History of Microbiology
- The existence of the microbial world was unknown until the invention of microscopes because microorganisms are too small to be seen clearly by the naked eye. The principle of the telescope and microscope was accidentally discovered in 1609 by Zacharias Jansen (Dutch spectacle maker). The first to apply this invention of the microscope to medicine was Professor Athanasius Kircher (1602- 1680).
- He examined the blood of patients suffering from plague and through a microscope saw countless masses of small worms invisible to the naked eye. He attributed these worms like structures to be the causative agents of the plague. In 1665, Robert Hooke (1635 – 1703) an English scientist used a simple lens that magnified objects. He examined the slices of cork from the bark of an oak tree.
He observed that the cork was made of tiny boxes that he referred to as ‘cells’. The credit for developing a compound microscope with multiple lenses and describing the world of microbes goes to Robert Hooke.
Antony Van Leeuwenhoek (1632-1723) was the greatest of all microscopists. He made many observations through the microscope. Leeuwenhoek’s microscope consists of a spherical lens mounted between two small metal plates. The culture or specimen was placed on the point of a blunt pin attached to the back plate and was brought into focus by manipulating the screw.
He was the first to observe and accurately describe the shape of human red blood cells as well as the little agents of disease ‘animalcules’.
He took the scarf from the root of the decayed tooth and mixed it with clean rainwater and saw the mobility in animalcules. In 1683, he described and sketched different forms of animalcules such as rods, spheres, and spiral shapes which are nothing but the morphological forms of bacteria. This was the first recorded observation of bacteria.
He discovered major classes of bacteria, protozoa, algae, yeasts, erythrocytes, and spermatozoa.
Antony Van Leeuwenhoek, (Dutch merchant) had little formal education but his keen interest in nature made him examine a variety of materials. Glass grinding and preparation of lenses was his hobby and this led him to assemble 250 simple microscopes.
He often used a magnifying glass to study the weaves of the various types of cloth. He observed and measured a large number of minute living organisms, including bacteria and protozoa, and communicated this to the Royal Society of London in 1684.
In his letter of 1684, he described his experiments on the inhibitory effect of acetic acid on microorganisms. Leeuwenhoek transmitted his discoveries to the Royal Society in the form of a long series of letters written in Dutch. Most of these letters were transmitted and published in English in the Proceedings of the Royal Society.
(01) Contributions of Louis Pasteur
- The credit for providing a foundation for bacteriology as science goes to Louis Pasteur (1822-1895), the French organic chemist.
Louis Pasteur was the first scientist who demonstrated that air contains microscopically observable organized structures. He passed a large quantity of air through a tube that contained a plug of guncotton to serve as a filter. The guncotton was then removed and dissolved in a mixture of alcohol-ether and the sediment was examined microscopically. He found that this sediment contained not only organic matter but also a large number of small microorganisms.
He performed a series of experiments to prove that although microorganisms were present in the air they were not spontaneously produced. Pasteur took boiled meat and infused it with several swan-neck flasks (Fig. 1.1). The flask’s openings were freely open to the air but curved so that gravity would cause any airborne dust particles to deposit in the lower part 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 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. In 1861, Pasteur finally resolved the controversy of spontaneous generation versus biogenesis and proved that microorganisms are not spontaneously generated from inanimate matter.
Pasteur also showed that microorganisms are not evenly distributed in the atmosphere and that their number varies from place to place. For this experiment, he took a large number of sealed flasks containing boiled and cooled infusions and opened a few at a time, for a short period, at various places and then resealed them.
Out of the 20 flasks which he opened and resealed on a dusty road, 8 showed spoilage, while out of the 20 that he opened on the top of a mountain, only 5 showed spoilage and out of the 20 that he opened near a glacier, only 1 showed spoilage. After incubation, only those flasks carrying micro-organisms from the air showed growth and spoilage. From these experiments, Pasteur concluded that air contained microorganisms, and their number varied from place to place.
Pasteur made an intensive study of beer and wine fermentation. He found that wine spoilage was caused by the growth of undesirable contaminating microorganisms. After some experimentation, he showed that wine did not undergo spoilage if it was held for a few minutes at 50 to 60°C. This gave rise to the new process of preserving wine, fruit juices, milk, etc., and was called ‘Pasteurisation’.
Pasteur studied several other fermentation processes and showed that not only each type of fermentation was accomplished by a specific type of microorganism but also that each fermentation required specific environmental conditions.
He showed that the agent responsible for alcoholic fermentation could flourish in an acid medium while microorganisms for lactic fermentation could grow best only at a neutral pH. During the studies on butyric acid fermentation, Pasteur discovered the existence of life in the absence of oxygen. He reasoned that oxygen was inhibitory to the growth of butyric acid bacteria.
Pasteur also found that many other microorganisms, including yeast, could grow either in the presence or in the absence of oxygen. He designated life in the presence of oxygen as ‘aerobic’, in the absence of oxygen as ‘anaerobic’, and in the presence or absence of oxygen as ‘facultative anaerobic’.
Pasteur opened the field of sterilization by stating that boiling rendered the fluids sterile. He soon found that ordinary water had some microorganisms even after boiling. Pasteur established the practice of heating fluid material to 120°C under the pressure of sterilization (autoclave). He also introduced the practice of sterilizing glassware by dry heat at 170°C. Pasteur’s achievements in sterilization were closely followed by many other researchers. An important investigation was made by Tyndall, who observed that actively growing bacteria were easily destroyed by boiling whereas, in the inactive phase, they were comparatively resistant. He introduced the method of sterilization by repeated heating with appropriate intervals. This method is known as Tyndallisation.
In 1880, Pasteur isolated the bacterium responsible for chicken cholera and grew it in pure culture. But on the day of his public demonstration, he failed to repeat his experimental results. The chicken survived after injecting the isolated bacterial culture and this result surprised him. In subsequent experiments, he concluded that on long preservation or subculturing, the virulence power of the pathogenic bacteria was destroyed.
Such microbial culture with decreased virulence was referred to as attenuated culture. Such attenuated culture does not cause infection but induces immunity in the host.
Pasteur further introduced a vaccine for anthrax and rabies. He knew that the causative agent of rabies attacked the brain and spinal cord. He took spinal cords from rabbits that died from the disease and suspended these cords in dry sterile air. Through this process, the rabies microorganisms in the nervous tissue lost their virulence and the emulsion of the spinal cord was used as a vaccine. It was found to be effective in dogs. Joseph Meister was the first boy cured of rabies by this vaccine.
(02) Robert Koch and Anthrax
The first direct demonstration of the role of bacteria in causing the disease was provided by Robert Koch (1843-1910).
He was a German physician, who first isolated Bacillus anthracis, the causative agent of anthrax. His method of preparing, fixing, and staining film preparation of bacteria using aniline dyes, opened a new area in bacteriological techniques. In 1881, he described his method of preparing cultures on solid media, and the isolation of pure strains of bacteria from single colonies was made possible.
Till 1881, solid media using potato and gelatin were in practice for the isolation of fungi. Robert Koch added nutrient agar, a solid transparent medium for the isolation of bacteria.
In 1882, he discovered Mycobacterium tuberculosis, and in 1883 Vibrio cholera. In 1905, Koch was awarded the Nobel Prize in Medicine for this discovery. “Koch phenomenon’ is the immunity to the second infecting dose of the tubercle bacillus. Koch was Director of the Prussian Institute of Infectious Diseases which was eventually renamed The Robert Koch Institute.
Their most notable contribution of Koch was the establishment of the causal relationship between microorganisms and a specific disease by applying a set of criteria referred to as Koch’s postulates. According to these postulates, a microbe may be accepted as a causative agent of an infectious disease only if it satisfies the following conditions.
The postulates are:
- The microorganisms must be present in every case of the disease.
- The microorganisms must be isolated from the diseased animal and grown in pure culture.
- The disease must be reproduced when a pure culture of the microorganisms is inoculated into a susceptible host.
- The same microorganism must be reisolated from the experimentally infected host.
(03) Germ Theory of Disease
Pasteur investigated silkworm disease and proved that the disease was due to a protozoan parasite. Even before Pasteur had proved by experiments that bacteria are the causative agent of some diseases: Girolamo Fracastoro 1546 wrote a treatise in which he said the disease was caused by minute “seed” and was spread from person to person. In 1762, Von Plenciz not only stated that living agents are the cause of diseases but suspected that different germs were responsible for different diseases. The first man to present real evidence that living organisms were the cause of the disease was Augustino Bassi (1773-1856). In 1836, he demonstrated that a fungus that caused disease in silkworms could be transmitted from one silkworm to another silkworm.
Lord Joseph Lister (1827-1912), a famous English surgeon in 1867, introduced antiseptics for the prevention and cure of wound infections. He developed a system of antiseptic surgery designed to prevent microorganisms from entering wounds by the application of phenol on surgical dressings. He also heats and sterilized the instruments to be used during surgery. Joseph Lister was the first to introduce aseptic techniques for the control of microbes by the use of physical and chemical agents. Because of his important contributions, Lister is known as the father of antiseptic surgery.
The confirmed evidence that microorganisms cause disease, came from experiments performed by Robert Koch. The proofs which support the Germ theory of disease became ‘Koch Postulates’. Richard Petri, Koch’s assistant designed a special plate for old solid culture media. This plate has great significance in microbiology and is referred to as the Petri plate’. The period from 1857 to 1914 is considered the ‘golden age of microbiology’ Because the most significant advances made during this period led to the establishment of microbiology as a science.
(04) Discovery of Chemotherapeutic Agents
Paul Ehrlich (1854-1915) 1904 found that the dye Trypan red was active against the trypanosome that causes African sleeping sickness. This dye with antimicrobial activity was referred to as a ‘magic bullet’. Subsequently, in 1910, Ehrlich in collaboration with Takahiro Hata, a Japanese physician, introduced the drug Salvarsan (arsenobenzol/ 606 arsenic) as a treatment for syphilis caused by Treponema pallidum. Ehrlich continued his experimentation until in 1912 he discovered neosalvarsan. This gave him the title “the father of chemotherapy. He coined the word ‘chemotherapy’ meaning the treatment of infectious diseases with chemicals that are antagonistic to the pathogen without damaging the host.
Ehrlich tested new dyes and aniline pigments as histological stains and showed that all dyes can be classified as acidic, basic, or neutral. He developed differential staining methods. for red and white blood cells laying the foundation of hematology. In 1882, Ehrlich developed a staining procedure to demonstrate the acid-fast property of tubercle bacilli which is known as the Ziehl- Nelson staining technique. Ehrlich propounded a new theory in the field of immunology, explaining the production of antibodies that he quantified in various sera and formulated the standardization of antitoxin into units related to a fixed and invariable standard. In 1908, Paul Ehrlich received the Nobel Prize in Medicine for his studies on immunity.