MICROBIOLOGY & ITS HISTORY

MICROBIOLOGY
Microbiology is the study of living organisms of microscopic size. The term microbiology was given by French chemist Louis Pasteur (1822-95). The word Microbiology is derived from three Greek wordsMikros (small), Bios (living) & Logos (science). Microbiology is said to have its roots in the great expansion and development of the biological sciences that took place after 1850. The term microbe was first used by Sedillot (1878). Microorganisms like all other living organisms possess the following characters: 1. 2. 3. 4. 5. Ability to reproduce Ability to ingest food materials Ability to excrete waste products Irritability Susceptibility to mutation

The discovery of microbiology as a discipline could be traced along the following historical eras:

Discovery Era

Transition Period

Golden Age

In 20th Century: Era of Molecular Biology

DISCOVERY ERA
This period concerns with the discovery of microbial world that has been dominated by Antony Van Leeuwenhoek.

Concepts of Aristotle: Aristotle (384 322 B.C.) believed that the animals might originate
spontaneously from the soil, plants, or other unlike animals. His influence was still strongly felt in the seventeenth century!!

Antony Van Leeuwenhoek (1632-1723):

Of Delft, Holland (Netherland) he was the first person to observe and accurately describe microorganisms (bacteria and protozoa) called 'animalcules' (little animals) in 1676. Actually he was a Dutch linen merchant but spent much of his spare time constructing simple microscopes composed of double convex lenses held between two silver plates. He constructed over 250 small powerful microscopes that could magnify around 50-300 times. Leeuwenhoek was the 1st person to produce precise and correct descriptions of bacteria and protozoa using microscope he made himself. Because of this extraordinary contribution to microbiology he is considered as the "Father of bacteriology and protozoology". He wrote over 200 letters which was transmitted as a series of letters from 1674-1723 to Royal society in London during a 50 years period.

Spontaneous Generation and Biogenesis:

Early belief that some forms of life could arise from vital forces present in nonliving or decomposing matter, abiogenesis. In other words, organisms can arise from non-living matter. The alternative hypothesis, that the living organisms arise from preexisting life, is called biogenesis.

TRANSITION PERIOD
Although, there were a number of significant developments in microbiology during Van Leeuwenhoeks time, Peoples were interested to correlate diseases with microbes. The main aspects were to solve the controversy over spontaneous generation which includes experimentations mainly of Francesco Redi, John Needham, Lazzaro Spallanzani and Nicolas Appert etc and to know the disease transmission which mainly includes the work of Ignaz Semmelweis and John Snow.

Francesco Redi (1626-1697): The ancient belief in spontaneous generation was first of all
challenged by Redi, an Italian physician, who carried out a series of experiments on decaying meat and its ability to produce maggots spontaneously. Redi was the first who put the theory of spontaneous generation to test by conducting a simple experiment in which he placed meat in three jars. One jar was covered with fine gauze, second was covered with paper and third was left uncovered. Flies entered the jar that was open to air i.e. left uncovered and landed on meat where they laid their egg that later developed into maggots. The other two pieces of meat did not produce maggots spontaneously. However flies were attracted to the gauze covered jars and laid their eggs on the gauze and maggots subsequently developed without access to the meat, indicating that maggots were the offspring of the flies and did not arise from some 'vital source' in the meat as previously believed.

Louis Jablot (1670):

In 1670, Jablot conducted an experiment in which he divided a hay infusion that had been boiled into two containers: a heated container that was closed to the air and a heated container that was freely open to the air. Only the open vessel developed microorganisms. This further helped to disprove abiogenesis.

John Needham (1713-1781): He was probably the greatest supporter of the theory of spontaneous
generation. He proposed that tiny organisms the animalcules arose spontaneously on his mutton gravy. In 1749, while experimenting with raw meat exposed to hot ashes, John Needham observed the appearance of organisms not present at the start of the experiment and concluded that the bacteria virtually originated from the raw meat itself. He proposed this observation as an example of spontaneous generation.

Lazzaro Spallanzani (1729-1799): He was an Italian Naturalist who attempted to refute

Needham's experiment. In 1749; he boiled beef broth for longer period, removed the air from the flask and then sealed the container. Followed incubation no growth was observed by him in these flasks. He showed that the heated nutrients could still grow animalcules when exposed to air by simply making a small crack in the neck. Thus Spallanzani disproved the doctrine of spontaneous generation. But he failed to convince Needham, who insisted that air was essential to the spontaneous production of microscopic beings and that it had been excluded from the flasks by sealing them. Nicolas Appert followed the idea of Spallanzani's work. He was a French wine maker who showed that soups and liquids can be preserved by heating them extensively in thick champagnes bottles. Ignaz Semmelweis and John Snow were the two persons who showed a growing awareness of the mode of disease transmission.

Experiment of Franz Schulze and Theodor Schwann: Two German scholars Schulze
(1815-1873) and Theodor Schwann (1810-1882) viewed that air was the source of microbes and sought to prove this by passing air through hot glass tubes or strong chemicals into boiled infusions in flasks. The infusion in both the cases remained free from the microbes. But the die-hard advocates of spontaneous generation were still not convinced. They said, acid and heat altered the air so that it would not support growth.

Experiment of H. Schroder and T. von Dusch: George Schroeder and Theodor Von Dusch
(1854) were the first to introduce the idea of using cotton plugs for plugging microbial culture tubes. They carried out a more logical and convincing experimental design by passing air via cotton fibers so as to prevent the bacterial growth ; and thus, it ultimately initiated and gave rise to a basic technique of plugging bacterial culture tubes with cotton plugs (stoppers), which technique being used still as to date. But the concept of spontaneous generation was revived for the last time by Felix-Archemede Pouchet, who published in 1859 an extensive report proving its occurrence. Darwin (1859) in his book, 'Origin of the Species' showed that the human body could be conceived as a creature susceptible to the laws of nature. He was of the opinion that disease may be a biological phenomenon, rather than any magic.

GOLDEN AGE OF MICROBIOLOGY (1857-1914)

The Golden age of microbiology began with the work of Louis Pasteur and Robert Koch who had their own research institute and lasted upto the works of Paul Ehrlich. More important there was an acceptance of their work by the scientific community throughout the world and a willingness to continue and expand the work. During this period, we see the real beginning of microbiology as a discipline of biology.

Louis Pasteur: Pasteur, a French microbiologist, performed a series of experiments to prove that
microorganisms were present in the air and were not spontaneously produced. Pasteur invented a swannecked flask in early 1800s. He filled several round bottomed flasks with nutrient solution and fashioned their openings into elongated, swan neck shaped tubes. The flask's opening were freely open to the air but curved so that gravity would cause any air borne dust particle to deposit in the lower parts of the neck. The flasks were heated to sterilize the broth and then incubated. No growth occurred even though the contents of the flasks were exposed to the air. Pasteur pointed out that no growth took place because dust and germs had been trapped on the walls of the curved necks but if the necks were broken off so that dust fell directly down into the flask, microbial growth commenced immediately. Some of these ingenious little flasks are still on display at the Pasteur Institute in Paris in their original sterile form. This experiment clearly showed that microorganisms present on or in non-living materials such as dust or water were responsible for the contamination of sterile solutions. Pasteur, thus in 1861 finally resolved the controversy of spontaneous generation versus biogenesis and proved that microorganisms are not spontaneously generated from inanimate matter but arise from other microorganisms.

John Tyndall (1820 - 1893): An English physicist, deal a final blow to spontaneous generation in
1877. He conducted experiments in an aseptically designed box to prove that dust indeed carried the germs. He demonstrated that if no dust was present, sterile broth remained free of microbial growth for indefinite period even if it was directly exposed to air. He discovered highly resistant bacterial structure, later known as endospore, in the infusion of hay. Prolonged boiling or intermittent heating was necessary to kill these spores, to make the infusion completely sterilized, a process known as Tyndallisation.

Fermentation and Pasteurization: Louis Pasteur began his brilliant career as professor of
chemistry at the University of Lille, France. At that time as a principal industry, France manufactured large variety of wines and beer. Pasteur played a critical and major role in the proper standardization of various processes and techniques intimately associated with the said two alcoholic beverages in order to obtain a consistently good product. Pasteur explored and exploited the unique capabilities of microbes in the fermentation industry exclusively using fruits and grains resulting in alcohol-based table wines, dry-wines, champagne, whiskies, etc. Pasteur meticulously isolated, typified, and characterized certain microbes exclusively responsible for the good batches predominantly in comparison to the ones found solely in the poor products. In fact, the overall net outcome of such extensive as well as intensive investigations helped in a long way for the assured and successful production of consistently good and uniform ultimate product. Pasteur vehemently argued and suggested that the unwanted/undesirable types of microbes must be destroyed and removed by heating not enough to alter the original and authentic inherent flavor/aroma of the fruit juice, but just sufficient to cause and afford the legitimate destruction of a relatively very high

percentage of the bad microbial population. This destructive microbial phenomenon could be accomplished successfully by holding the juices at a temperature of 145F ( 62.8C) for a duration of 30 minutes (Pasteurization). Pasteurization is the application of a high heat for a short time to destroy microorganisms. Pasteurization was introduced into the United States on a commercial basis in 1892. His work led to the development of the germ theory of disease.

Germ Theory: Microbes (or bacteria) happen to be the root cause of several human dreadful
diseases Germ theory. Various scientists supported and proved the aforesaid germ theory in one way or the other as stated under: Girolamo Fracastro (14831553): He advocated that certain diseases might be caused by virtue of invisible organisms transmitted from one subject to another. Plenciz (1762): He stated that the living microbes (or agents) are the ultimate cause of disease but at the same time aired his views that different germs were responsible for different ailments. Oliver Wendell Holmes (18091894): He suggested that puerperal fever was highly contagious in nature; besides, it was perhaps caused by a germ carried eventually from one mother to another either by midwives or physicians. Ignaz Philipp Semmelweis (18181865): He pioneered the usage of antiseptics specifically in the obstetrical practices.

Lord Joseph Lister (1827-1912): This man, a famous English surgeon is known for his notable
contribution to the antiseptic treatment for the prevention and cure of wound infections. Lister concluded that wound infections too were due to microorganisms. In 1867, he developed a system of antiseptic surgery designed to prevent microorganisms from entering wounds by the application of phenol on surgical dressings and at times it was sprayed over the surgical areas. He also devised a method to destroy microorganisms in the operation theatre by spraying a fine mist of carbolic acid into the air, thus producing an antiseptic environment. Thus Joseph Lister was the first to introduce aseptic techniques for control of microbes by the use of physical and chemical agents which are still in use today. Because of these notable contributions, Joseph Lister is known as the Father of Antiseptic surgery.

Robert Koch (18431910): Robert Koch gave the first direct demonstration of the role of bacteria in
causing disease. He was a German physician who first of all isolated anthrax bacillus (Bacillus anthracis, the cause of anthrax) in 1876. He perfected the technique of isolating bacteria in pure culture. He also introduced the use of solid culture media in 1881 by using gelatin as a solidifying agent. In 1882 he discovered Mycobacterium tuberculosis. Koch adopted the following steps to isolate microbes causing anthrax: (1) First of all these bacteria were duly grown in cultures in the laboratory. (2) Bacteria examined microscopically so as to ascertain only one specific type was present. (3) Injected bacteria into other animals to observe whether they got also infected, and subsequently developed clinical symptoms of anthrax. (4) Isolated microbes from experimentally infected animals squarely matched with those obtained originally from sheep that died due to infection of anthrax. He proposed Koch postulate which were published in 1884 and are the corner stone of the germ theory of diseases and are still in use today to prove the etiology (specific cause) of an infectious disease.

Kochs postulates
1. The microorganism must be present in every case of the disease but absent from healthy organisms. 2. The suspected microorganisms must be isolated and grown in a pure culture. 3. The same disease must result when the isolated microorganism is inoculated into a healthy host.

Experiment
Koch developed a staining technique to examine human tissue. M. tuberculosis cells could be identified in diseased tissue. Koch grew M. tuberculosis in pure culture on coagulated blood serum. Koch injected cells from the pure culture of M. tuberculosis into guinea pigs. The guinea pigs subsequently died of tuberculosis. Koch isolated M. tuberculosis from the dead guinea pigs and was able to again culture the microbe in pure culture on coagulated blood serum.

4. The same microorganism must be isolated again from the diseased host.

Classical Laboratory Method and Pure Culture:

Laboratory Methods: Well defined, articulated, and explicit laboratory methods have been adequately developed which enable it to isolate a host of microorganisms representing each species, besides to cultivate each of the species individually.

Pure Culture: Pure culture may be defined as the propagation of microorganisms or of living tissue cells
in special media that are conducive to their growth. In other words it may also be explained as the growth of mass of cells belonging to the same species in a laboratory vessel (e.g., a test tube). It was indeed Joseph Lister, in 1878, who first and foremost could lay hand on pure cultures of bacteria by the aid of serial dilution technique in liquid media. Example: Lister diluted milk, comprising of a mixture of bacteria, with a specially designed syringe until a single organism was strategically delivered into a container of sterile milk. The container on being subjected to incubation for a definite period gave rise to bacteria of a single type, very much akin to the parent cell. Lister termed it as Bacterium lactis. Colonies: For the specific study of microorganism, Koch smeared bacteria on a sterile glass slide, followed by addition of certain specific dyes so as to observe the individual cells more vividly under a microscope. Koch carefully incorporated some specific solidifying agents, such as: gelatin, agar into the media in order to obtain characteristic isolated growths of organisms usually called as colonies. Importantly, each colony is essentially comprised of millions of individual bacterial cells packed tightly together. Now, from these identified colonies one may transfer pure cultures to other sterile media. However, the development of a liquefiable solid-culture medium proved to be of immense fundamental importance. Example: Koch thoroughly examined material obtained from subjects suffering from pulmonary tuberculosis, and succeeded in the isolation of Mycobacterium tuberculosis.

Importance of Pure Culture:

1. 2. 3. 4. 5. 6. Microorganisms causing a large number of infections, Certain specific fermentative procedures, Nitrogen-fixation in soil, High-yielding alcohol producing strains from malt wart, and molasses, Selected good cultures for making top-quality wines, & Specific cultures for manufacturing dairy products viz., cheeses, yogurt.

Immunity:
Immunity refers to the state of being immune to or protected from a disease, especially an infectious disease. This state is invariably induced by having been exposed to the antigenic marker on an organism that invades the body or by having been immunized with a vaccine capable of stimulating production of specific antibodies. Interestingly, Pasteurs practical aspects and Kochs theoretical aspects jointly established the fact that the attenuated microorganisms retained their capacity and capability for stimulating the respective host to produce certain highly specific substances i.e., antibodies which critically protect against subsequent exposure to the virulent organisms.

Edward Jenners successful cowpox vaccine (1798): An English physician was the first
to prevent small pox. He was impressed by the observation that countryside milk maid who contacted cowpox (Cowpox is a milder disease caused by a virus closely related to small pox) while milking were subsequently immune to small pox. On May 14th, 1796 he proved that inoculating people with pus from cowpox lesions provided protection against small pox. Jenner in 1798 published his results on 23 successful vaccinators. Eventually this process was known as vaccination, based on the Latin word 'Vacca' meaning cow. Thus the use of cow pox virus to protect small pox disease in humans became popular replacing the risky technique of immunizing with actual small pox material.

Pasteurs successful rabies vaccine: Jenner's experimental significance was realized by

Pasteur who next applied this principle to the prevention of anthrax and it worked. He called the attenuated cultures vaccines (Vacca = cow) and the process as vaccination. Encouraged by the successful prevention of anthrax by vaccination, Pasteur marched ahead towards the service of humanity by making a vaccine for hydrophobia or rabies (a disease transmitted to people by bites of dogs and other animals). As with Jenner's vaccination for small pox, principle of the preventive treatment of rabies also worked fully which laid the foundation of modern immunization programme against many dreaded diseases like diphtheria, tetanus, pertussis, polio and measles etc. Edwin Klebs (1883) and Frederick Loeffler (1884): They discovered the diphtheria bacillus, Corynebacterium diphtheriae and showed that it produced its toxins (poisons) in a laboratory flask. Emile Roux (1853-1933) and Alexandre Yersin: The two notable French bacteriologists demonstrated the production of toxin in filtrates of broth cultures of the diphtheria organism. Emil von Behring (18541917) and Shibasaburo Kitasato (18521931): They injected inactivated toxin into rabbits, inducing them to produce an antitoxin, a substance in the blood that would inactivate the diphtheria toxin and protect against the disease. A tetanus antitoxin was then prepared, and both antitoxins were used in the treatment of people. The antitoxin work provided evidence that immunity could result from soluble substances in the blood, now known to be antibodies (humoral immunity). The discovery of toxinantitoxin relationship was very important to the development of science of immunology. Shibasaburo Kitasato and Emil von Behring: In 1890,they cultivated (grown) the microorganism responsible for causing tetanus (lockjaw), Clostridium titani ; and Behring prepared the corresponding antitoxin for the control, prevention, treatment, and management of this fatal disease. Von Behring in 1890 reported on immunization against diphtheria by diphtheria antitoxin. (Emil von Behring bagged the Nobel Prize in 1901 in physiology or medicine.) De Salmon and Theobald Smith: They proved amply that immunity to a plethora of infectious diseases may be produced quite effectively and efficiently by proper timely inoculation with the killed cultures of the corresponding microorganisms.

Elie Metchnikoff: He described for the first time the manner certain specific leukocytes (i.e., white
blood cells) were able to ingest (eat up) the disease-producing microorganisms present in the body. He

baptized these highly specific defenders and crusaders against bacterial infections known as phagocytes (eating cells), and the phenomenon is termed as phagocytosis. Thus human blood cells also confer immunity, referred to as cellular immunity. Metchnikoffs Theory: Based of the aforesaid explanations Metchnikoff put forward a theory that the phagocytes were the bodys first and most important line of defense against a variety of infection.

Paul Ehrlich (1854-1915): (Robert Kochs brilliant student)

In 1904, he found that the dye Trypan Red was active against the trypanosome that causes African sleeping sickness and could be used therapeutically. This dye with antimicrobial activity was referred to as a 'magic bullet'. Subsequently in 1910, Ehrlich in collaboration with Sakahiro Hata, a Japanese physician, introduced the drug Salvarsan (arsenobenzol) as a treatment for syphilis caused by Treponema pallidum. Ehrlich's work had laid important foundations for many of the developments to come and the use of Salvarsen marked the beginning of the eni of chemotherapy and the use of chemicals that selectively inhibit or kill pathogens without causing damage to the patient. Paul Ehrlich put forward two altogether newer concepts with regard to the modus operandi whereby the body aptly destroys microorganisms (bacteria), namely: (a) Antibody & (b) Chemotherapy and Antibiotics

Gerhard Domagk: Of Germany in 1935, he experimented with numerous synthetic dyes and reported that Prontosil, a red dye used for staining leather, was active against pathogenic, Streptococci and Staphylococci in mice even though it had no effect against that same infectious agent in a test tube. Jacques and Therese Trefonel: In the same year two French scientists Jacques and Therese Trefonel showed that the compound Prontosil was broken down within the body of the animal to sulfanilamide (Sulfa drug) the true active factor. Domagk was awarded Nobel Prize in 1939 for the discovery of the first sulpha drug.

Alexander Fleming: Alexander Fleming of England, a Scottish physician and bacteriologist. Fleming
had been actually interested in searching something that would kill pathogens ever since working on wound infections during the First World War (1914-1918). One day in 1928 upon his return from a week's vacation, Fleming observed that a plate of Staphylococcus aureus had become contaminated with a green mold Penicillium notatin which had accidentally fallen in plate. Observing this plate, Fleming noted that the colonies of Staphylococcus bacterium were evidently being destroyed by the nearby Penicillium colonies. Rather than discarding the contaminated plate, he speculated that the mold was producing a diffusible substance that inhibited the bacterial growth. Fleming isolated and subcultured the mold for further study. He extracted from the fungus a compound which he called penicillin, after the name of the producer organism Penicillium notatium that could destroy several pathogenic bacteria. Thus, Sir Alexander Fleming in 1929 discovered the first antibiotic (Gr. Anti = against + bios = life, the microbial products that can kill susceptible microorganism and inhibit their growth) penicillin. The commercial production of penicillin in the USA began in 1941 Fleming, Florey and Chain shared the Nobel Prize in 1945 for the discovery and production of penicillin. Penicillium notatin has been replaced with Penicillium chrysogenum for the commercial production of penicillin.

IN 20~ CENTURY: ERA OF MOLECULAR BIOLOGY

By the end of 1900, science of microbiology grew up to the adolescence stage and had come to its own as a branch of the more inclusive field of biology. In the later years the microorganism were picked up as ideal tools to study various life processes and thus an independent discipline of microbiology, molecular biology was born. The relative simplicity of the microorganism, their short life span and the genetic homogeneity provided an authentic simulated model to understand the physiological, biochemical and genetical intricacies of the living organisms.