. Drebbel. This type of microscope was later used to study the cells of plant and animal tissues and also minute organisms. The remarkable and pioneer discovery was made by the Dutch cloth merchant Antony van Leeuwenhoek ( ). He himself made simple lenses which magnified 160 to 300 fold. In 1678, Antony van Leeuwenhoek published his work on animalicula viva, live animalcules, which he observed in water, various infusions, faeces and teeth scrapings along with the meticulous drawings. The pioneer discovery made by Antony van Leeuwenhoek stimulated a live interest among scientists. This also became the starting point for the systematic study of the microbial world and formed the foundation stone for the science of new biological science called microbiology. The optical device which Antony van Leeuwenhoek made formed the forerunner for the modern world microscope. The animalicula viva (living animals), which Antony van Leeuwenhoek observed, are now considered as bacteria and protozoa. The development of microbiology as a branch of science is considered in the following three era. The summary of some of the major events with respect to microbiology is represented in Table 1.1. Table 1.1 A few important events in the development of microbiology Year Microbiological Events 1546 Fracastoro suggests that invisible organisms cause disease Jansen develops first useful compound microscope Leeuwenhoek discovers animalcules Schwann and Schleiden proposed cell theory Pasteur showed lactic acid fermentation by microbes Pasteur develops rabies vaccine Ivanovsky proved viruses causation of tobacco mosaic disease Ehrlich develops chemotherapy for syphilis D Ilerelle and Twort discovered bacteriophages First edition of Bargey s manual Fleming discovers penicillin Waksman discovers streptomycin Watson and Crick discovered DNA structure Arber and Smith discovered restriction endonucleases.

6 DEFINITION AND HISTORY OF MICROBIOLOGY Gallo and Montagnier isolated and identified HIV First genetically engineered Hepatitis vaccine approved for human use Haemophilus influenzae genome sequenced Methanococcus jannaschii genome sequenced Escherichia coli genome sequenced Mycobacterium tuberculosis genome sequenced Vibrio cholerae genome sequenced Mycobacterium leprae genome sequenced Plasmodium falciparum genome sequenced. ERA OF MICROBIAL DISCOVERY Antony van Leeuwenhoek ( ) Antony van Leeuwenhoek (Fig. 1.3) was born to Dutch couple in 24th October, 1632 in Delft, Netherlands (earlier known as Holland). He had a formal primary education in school in the Warmond town. During 1654 his family shifted back to Delft and he spent entire life in the same city. He started his own business and started different hobbies. He was a draper (a cloth merchant) and he sold clothes to the wealthy ladies of the city. Antony van Leeuwenhoek was also a qualified surveyor, official wine taster of the city and a minor city official. During 1666, he started grinding lenses as his hobby and constructed his own simple microscopes. He used such simple microscopes for observing the weaving pattern of various types of cloths. For the curiosity, he started observing the things surrounding him. He was also inspired by the work of Robert Hooke and his illustrated book Micrographia. Later, he took up lense grinding work completely and started making more perfect lenses of various types. Fig. 1.3 Antony van Leeuwenhoek ( ).

7 8 BIOTECHNOLOGY-II During his entire life time Antony van Leeuwenhoek made nearly 500 microscopes of different magnifications. He was so involved in grinding the lenses that he entirely gave up the cloth business and took the lense making work as full time job. He designed the microscope in such a way that all were simple but had highly powerful magnifying optical devices. When compared to the present day microscopes they were very simple and around 4 inches in length. It consisted of one lense (a), which was mounted in a small hole in the brass plate that formed the body of the device (See Chapter 5 Fig. 5.3A). The specimen was placed at the tip of blunt pin (b) attached to back plate. The pin tip was brought into focus by adjusting the two screws (c) and (d). This device was held very close to eye during the observation. The main disadvantage of his microscopes was that the magnifying power of each microscope could not be increased. Antony van Leeuwenhoek had great curiosity and by using his own microscopes he observed variety of materials. He observed seeds, embryo, leaves, plant tissues, hair, insects eye, crystals, different types of fluids (rainwater, pond water, blood). He also used scrapings from his teeth, tail fins of fishes for his observations. Even though Antony van Leeuwenhoek did not have formal university education, he had kept all his observations recorded. Each recorded observation was supported with the accurate drawings. He made observation on blood and found tiny rounded objects which are considered as red blood cells. Leeuwenhoek also observed yeasts and said that they were made of small round particles. Antony van Leeuwenhoek s fame in the field of microbiology came from his discovery and explanation of microbes which he called animalcules (tiny animals). He observed them in different sources such as pond water, stagnant water, rainwater, fruits and seeds infusion, scrapings from his teeth and others. He was very much excited to see them living, showing movement in the microscopic field. The different types of main unicellular microorganisms that we know today algae, bacteria, protozoa and yeasts, were first described by Antony van Leeuwenhoek with great accuracy in the early years of Antony van Leeuwenhoek started communicating his observations to the Royal Society of England, which was established for the communication and publication of the scientific work. In the first letter of 7th September 1674, he gave explanation to the tiny animals animalcules. These animalcules are now considered as protozoa. In his eighth letter written on 9th October 1676, he gave a detailed description of the microbial forms. All the descriptions were appropriately supported by the accurate drawings. In 1680, he was elected as Fellow of Royal Society. Antony van Leeuwenhoek, during explaining the microorganisms, said about these organisms as, in his own writings... in the year 1675, I discovered living creatures in rainwater which had stood for a few days in a new earthen pot, glazed blue within. This invited me to view this water with great attention, especially those little animals appearing to me ten thousand times less than those... which may be perceived in the water with naked eye. He was very meticulous and accurate in his descriptions and it is now clear that what he had observed must have been different types of algae, bacteria, fungi, protozoa and others. On 16th June 1675, during the observation of well water into which he had added a pepper grain, he said

8 DEFINITION AND HISTORY OF MICROBIOLOGY 9 I discovered in a tiny drip of water, incredibly very little animalcules and these were of diverse sorts and sizes. They moved with bending, as an eel always swims with its head in front and never, tail first; yet these animalcules swam as well backwards as forwards, though their motion was very slow. Obviously what Leeuwenhoek saw were the motile bacteria. In 1683, he made observations and explained different forms of animalcules such as rods, spheres and spiral shapes which were nothing but the different morphological forms of bacteria. This was the first recorded information of bacteria and even today this morphological classification of animalcules is known to be perfect for bacteria. Antony van Leeuwenhoek had not proceeded further beyond describing the microbes. He never tried to associate them with their surroundings as causative agents. It is also said that Antony van Leeuwenhoek was a secretive person and invited none to work with him, nor did he reveal the secret of his technique of grinding lenses. During the time of Leeuwenhoek and even later, microscopic observation was regarded as an idle man s job with no practical relevance. He also made observation on spermatozoa, small invertebrate animals and red blood cells (thus he was also considered as founder of histology) and he also described capillary circulation of blood. ERA OF TRANSITION Leeuwenhoek s discovery of microbial world took long period of time to get its importance in the science. This was due to optical defect, which limits the observation of microorganisms by using compound microscopes. Leeuwenhoek gave a clear indication of microbial diversity along with its incredible abundance in the nature. This made scientists to think about their origin. There were two schools of thought. One group believed that animalcules were formed spontaneously from the non-living materials and this is known as spontaneous generation or abiogenesis. But, others including Leeuwenhoek believed that they were formed from the germs or seeds of these animalcules, which were always present in the air. This was referred as biogenesis. This raised the controversy over abiogenesis and biogenesis and abiogenesis has had a long acceptance. Scientists at that time believed this, because meat kept open for longer period of time produced maggots and mushrooms suddenly appeared on rotten wood. Later, due to increased knowledge of living organisms, abiogenesis gradually lost its acceptance. Doubtfulness about the abiogenesis started by meticulously performed experiment by an Italian physician Francesco Redi in Francesco Redi ( ) Theory of abiogenesis was first challenged by Francesco Redi with experimental proof. He placed fresh meat in three glass containers. One was kept open, the second was covered with paper, and the third was covered with a fine gauze that would not allow flies to enter the container. After some time flies laid eggs on the meat that was not covered. In the other two containers maggots were not produced on the meat. But, flies moved towards gauze covered container and laid their eggs on the gauze itself (as they could not enter the container).

9 10 BIOTECHNOLOGY-II Later these eggs produced maggots. This clearly indicated that the meat did not spontaneously produce maggots, but they were produced from the eggs that were laid by the flies on the meat. This was not considered true for the microorganisms and they said microorganisms arose spontaneously. To support this John Needham performed the experiment. John Needham ( ) John Needham, an English priest, in 1748 did experiments to support the views of abiogenesis. He boiled the mutton broth and then covered the mouth of the flasks tightly. Later these flasks became turbid and consisted of microorganisms. He thus proposed that organic matter contains vital force that converts a non-living material into living ones. A few years later this was again questioned by the experiments of another priest, Lazzaro Spallanzani. Lazzaro Spallanzani ( ) Lazzaro Spallanzani was an Italian priest and naturalist and by his experiment strongly argued that microorganisms were not spontaneously generated. He kept seeds and water in glass flasks. Then it was sealed and boiled for ¾ of an hour. In these flasks no growth of microorganisms was noticed until the seals were broken. From this he proposed that air carried microbes to the broth medium and the said air is required for the growth of microorganisms. But supporters of abiogenesis claimed that heating the air in sealed container completely destroyed the ability of air to support the life forms, because during this period Priestley and Lavoisier clearly indicated that oxygen (air) is essential to life forms. Many workers later tried to give a proper answer to these arguments in support of biogenesis. The main scientists were Theodore Schwann, Friedrich Schroder, von Dusch, Pasteur, Tyndall and others. Theodore Schwann ( ) Theodore Schwann by passing air through red-hot tube into sterile culture medium showed that even after providing the air microbes did not appear spontaneously in the broth medium. This work was further improved by George Friedrich Schroder and Theodore von Dusch. They passed the air through sterile cotton wool into sterile culture medium and allowed for growth of microorganisms. No growth was noticed even though the air had not been heated. This paved the way for practice of plugging the culture vessels with cotton plugs in all culture works. But French naturalist Felix Pouchet still argued that microbial growth can occur without air contamination. This argument attracted French scientist Louis Pasteur who decided to settle the matter towards biogenesis. Edward Jenner ( ) Edward Jenner (Fig. 1.4) was an English physician, who, during his treatment practice, discovered the method of safe and proper immunity protocol for smallpox disease. He successfully developed efficient method of vaccination for the first time against viral disease even though its pathogenic agent was not isolated in pure form. The vaccination (Latin vacca means cow) is a term later adopted and coined by the Pasteur in the recognition and honor of the work contributed by Edward Jenner. Vaccination is the process of protection of an individual by the preparation of microbes or its contents.

10 DEFINITION AND HISTORY OF MICROBIOLOGY 11 Fig. 1.4 Edward Jenner ( ). During Jenner s period cow pox was a common disease and was caused by cow pox virus (vaccinia virus). This disease is known to cause small pustules on the teats in the udder. Sometimes similar pustules were also observed on the milkmen s hands who generally handled infected cows for milking. Such cow pox lesions were very much similar in character to smallpox. But, they were restricted, localized and known to cause mild symptoms without complications. Cow pox was also not able to disperse from one person to another person (noncommunicable). It were also observed that people who were infected with cow pox showed complete resistance against the smallpox. The cow pox pustules by some phenomena protected the individuals from the infection by smallpox. These facts made Edward Jenner to take up serious observation because he was a medical student. Later, as a physician, he made detailed investigation and recorded all his methods of practice. By the repeated experiment for many years he considered that cow pox lesions gave protection against smallpox disease. During his studies Jenner collected the pus from a cow pox lesion from the hand of a dairy maid (called Sarah Nelms) in Then he rubbed it on the skin surface of a healthy boy (called James phillip). Later the boy developed a typical scar on the skin. After a few weeks the boy was inoculated with the pus taken from the smallpox pustules of a diseased person. But the boy could not show any disease of smallpox. Hence, boy showed the complete immunity to the disease. From experiments performed later and scientific accumulation of data of his studies, he concluded that inoculation of a person with cow pox pus could offer immunity from the smallpox disease. Later, in June 1798, he published his findings in An Inquiry into the Cause and Effect of Variole Vaccinae.

11 12 BIOTECHNOLOGY-II This achievement is considered as one of the greatest achievements ever made by man in his quest for conquering disease and is also heralded as a significant milestone in the history of microbiology. Though vaccination was met with greater opposition in the beginning, it has stood the test of time and proved its advantage in medical field in later dates. Through this process of vaccination the smallpox virus and the disease have been eradicated from the world population and now vaccination against this disease is considered as not that necessary. GOLDEN ERA OF MICROBIOLOGY The golden era of microbiology began with brilliantly planned experiments of Louis Pasteur, Robert Koch, John Tyndall, Elie Metchnikoff and others. They provided the sound scientific principles without debatable answers to the abiogenesis phenomena. This was greatly accepted by the scientific community of that age. The majority of the microbial discoveries, their scientific principles and advancement were made during this period of time. These paved the way for development of microbiology as a very important branch of science. Louis Pasteur ( ) Louis Pasteur (Fig. 1.5) was a French scientist, chemist and microbiologist and is linked with most important discoveries in the field of microbiology. Louis Pasteur was born on 27th December 1822, in the countryside of Dole, France. He was first involved in the study of chemistry and obtained the doctorate in Chemistry from the Norman School in Paris and became Professor of Chemistry at Paris. Fig. 1.5 Louis Pasteur ( ).

12 DEFINITION AND HISTORY OF MICROBIOLOGY 13 Death blow to the theory of abiogenesis Louis Pasteur performed many experiments in 1860 to give a death blow to the theory of spontaneous generation of life (abiogenesis). Pasteur first filtered air through sterile cotton and then the sterile cotton was placed in a sterile medium. This showed the growth of microorganisms and indicated that air contained these microorganisms. Later, he poured sterile nutrient medium in specially designed flasks called swan-necked flasks. These are round-bottom flasks having curved necks of various types with open ends. He then boiled the medium to free the contamination and allowed to cool. In this, growth was not observed even though the curved necks had opening to surrounding atmosphere. Pasteur thus opined that no growth was seen because dust and germs had been trapped by the walls of the curved necks. But, growth would be noticed if the curved necks were broken because the air could enter directly into the flasks with depositing the germs and dust on the neck walls. However, Pasteur not only conclusively disproved the theory of spontaneous generation or abiogenesis in 1861, but also showed how to maintain any solution in sterile condition for longer period. Later, this was also supported by the work of John Tyndall, which even could destroy the bacterial endospore (Tyndallization the process of repeated heating and cooling of the medium that can completely destroy all living germs in the solution) that are highly resistant to regular boiling. Pasteur with his knowledge in chemistry came into the limelight while working on variety of biological fields including medicine because he had great insight into the problems which he came across. Thus, he was called for the majority of the unsolved problems in the different fields. The major contribution of Pasteur to the field of microbiology in particular and to the science in general is listed below. Tartaric acid isomers Tartaric acid is an organic compound which has the capacity to get crystallized. When working with tartaric acid crystals he found out two types of crystals based on their capacity to refract light in different directions called polarization. One type of crystals refracted light (polarized) towards right and others towards left. These were microscopically separated into two different types of crystals and were called isomers. Microbial fermentation The process of conversion of carbohydrates (sugar) into alcohol is known as fermentation. Large numbers of people in France are involved in wine production. In the meantime Theodore Schwann (1837) showed that yeast cells are responsible for the process of fermentation. But German chemist Justin Liebeg proposed that fermentation was a chemical process. During this period distillers of France faced a problem of their alcohol turned into sour taste. This totally hampered the entire industry in France and they approached Pasteur to solve this problem. Pasteur took up this challenge and started observation on wine preparation in greater detail. He could observe yeast cells and bacteria under microscopic field. Pasteur considered that yeast cells were responsible for the fermentation process. By setting up a number of conclusive experiments he demonstrated that yeast cells turned grape juice (sugar) into alcohol. By the application of heat Pasteur destroyed the yeast cells in the grape juice mixture. In this case no alcohol was formed and alcohol formation could be regained when the fresh yeast cells were added into the grape juice.

13 14 BIOTECHNOLOGY-II He also showed that bacteria were responsible for sour taste of the wine. This sour taste is formed due to the growth of the specific type of bacteria and their fermentation in the mixture. The basic discovery of microbial fermentation was remarkable in microbiology, because up to that time microbes were considered as the things of curiosity and none could link them with any biochemical activity of significance. Pasteur published a research paper on fermentation that formed the basis of emergence of microbiology as a branch of Biology. This could also gave a solution to the problem which occurred in the French distilleries. Aerobes and anaerobes discovery Priestley showed that organisms required air (oxygen) for the growth and development. Hence, scientists during 19th century believed that microorganisms also needed oxygen for the growth and development. Microorganisms that require oxygen for the growth are referred as aerobes or aerobic microorganisms (E.g.: Bacillus, Pseudomonas). Pasteur during his studies on butyric acid fermentation showed that some of the microorganisms did not require oxygen. Observations of the fermented mixture under the microscope indicated that large numbers of microbes were actively showing the motility. These microbes could not show the motility when exposed to the air. He also showed that fermentation of the mixture could be stopped by aeration of the broth. Pasteur named those groups of microorganisms that did not require oxygen for their growth as anaerobes or anaerobic microorganisms (E.g.: Clostridium). Process of pasteurization The process of heating liquids to destroy microbes which are harmful in nature is known as pasteurization. This was first adopted for the wine and beer industry by Pasteur to kill the undesirable microbes present in the grape juice. These microorganisms produced lactic acid and acetic acid in the fermentation mixture. By this process Pasteur showed that spoilage of taste of wine (due to the presence of acid) can be prevented by exposing fermentation mixture to low heating at 60 C for a brief period instead of complete boiling. This temperature was more than enough for the killing of undesirable bacteria which acted as contamination in the juices. This pasteurization process is now employed for a number of liquids such as beer, milk, milk products and other beverages. This process has become one of the necessary processes in the food industry and others. Present day pasteurization temperatures are heating of liquid at 62.8 C for 30 minutes (slow pasteurization) or 72 C for 15 seconds (flash pasteurization or high-temperature short-term [HTST] pasteurization). Work on pebrine disease The southern parts of France was the major area for the silk industry that produced the large economy to the France. During 1865 this silk producing area suffered a disastrous economic loss for the country. This was due to the pebrine disease affecting the silk worms and resulted in large scale spread of the disease in majority of the silk worm rearing areas. Then Ministry of Agriculture appointed Pasteur as a major person to study and find the proper solution for the disease. Pasteur after detailed investigation in the affected area for three years was able to find out the causative agent of the disease as a protozoa member (Nosema bombysis). He also showed the farmers to get rid off the disease by using the disease-free eggs for rearing and

14 DEFINITION AND HISTORY OF MICROBIOLOGY 15 destroying the affected eggs and older leaves that were used as silk worms feed. Thus by using hygienic source material the disease could be completely eliminated. The silk industry of France was thus saved from the major economic loss. This path-breaking discovery led him to become a great patriot apart from being an eminent scientist and scholar. Work on anthrax In France anthrax was a fatal disease and affected the sheep industry. The same type of disease was also observed in the other animals that became a major problem for the Government economy. In the meantime, work done by the German physician Robert Koch revealed that the anthrax bacterium (Bacillus anthracis) was associated with the disease but could not establish the relationship with the disease causing ability of the bacterium. It was Pasteur s idea which proved that the bacteria present in the culture medium was causing the disease in healthy animals. Pasteur also got a clue on the process of vaccination or protective inoculation of healthy host to get resistance against disease. He successfully demonstrated that inoculation of weakened microbes to animals could give them immunity and ward-off from the disease. Through this, Pasteur discovered the effective vaccine for the anthrax. This created the curiosity and interest in many of the contemporary scientists towards vaccination. Pasteur developed anthrax vaccine along with his associate Charles Chamberland ( ). Charles Chamberland also developed the bacteriological filters. The most famous and remarkable discovery in the field of medicine came from the research work of Pasteur through the work on rabies. Rabies or hydrophobia is a viral (Rhabdovirus group) disease that comes to humans through mad dog bite. Even though Pasteur was not able to observe or isolate the causative agent of the disease under microscope, he successfully developed the treatment or protection against the disease. Pasteur first started his work on the animals on a trial basis. By observing the observation of Edward Jenner, he planned to place an avirulent (non-virulent; made by growing virus in an abnormal host, the rabbit) mixture of dried, grinded spinal cord under the dog skin surface. This process continued for a few days with increased virulent strain preparation from spinal cord extract. In the end of the experiment, the animal successfully got resistance against the disease. The rabid dog bite and application of most potent extracts to brain of healthy animal failed to induce rabies. With this success in the case of animals, he also explained the need for further research work in the application of this process directly to human beings. Before the further research work could occur the situation had aroused which led Pasteur to practice it even on humans. During the course of his experiments, Joseph Meister, a nine year old boy who had been bitten by a rabid dog, was brought to the observation of Pasteur. As boy s death was certain in the absence of treatment, Pasteur considered for trying vaccination. The boy was vaccinated 13 times over the next 10 days with increasingly virulent preparations of the attenuated strain of virus. To the surprise the boy survived with the vaccination and later became the faculty in Pasteur Institute. In the gratitude for Pasteur s development of vaccination process, people from different parts of the world immensely contributed to initiation of research work in a separate institute called Pasteur Institute in Paris, France. He died in Paris, on 28th September, His tomb was constructed in the basement of the Pasteur Institute, Paris.

15 16 BIOTECHNOLOGY-II Joseph Lister ( ) Joseph Lister (Fig. 1.6), was an English surgeon born in Upton, England on 5th April, In 1852 he earned his degree in medicine. Lister initiated the study on the coagulation of blood and inflammation that occurred after injuries and surgical wounds. He became Professor of Surgery at Glasgow and Edinburgh. Lister started concentrating to reduce the gangrene and bacterial infections. During his time gangrene was considered to be caused by the bad quality air. Even after maintaining the cleanliness in surgical materials and rooms, the death rate was very high (50%). Lister made several contributions for the improvement of the microbiology as a whole and medicine in particular. Fig. 1.6 Joseph Lister ( ). Antiseptic technique Lister had contributed to surgery in many ways, but his major contribution has been in preventing wound infection after surgery by following antiseptic methods. As a great observer and surgeon, Lister was attracted towards post-operative complications in patients due to wound infections. He made detailed study the cause of such wound infections. He observed a similarity between the wound inflammation, fermentation and putrefaction described by Pasteur, which was caused due to microorganisms. This made him to believe that the microbes entered the wound through air, hands, surgical instruments or bandage cloths and thus resulted in sepsis. When he protected the wounds with dressing process that killed microbes, inflammation never resulted. Thus later this formed the basis for antiseptic technique.

16 DEFINITION AND HISTORY OF MICROBIOLOGY 17 Lister by his observations came to the practice of applying carbolic acid as a microbicidal chemical agent. He covered all the wounds with the carbolic acid-soaked bandage cloths. This resulted in quick healing of the wounds which generally caused the fatal inflammations. Later he made similar applications to many deadly wounds and observed the same results. This made him to continue it as a routine for all the operation procedures and that came to be known as antiseptic system of operation or antiseptic surgery. Pure culture technique Microorganisms are omnipresent in nature. They live in extremely large populations made up of millions of cells from different species. For the study of a particular species characteristics it is necessary to separate it from all other species present in the mixed population. Lister in 1878, by using serial dilution technique for the first time, obtained pure culture of bacteria in liquid media. By using a specially designed syringe, he diluted curdled milk containing a mixture of bacteria until a single bacterium was delivered into a container of sterile milk. After incubation he observed the bacteria in that container. They were all of a single kind and rod shaped bacteria. Lister coined them as Bacterium lactic. Robert Koch ( ) Robert Koch (Fig. 1.7) was born on 11th December, 1843 in Germany. In 1866 he obtained his medical degree from the University of Gottingen and started as medical practitioner. Later Robert Koch switched over to microscopic studies that he engaged in his full life time. Then he became Professor of Hygiene and headed as Director in the Institute of Infective Diseases at Berlin, Germany. Robert Koch is considered as one of the main founders of modern bacteriology. Robert Koch s major contributions to the development of microbiology are as follow: Fig. 1.7 Robert Koch ( ).

17 18 BIOTECHNOLOGY-II 1. Germ theory of diseases Robert Koch by his experiments proved that microbes are responsible for the diseases in animals. During the study of anthrax disease in cattle, Koch had noticed rod shaped microorganisms in the blood of diseased animals. For testing if these microorganisms were the causative agents of anthrax disease, he injected a healthy mice with the blood from a diseased animal. He found that disease was transmitted to the mice which resulted in death. Later Robert Koch took the blood of the dead mice and injected it into another healthy mouse and again the mouse died due to disease. Koch repeated this experiment for 20 times and each time the healthy mice died after inoculation. In the laboratory Robert Koch cultured these bacteria in the aqueous humour from the eyes of a cow. He was able to reproduce the disease every time through inoculating these microorganisms into healthy animals. With his experimental procedures he laid down certain basic criteria for considering microorganisms as causative agents of the disease. These criteria are referred as Koch s postulates and are: (a) The causative agent of the disease must be present in every diseased individual. (b) (c) The causative agent must be isolated from the diseased individual and grown in pure culture. The isolated infectious microorganism must reproduce the disease when inoculated into a healthy individual, and (d) The same infectious agent must be reisolated from the experimentally induced diseased individual. These postulates of Koch continue to be useful even today with some modification. 2. Discovery of Mycobacterium tuberculosis After the anthrax work Koch concentrated towards widespread tuberculosis disease. With the intensive research work on tuberculosis, Robert Koch discovered and proved in 1882 that all forms of infectious tuberculosis were caused by the bacillus forms called Mycobacterium tuberculosis. He showed method of culturing these bacilli in pure form and made detailed study on the Mycobacterium tuberculosis. 3. Preparation of smears As microorganisms occurred in groups of millions of cells, it was very difficult to study the bacteria under microscope. Hence, Robert Koch showed for the first time the technique of making smears of bacteria on glass slides and staining them with aniline dyes to observe them more clearly under the microscope. Making of smears on the slides distributed the microorganisms in thin layer without overlapping and gave the access for the proper staining. 4. Plating method for isolation of pure culture In his laboratory Robert Koch accidentally observed that a slice of potato had many colonies of bacteria growing distinctly from one another. However, the potato had certain disadvantages such as the moist surface of potato allowed motile bacteria to spread on entire surface. As the surface of potato is opaque it was very difficult to observe bacterial colonies and it is not a good nutrient medium for large number of bacterial strains.

18 DEFINITION AND HISTORY OF MICROBIOLOGY 19 Fi Owing to above mentioned disadvantages, Koch planned that if a liquid nutrient medium could be solidified by the addition of a solidifying agent such as gelatin, then it could be used for the isolation of bacteria in the laboratory. He poured the molten gelatin medium on glass plates and allowed it to solidify. After the gel had solidified, the surface was streaked with a platinum wire dipped into a bacterial suspension. These plates were then covered and incubated. After incubation period the growth was found all along the streaking line and single colonies were found at the end of the streak. These single colonies contained only one type of bacteria and this formed the pure cultures of bacteria. Thus, the use of gelatin for the preparation of a solid medium and the streak plate method for the isolation of pure bacterial cultures came into existence. Koch s another associate, Walter Hesse gave a clue of using agar as solidifying agent for its advantage over gelatin. Richard Petri, a student of Robert Koch in 1887, discovered usage of the petri dishes in the laboratory for the culturing purpose. This enables the microbiologists to handle large number of microbes in the laboratory without the problem of contamination. The discovery of acellular particles, viruses and their role in disease development was made possible when Charles Chamberland (Pasteur s colleague) developed porcelain bacterial filters in This made the microbiologists to use such filters and study the viruses in greater detail. During this period development was also taking place in determining how animals resisted disease and in understanding immunity. After the discovery of diphtheria toxin, Emil von Berhring ( ) and Shibasaburo Kitasato ( ) inoculated inactivated toxin into rabbits. Later a substance known as antitoxin (antibody against diphtheria toxin) was produced in the blood and this protected against the disease by inactivating the toxin. Fig. 1.8 Elie Metchnikoff ( ).

19 20 BIOTECHNOLOGY-II The antitoxin work provided evidence that immunity could result from soluble substances present in the blood. This is now well known that these substances are antibodies that are responsible for the humoral immunity (antibody-induced immunity). Later, Elie Metchnikoff ( ) (Fig. 1.8) discovered that blood leukocytes (WBC s) were also responsible for the immunity. He referred these cells as phagocytes and the process as phagocytosis. Thus, was opened a new branch of immunity named cellular immunity. Alexander Fleming ( ) Alexander Fleming (Fig. 1.9), a British scientist, was born on 6th August, 1881, at Lochfield in Scotland. He studied medicine and obtained his degree from St. Mary s Medical School, London University. Later, he joined the military service and worked as captain in Army Medical Corps throughout First World War. During 1921, working with blood and its action on bacteria he discovered the bacteriolytic properties in the body tissues and secretions. For these substances he coined the term Lysozyme. In 1928, Alexander Fleming, a student of Almroth Wright (discoverer of opsonins) and an excellent bacteriologist working at St. Mary s Hospital, in London, discovered Penicillin the first wonder drug. Fig. 1.9 Alexander Fleming ( ). When he was working in the laboratory, one of the petri plates containing the bacterial culture (Staphylococcus aureus) attracted his attention. In his laboratory, Fleming was performing experiments in search of novel bactericidal agents, particularly against wound infections. During the course of experiments, he observed that a culture plate of Staphylococcus aureus was contaminated by a blue-green coloured fungal growth. The area surrounding the edges of the fungal colony was clear. The growth of Staphylococcus aureus near the fungal colony was completely inhibited or was killed by the fungus. Fleming by careful examination

20 DEFINITION AND HISTORY OF MICROBIOLOGY 21 opined that something that is excreted by the fungal colony proved to be fatal to Staphylococcus cells. The contaminated fungal colony was then isolated and identified as a common bluegreen fungal member Penicillium notatum. Later, Fleming cultured this fungal colony on broth medium in a separate flask. Then from the broth medium he obtained the exudates content. In order to test his hypothesis that the fungal secretions could destroy Staphylococcus he inoculated this extraction into laboratory mice infected with Staphylococcus, Streptococcus and Pneumococcus. The mysterious compound present in this extraction killed all the microorganisms in the body of the mice as it had done similar thing on Staphylococcus aureus in the petri plate. Then, Fleming named this mysterious compound as Penicillin. Fleming, in 1929, published a research article in the British Journal of Experimental Pathology. This work later drew the attention of Sir Howard W. Florey and Ernst B. Chain at Oxford University. In due course Howard Florey and Ernst Chain successfully purified the penicillin from the culture filtrate of Penicillium notatum. This meant that the penicillin could be used for the large scale purpose. During 1941 industrial production of penicillin had started and proved efficient against bacterial infection during First World War. In Medicine category Fleming shared the Nobel Prize along with H.W. Florey (Australia) and E.B. Chain (Great Britain) in FURTHER DEVELOPMENT OF MICROBIOLOGY Ecological role of microorganisms was studied by a few of the early microbiologists. Particularly they studied by taking soil and aquatic habitats as the environment and worked in detail microbial involvement in the carbon, nitrogen and sulfur cycles. Sergei N. Winogradsky ( ) and Martinus W. Beijerinck ( ) are the two pioneers in this category. Fig Sergei N. Winogradsky ( ).

21 22 BIOTECHNOLOGY-II Sergei N. Winogradsky (Fig. 1.10), a Russian microbiologist, made several important contributions to the soil microbiology. Winogradsky discovered that soil bacteria are involved in oxidization of iron, sulfur and ammonia to get their energy requirement. He also discovered that photosynthetic bacteria, similar to plants, can also use CO 2 to produce organic materials. Another new groups of microorganisms were discovered by Winogradsky including anaerobic nitrogen-fixing microorganisms. He isolated thems from the soil habitat and also studied in detail the decomposition of cellulose in the soil. Martinus W. Beijerinck (Fig. 1.11), another Russian microbiologist also made many basic contributions in the development of microbial ecology and other branch of microbiology. Beijerinck isolated the Azotobacter, an aerobic nitrogen-fixing bacterium (soil bacterium); Rhizobium, a root nodule forming symbiotic bacterium capable of fixing nitrogen and many sulfate-reducing bacteria from soil. Fig Martinus W. Beijerinck ( ). Later Beijerinck and Winogradsky together developed the enrichment technique of culturing of microbes and the use of selective media in the growth and isolation of pure culture. This became a great importance in the microbiological field. Heinrich Anton de Bary ( ), a German doctor, made large number of contributions to the fungi and mycology and established Phytophthora infestans as cause of the late blight of potato. Anton de Bary (Fig. 1.12) laid clear foundations of mycology and hence he is commonly known as founder of modern mycology or father of plant pathology. Dmitry Ivanovsky ( ) (Fig. 1.13) found a new field of microbiology called virology by discovery of tobacco mosaic disease, causative agent, a virus (later named as Tobacco mosaic virus). This gave way for observing the organisms that are not easily studied by using conventional microscopes and started virology as a major field in microbiology.

22 DEFINITION AND HISTORY OF MICROBIOLOGY 23 Fig Heinrich Anton de Bary ( ). Fig Dmitry Ivanovsky ( ). Selman Waksman ( ) working at Rutgers University in US isolated a soil actinomycetes bacterium Streptomyces and discovered streptomycin, a major broad spectrum antibiotic. Selman Waksman (Fig. 1.14) was the person who coined the word antibiotic and won the Nobel Prize in 1952 for the same.

23 24 BIOTECHNOLOGY-II Fig Selman Waksman ( ). David Hendricks Bergey ( ) was the person who headed the manual preparation team for the entire bacterial organisms identifications called Bergey s Manual. This is most important for Bary proper identification and placing of newly discovered bacterium. David Hendricks Bergey s (Fig. 1.15) name is still used in the recent edition of the manual for his great work done in bacteriology. Fig David Hendricks Bergey ( ). In the early part of twentieth century, microbiology developed independently of other biological disciplines due to difference in focus. During this period microbiologists were more