The following points highlight the ten important landmarks in the history of microbiology. Some of the landmarks are: 1. Discovery of Microscope 2. Discovery of Microbial Life 3. Abiogenesis Versus Biogenesis 4. Fermentation; Pasteurization 5. Germ Theory of Disease 6. Pure Culture Concept and Other Microbial Techniques 7. Growth of Medical Microbiology and Others.
Landmark # 1. Discovery of Microscope:
The fascinating microbial world would have remained unknown had the microscope not been invented. It was Roger Bacon (1267) who developed a lens for the first time. Jansen and Jansen (1590), about 300 years later first produced a crude type of microscope by placing two lenses together without any provision for focusing’ Galileo Galilei (1610) prepared a microscope with a focusing device called ‘occiale’.
Till then, the name ‘microscope’ had not been in use and it was first proposed by Faber (or Fabri) in 1625. However, the advent of such optical lens systems did not reveal the existence of microorganisms.
It was not until the mid-17th century when further development of the optical lens systems to definite microscope permitted the visualization of microorganisms that the great diversity of the microbial world began to be recognized.
Robert Hooke (1635-1703) made and used a compound microscope in the 1660s and described his fascinating explorations of the newly discovered universe of microscopic creatures in his classic “Micrographia” (1665).
Although Hooke’s highest magnifications were possibly enough to reveal bacteria, he apparently could not see them probably because he studied mainly opaque objects in the dry state by reflected light, conditions that are not optimal for observing bacteria. However, his pictures of “white moulds” (probably a Mucor species) are very informative and accurate (Fig. 1.1).
Landmark # 2. Discovery of Microbial Life:
The exact beginning of the knowledge about the existence of microorganisms can be traced back only to the latter part of the seventeenth century when Antony van Leeuwenhoek (1677) first recorded observations of microorganisms (bacteria, yeasts, and protozoa) seen in water, faeces, teeth scrappings etc. under his own microscopes (Fig. 1.2) which were not compound.
Leeuwenhoek (1632-1723) was basically a cloth maker and tailor by trade, was also a surveyor and the official wine taster of Defft, Holland and his interest in microscopes was probably related to the use of magnifying glasses to examine fabrics. He transmitted his findings in a series of more than two hundred letters to the Royal Society of London during his lifetime.
He described such tiny creatures as “dierkens” or “animalcula viva” which were translated in English as “animalcules” by the Royal Society. Leeuwenhoek was later elected a fellow of the Royal Society.
Although there are reports of works on microorganisms, O.F. Muller gave first classification of bacterial microbes in 1773 and 1788, and coined the terms “Vibrio and “Monas” for certain forms; Ehrenberg established a new genus ‘Bacterium’ in 1829. Leeuwenhoek’s animalcules took two centuries to cause any spurt among the scientists when their importance was realized in different areas of human affairs.
Landmark # 3. Abiogenesis Versus Biogenesis (Microbes and the Origin of Life):
1. Spontaneous Generation Doctrine (or Abiogenesis):
Men of ancient times (Thales, 624-548 B.C.; Anaximander, 611-547 B.C.; Anaximenes, 588-524 B.C.; Empedocles, 504-433 B.C.; Aristotle, 384-322 B.C.; Epicurus, 341-270 B.C.; and Socretius 99-55 B.C.) knew nothing of microorganisms, of evolution, or of the fact that only living things could beget living things.
They believed that all living organisms could spring forth spontaneously from non-living matter. This belief has been referred to as Doctrine of Spontaneous Generation or Abiogenesis (Gr. a = not; bios = life; genesis = origin). They believed that frogs, snakes and mice could be born of moist soil, that flies could emerge from manure, and that maggots could arise from decaying corpses.
The idea of spontaneous generation was supported even 2000 years later. Van Helmont (1577-1644) devised a method for manufacturing mice. He recommended putting some wheat grains with soiled linen and cheese into an appropriate receptacle and leaving it undisturbed for a time in an attic or stable.
Mice would then appear. However, the idea of spontaneous generation continued until the mid-19th century with great oppositions against it.
2. Controversy over Spontaneous Generation:
Actually, it was the discovery of microorganisms and improvements in microscopy that enabled scientists to think seriously about the origin of life.
Francesco Redi (1626-1679), an Italian physician, demonstrated during mid-17th century by simple experiments (Fig. 1.3) that spontaneous generation (abiogenesis) does not exist. He took rotting meat pieces and placed them in jars. He sealed some of these jars tightly and left others open.
In a few days, maggots appeared in open jars in which the flies went freely in and out and laid their eggs on meat. Contrary to it, the sealed jars in which the flies could not enter did not show any maggots. From these observations Redi concluded that the maggots arise from the eggs laid down by the parent flies and that the maggots cannot appear spontaneously.
Still, the supporters of abiogenesis did not agree with Redi and argued that the free air, which was considered as “vital force” necessary for spontaneous origin of life, was not allowed to reach the meat placed in sealed jars.
So Redi set up new set of experiment in which he covered jars with fine muslin cloth or gauze instead of sealing them tightly and thus allowed free air to go in and out of the jars. Even after doing so the maggots appeared only in those jars in which flies were allowed free to go in and lay their eggs on the meat.
Even after Reid’s convincing demonstration, abiogenesis versus biogenesis controversy continued. John Needham (1745) advocated that even after he heated chicken broth and corn infusions (nutrient fluids) before pouring them into covered flasks, the cooled solutions showed existence of tiny organisms in them and thus he claimed that the organisms originated spontaneously from the nutrient fluids.
We shall see later that this result was due to insufficient heating which failed to kill heat-resistant forms of bacteria containing endospores. But nothing was known about endospores at that time. In the year 1765, twenty years later Lazzaro Spallanzani demonstrated that nutrient fluids of Needham did not contain microorganisms when they were subjected to prolong heating after being sealed in flasks.
He explained that the microorganisms from air probably had entered Needham’s solutions after they were boiled. Needham responded to it and said that the free air, the “vital force”, present inside Spallanzani’s scaled flasks had been destroyed by heating and, therefore, microorganisms did not appear in nutrient fluids in absence of the “vital force”.
3. End of the Debate:
Irritated by continuous advocacy in favour of spontaneous generation even by nineteenth century scientists, Louis Pasteur (1861) conducted series of experiments to prove that if the solutions are made microbe-free by boiling and they are provided with microbe-free air (the -vital force” for spontaneous generation), they do not show any sign of spontaneous origin of microbial life in them.
In his swan-necked flask experiment (Fig. 1.4), he took various type of broths (yeast water, sugared yeast water, urine, sugar beet juice etc.) in long-naked flasks and, then, softened the neck of the flasks under a flame and drew it out in the shape of ‘S’ looking like the neck of the swan. The broths of these flasks were boiled until they steamed through the necks, and then cooled.
The broths so treated in the flasks did not decay, and there were no signs of microorganisms in them after days, weeks and even months though they were open to free-air.
Pasteur’s unique swan-necks of the flasks trapped air-borne microorganisms before they could reach the broth and flourish in it. The broths in the flasks open to free air but free of microbes for very long periods, therefore, definitely discredited the doctrine of spontaneous generation.
Despite Pasteur’s successful demonstrations against spontaneous generation, attempts to repeat his experiments occasionally failed because, after some time, existence of microbes was evident in some broths of swan-necked flasks. This created doubt in the minds of many. But, this problem was soon solved by John Tyndall, an English physicist, in the year 1877.
He explained that bacteria exist in two forms: Heat-labile forms (thermolabile) which could be killed by exposure to high temperatures, and heat-resistant forms which could not be killed by continuous boiling of the broth and, after the broth has cooled, they resulted in microbial growth in such broths.
He further stated that if such broths are subjected to intermittent boiling (discontinuous boiling) on successive occasions, a process now popular as tyndallization, the heat-resistant forms of bacteria will be killed and the broths become completely free of them, and do not show any microbial growth. It so happens because the first boiling kills vegetative cells of bacteria but endospores remain as such.
The endospores now germinate in cooled broth and produce new bacterial cells which are killed during further boiling and so on. In this way, Tyndall validated Pasteur’s results and helped ending the debate on abiogenesis versus biogenesis.
Landmark # 4. Fermentation (A biological Process); Pasteurization (Heating Destroys Microbes):
Fermentation is a process that breaks down carbohydrates into alcohols and organic acids. Earlier it was believed that the fermentation is purely a chemical process.
But, it was Louis Pasteur, a chemist by training who convinced in 1857 the scientific world of his time that all fermentative processes are the results of microbial activity. This he did by showing that fermentation is invariably accompanied by the development of microorganisms.
Pasteur studied various types of fermentations and demonstrated that each particular type of fermentation occurs by the act of specific type of microorganism.
During his work on butyric fermentation Pasteur discovered another fundamental biological phenomenon: the existence of forms of life that can live only the absence of free oxygen (anaerobic life) and he introduced the terms ‘aerobic’ and ‘anaerobic’ to designate, respectively, life in the presence of and in the absence of oxygen.
It was found that during fermentation processes for desired products certain undesired microbes grew in “ferments” and resulted in undesired products. For convenience, rod-shaped bacteria grew in certain wine-vats and produced lactic acid that caused souring of wine.
To solve this problem, Pasteur (1860) suited that heating could be used to kill undesirable type of microbes growing in “ferments”. Pasteur’s this suggestion later came to be recognized as pasteurization, i.e., heating at moderate temperature to kill high percentage of microbial population. Today pasteurization is widely used in the fermentation industries especially the dairy industry.
Landmark # 5. Germ Theory of Disease:
Though strong arguments for the germ theory of disease (diseases are caused by living organisms) were given by many earlier workers but all were mostly speculative. The first satisfactory demonstration of the probable causal relationship of organisms to disease was given by Benedict Prevost (1807) in plants He proved conclusively that bunt disease of wheat is caused by a fungus.
Though Prevost’s experiments provided the first proof and interpretation of the role of a microorganism in the causation of a disease (i.e. the germ theory of disease), his findings were ahead of his time and were rejected by almost all his contemporaries who believed in spontaneous generation of microorganisms and of disease and that the microorganisms and their spores were the result rather than the cause of disease.
The discovery that bacteria can act as specific agents of infectious disease in animals was made through the study of anthrax disease. C.J. Davaine (1863 and 1868) showed that bacteria are invariably present.
In diseased animals but are undetectable in healthy ones and that the disease can be transmitted to healthy animals by inoculation with blood containing these bacteria but he could not prove whether these bacteria were the cause or the result of the disease.
Somewhat later, Robert Koch (1876) solved the problem and confirmed the germ theory of disease by conclusive demonstration of the bacterial causation or etiology of anthrax disease.
By a series of experiments on anthrax disease of cattle, he showed that the spores of anthraxbacilli isolated from pure cultures could infect animals and thus demonstrated that germ grown outside a body could cause disease and that specific microorganisms caused specific diseases.
Finally in 1882, to establish cause and effect relationship between a given microorganism and a specific disease, Koch described some steps necessary to identify the causative agent of a disease.
These steps are popularly known as Koch’s Postulates which is; even today used in animal and plant pathology.
In fact the “steps” were the ideas of Jacob Henle who proposed during 1840s that to establish the etiology of a specific disease, the agent would have to be found regularly in the host during the disease, the agent would have to be isolated, and the isolated agent would have to be shown capable of producing the disease. Koch, however, first applied these ideas experimentally and established their validity.
Landmark # 6. Pure Culture Concept and Other Microbial Techniques:
Around 1870 it began to be realized that a sound understanding of the form and function of microorganisms could be obtained only if they are isolated and grown in pure culture form. A pure culture is one that contains only a single kind of microbial population grown from a single cell.
Much of the pioneer work on pure culture technique was done by B. Brefeld (a mycologist) but his methods worked well for fungi and were found to be unsuitable for bacteria.
It was Joseph Lister (1878) who first obtained pure culture of bacteria using serial dilutions in liquid media. He took a fluid containing a mixture of bacteria, diluted it with sterile medium, and delivered it into a container of sterile milk by a specially constructed syringe.
After incubation, he found that there were single kind of bacteria growing in container identical to their parent cell. In practice, serial dilution method proved to be tedious, difficult and uncertain for routine use. It also proved to be disadvantageous because one could only isolate in pure form the microbes that predominated in the original mixture. Therefore, another promising device needed to be investigated.
Robert Koch was particularly concerned with this problem and, at first, he cultured bacteria on solid fruits and vegetables such as slices of boiled potato but many bacteria did not grow on such substrates. Then he perceived that it would be far better if a well-tried liquid medium could be solidified with some clear substance.
Koch (1881) tried gelatin as a solidifying agent and succeeded in developing solid culture media, but gelatin, the first solidifying agent used, had serious disadvantage of becoming liquid above 28-30°C which is below the optimum temperature for the growth of human disease producing bacteria.
However, Koch replaced gelatin by agar in 1883-84 on the recommendation of F.E. Hesse, a German housewife, who had gained experience with the characteristics of agar in the process of making jelly.
Agar is still frequently used as solidifying agent in microbiological laboratories. The development of solid culture media to grow pure culture was of fundamental importance and may be considered one of the Koch’s greatest contributions.
Besides developing solid culture media using gelatin and agar, Koch also evolved methods to place microbes on glass slides and colour them with analine dyes (stains) so that the individual cells could be seen more clearly under the microscope.
Landmark # 7. Growth of Medical Microbiology:
1. Causation of Diseases:
Reports regarding causative agents of animal and human diseases started pouring in during the second quarter of the nineteenth century. A. Bassi (1836) recognised that the disease of silkworms may be caused by a fungus. J. Schoelein (1839) established that ‘favus’ is caused by a pathogenic fungus. D. Gruby (1843) revealed the causative agent of trichophytosis (ringworm).
In the second half of the nineteenth century due to availability of better microscopes and abandonment of spontaneous generation, Pasteur and his contemporary workers turned towards this area of microbiology which successfully led to several important discoveries regarding causative agents of animal and human diseases with certainty (Table 1.3).
Immunization is the artificial induction of immunity to disease. The practice of immunization was used in Asia for centuries to produce immunity to smallpox before it was introduced into England in 1718 by Lady Mary Montagu but she had no explanation of how or why it worked.
The procedure of immunization against smallpox was quite simple; material from a pustule of an infected person was scratched into the skin of the person to be immunized.
In most cases this resulted in a mild case of smallpox without the scarring that was common in naturally acquired cases. E. Jenner (1749-1823) in 1798 reported to Royal Society in London the value of immunization with cowpox as a means of protecting against smallpox; a clear case of vaccination.
This he did on the basis of the fact that when he inoculated a 8- years old boy, James Phipps, with cowpox virus content, the boy escaped from small pox infection.
Jenner’s explanation regarding cowpox vaccination against small pox established the scientific credibility of vaccination to prevent disease and was accepted by the scientists and physicians of the time.
Jenner, therefore, is credited to have develop first vaccine from cowpox (Latin name Vaccinia). Later on, Pasteur developed vaccines (attenuated microorganisms) against chicken cholera and anthrax in 1880, and against rabies in 1885.
3. Surgical Antisepsis:
Joseph Lister (1827-1912), a British surgeon, reasoned that the post-surgery infections might well result due to microorganisms present in air that enter the tissues exposed during operation, and revolutionized surgical practice in 1867 by introducing antiseptic principles. He used carbolic acid (a phenol) as a disinfectant and adopted several other antiseptic procedures which are in practice still today.
4. Rise of Chemotherapy:
For many years even after the discovery of the role of microorganisms as the causative agents of infectious diseases, their control was largely preventive, exclusively based on the use of vaccines and antisera and there were no scientific approach to cure them after they had appeared in an organism.
Paul Ehrlich (1854-1915), a German physician-chemist, undertook extensive studies to search synthetic chemicals having curative properties for pathogenic microorganisms and coined the term ‘chemotherapy’ to describe this approach to control infectious diseases.
During his research between 1880-1910 he developed almost 1000 new derivatives of an arsenical chemical, the atoxyl, and found that the derivative no. 606 called salvarsan proved to be effective in treating syphilis. In this way, Paul Ehrlich, the founder of modern chemotherapy, opened a new way of chemical treatment of infectious diseases.
Landmark # 8. Growth of Plant Pathology:
It has been shown during the early decades of nineteenth century that specific fungi can cause diseases of wheat and rye, and in 1845 A.J. Berkeley stated that the blight disease of potato in Northern Europe during 1840s was caused by a fungus.
The blight disease of potato turned to epiphytotic form in the mid-1840s particularly in Ireland resulting in death and migration of great number of people and tragically dramatized the importance of plant diseases.
Extensive studies started and, finally, de Bary (1861, 1863) proved experimentally that the causal agent of blight of potato was a fungus which he named Phytophthora infestans de Bery thus pioneered the scientific approach in the area of plant diseases and is rightly called the “Founder of Experimental Plant Pathology”.
Burill (1878) for the first time showed that the fire blight disease of pears was caused by a bacterium, Erwinia amylovora. Infect, it was the discovery of phytopathogenic nature of bacteria. This achievement of Burill was established beyond any doubt with numerous and excellent contributions on the study of bacterial disease of plants by E.F. Smith from 1895 onwards.
Landmark # 9. Discovery of Viruses:
The tobacco crop in Holland was struck by a severe disease around 1870. Adolf Mayer, Director of Agricultural Experimental Station, Wageningen, began his studies on this disease about 1880 and published his results in 1886.
Mayer christened the disease as “MOSAIKKRANKHEIT” (mosaic-like), from the mosaic-like pattern on leaves of diseased plants and succeeded in reproducing the disease by infecting juice extracted from infected tobacco leaves onto healthy ones; he could not succeed in identifying the real agent that caused the disease.
However, Mayer’s contribution will always be remembered as he was the first person who put first step forward in the development of a new discipline later recognised as ‘Virology’.
A later in the year 1892, D. Ivanowski first successfully experimentally demonstrated that the tobacco mosaic disease has been caused by agents which successfully passed the Chamberland-filter that retains even the smallest bacteria.
It was an important clue, but contrary to his experimental result and despite his inability to isolate any bacterium. Ivanowski still maintained that either the ‘pathogenic bacterium’ somehow passed through the filter or a ‘toxin’ secreted by them passed through the filter and made the filtrate infectious. Within six years after the experiments of Ivanowski.
M. Beijerinck (1898) confirmed by repeating the same experiments and found that the tobacco mosaic disease was caused not by any pathogenic bacteria or toxin but rather by some new type of pathogenic agents which he called “contagium vivum fluidum” (infectious living fluid) and referred subsequently to it as a “virus” (poison). He also said that the viruses multiply only inside the living cell.
Landmark # 10. Other Important Contributions:
The methyl violet dye was first used by Weigert (1875) for staining bacteria. Christian Gram (1884) introduced the ‘Gram Staining Technique’ to stain bacteria.
We still use this technique for identifying and classifying bacteria. H.C. Hansen (1842-1909) opened the way to study of industrial fermentations as he developed the pure culture study of yeast and bacteria used in vinegar manufacture and named them as “starters”.
Adametz (1889), for example, used pure cultures in cheese manufacture, and Conn and Weigmann developed pure culture starters for butter production (1890-1897).
Thus the science of microbiology grew up profoundly within a period of forty years (1860-1900). Its early years were tempestuous, but by the first years of twentieth century the man in the street and on the farm knew of bacteria. Man, by this time, also knew that the bacteria could do good for him, and resolved in learning to control them from causing diseases to plants, animals, and humans.
Many landmark discoveries in the field of microbiology have been made in 20th century, the modern period of grow of microbiology. However, the landmark events in the development of microbiology are given in Table 1.4.