3 Pioneers in the Science of Microbiology

Bacteria and protozoa were the first microbes to be observed by humans. It then took about 200 years before a connection was established between microbes and infectious diseases. Among the most significant events in the early history of microbiology were the development of microscopes, bacterial staining procedures, techniques that enabled microorganisms to be cultured (grown) in the laboratory, and steps that could be taken to prove that specific microbes were responsible for causing specific infectious diseases. During the past 400 years, many individuals contributed to our present understanding of microbes.

Anton van Leeuwenhoek (1632–1723) 

Because Anton van Leeuwenhoek was the first person to see live bacteria and protozoa, he is sometimes referred to as the “Father of Microbiology,” the “Father of Bacteriology,” and the “Father of Protozoology.” Interestingly, Leeuwenhoek was not a trained scientist. At various times in his life, he was a fabric merchant, a surveyor, a wine assayer, and a minor city official in Delft,  Holland. As a hobby, he ground tiny glass lenses, which he mounted in small metal frames, thus creating what today are known as single-lens microscopes or simple  microscopes. During his lifetime, he made more than 500 of these microscopes. Leeuwenhoek’s fine art of grinding lenses that would magnify an object to 200 to 300 times its size was lost at his death because he had not taught this skill to anyone during his lifetime. In one of the hundreds of letters that he sent to the Royal Society of London, he wrote:
My method for seeing the very smallest animalcules I do not impart to others; nor how to see very many animalcules at one time. This I keep for myself alone.
Portrait of Anton van Leeuwenhoek by Jan Verkolje.
Fig.1: Portrait of Anton van Leeuwenhoek by Jan Verkolje (first pioneer).
Apparently, Leeuwenhoek had an unquenchable curiosity, as he used his microscopes to examine almost anything he could get his hands on (Fig. 1). He examined scrapings from his teeth, water from ditches and ponds, water in which he had soaked peppercorns, blood, sperm, and even his own diarrheal stools. In many of these specimens, he observed various tiny living creatures, which he called “animalcules.” He recorded his observations in the form of letters, which he sent to the Royal Society of London. The following passage is an excerpt from one of those letters (Milestones in Microbiology, edited by Thomas Brock. American Society for Microbiology, Washington, DC, 1961):
Tho my teeth are kept usually very clean, nevertheless when I view them in a Magnifying Glass, I find growing between them a little white matter as thick as wetted flower. . . . I therefore took some of this flower and mixt it . . . with pure rain water wherein were no Animals. and then to my great surprize perceived that the aforesaid matter contained very many small living Animals, which moved themselves very extravagantly. . . . The number of these Animals in the scurf of a mans Teeth, are so many that I believe they exceed the number of Men in a kingdom. For upon the examination of a small parcel of it, no thicker than a Horse-hair, I found too many living Animals therein, that I guess there might have been 1000 in a quantity of matter no bigger than the 1/100 part of a sand.
Leeuwenhoek’s letters finally convinced scientists of the late 17th century of the existence of microbes. Leeuwenhoek never speculated on their origin, nor did he associate them with the cause of disease. Such relationships were not established until the work of Louis Pasteur and Robert Koch in the late 19th century.

The following quote is from Paul de Kruif’s book, Microbe Hunters, Harcourt Brace, 1926:
[Leeuwenhoek] had stolen and peeped into a fantastic sub-visible world of little things, creatures that had lived, had bred, had battled, had died, completely hidden from and unknown to all men from the beginning of time. Beasts these were of a kind that ravaged and annihilated whole races of men ten million times larger than they were themselves. Beings these were, more terrible than fire-spitting dragons or hydra-headed monsters. They were silent assassins that murdered babes in warm cradles and kings in sheltered places. It was this invisible, insignificant, but implacable—and sometimes friendly—world that Leeuwenhoek had looked into for the first time of all men of all countries.
Once scientists became convinced of the existence of tiny creatures that could not be observed with the naked eye, they began to speculate on their origin. On the basis of observation, many of the scientists of that time believed that life could develop spontaneously from inanimate substances, such as decaying corpses, soil, and swamp gases. The idea that life can arise spontaneously from nonliving material is called the theory of spontaneous generation or abiogenesis. For more than two centuries, from 1650 to 1850, this theory was debated and tested. Following the work of others, Louis Pasteur (discussed later) and John Tyndall finally disproved the theory of spontaneous generation and proved that life can only arise from preexisting life. This is called the theory of biogenesis, first proposed by a German scientist named Rudolf Virchow in 1858. Note that the theory of biogenesis does not speculate on the origin of life, a subject that has been discussed and debated for hundreds of years.

Although Leeuwenhoek was probably the first person to see live protozoa, he may not have been the first person to observe protozoa. Many scholars believe that Robert Hooke (1635–1703), an English physician, was the first person to observe and describe microbes, including a fossilized protozoan and two species of live microfungi.

Louis Pasteur (1822–1895)

Louis Pasteur (Fig. 2), a French chemist, made numerous contributions to the newly emerging field of microbiology, and, in fact, his contributions are considered by many people to be the foundation of the science of microbiology and a cornerstone of modern medicine. Listed below are some of his most significant contributions:
Pasteur in his laboratory. A 1925 wood engraving by Timothy Cole. (From Zigrosser C. Medicine and the Artist [Ars Medica]. New York: Dover Publications, Inc.; 1970. With permission from the Philadelphia Museum of Art.)
Fig.2: Pasteur in his laboratory. A 1925 wood engraving by Timothy Cole. (From Zigrosser C. Medicine and the Artist [Ars Medica]. New York: Dover Publications, Inc.; 1970. With permission from the Philadelphia Museum of Art.)
  • While attempting to discover why wine becomes contaminated with undesirable substances, Pasteur discovered what occurs during alcoholic fermentation. He also demonstrated that different types of microbes produce different fermentation products. For example, yeasts convert the glucose in grapes to ethyl alcohol (ethanol) by fermentation, but certain contaminating bacteria, such as Acetobacter, convert glucose to acetic acid (vinegar) by fermentation, thus, ruining the taste of the wine.
  • Through his experiments, Pasteur dealt the fatal blow to the theory of spontaneous generation.
  • Pasteur discovered forms of life that could exist in the absence of oxygen. He introduced the terms “aerobes” (organisms that require oxygen) and “anaerobes” (organisms that do not require oxygen).
  • Pasteur developed a process (today known as pasteurization) to kill microbes that were causing wine to spoilan economic concern to France’s wine industry. Pasteurization can be used to kill pathogens in many types of liquids. Pasteur’s process involved heating wine to 55°C and holding it at that temperature for several minutes. Today, pasteurization is accomplished by heating liquids to 63°C to 65°C for 30 minutes or to 73°C to 75°C for 15 seconds. It should be noted that pasteurization does not kill all of the microbes in liquids—just the pathogens.
  • Pasteur discovered the infectious agents that caused the silkworm diseases that were crippling the silk industry in France. He also discovered how to prevent such diseases.
  • Pasteur made significant contributions to the germ theory of disease—the theory that specific microbes cause specific infectious diseases. For example, anthrax is caused by a specific bacterium (Bacillus anthracis), whereas tuberculosis is caused by a different bacterium (Mycobacterium tuberculosis).
  • Pasteur championed changes in hospital practices to minimize the spread of disease by pathogens.
  • Pasteur developed vaccines to prevent chicken cholera, anthrax, and swine erysipelas (a skin disease). It was the development of these vaccines that made him famous in France. Before the vaccines, these diseases were decimating chickens, sheep, cattle, and pigs in that country—a serious economic problem.
  • Pasteur developed a vaccine to prevent rabies in dogs and successfully used the vaccine to treat human rabies.
To honor Pasteur and continue his work, especially in the development of a rabies vaccine, the Pasteur Institute was created in Paris in 1888. It became a clinic for rabies treatment, a research center for infectious diseases, and a teaching center. Many scientists who studied under Pasteur went on to make important discoveries of their own and create a vast international network of Pasteur Institutes. The first of the foreign institutes was founded in Saigon, Vietnam, which is today known as Ho  Chi Minh City. One of the directors of that institute was Alexandre Emile Jean Yersin—a former student of Robert Koch and Louis Pasteur—who, in 1894, discovered the bacterium that causes plague.

An Ethical Dilemma for Louis Pasteur 

In July 1885, while he was developing a vaccine that would prevent rabies in dogs, Louis Pasteur faced an ethical decision. A 9-year-old boy, named Joseph Meister, had been bitten 14 times on the legs and hands by a rabid dog. At the time, it was assumed that virtually anyone who was bitten by a rabid animal would die. Meister’s mother begged Pasteur to use his vaccine to save her son. Pasteur was a chemist, not a physician, and thus was not authorized to treat humans. Also, his experimental vaccine had never been administered to a human being. Nonetheless, 2 days after the boy had been bitten, Pasteur injected Meister with the vaccine in an attempt to save the boy’s life. The boy survived, and Pasteur realized that he had developed a rabies vaccine that could be administered to a person after he or she had been infected with rabies virus.

Robert Koch (1843–1910) 

Robert Koch (Fig. 3), a German physician, made numerous contributions to the science of microbiology. Some of them are listed here:
Fig.3 : Robert (widroid archive) Koch
Fig.3 : Robert Koch
  • Koch made many significant contributions to the germ theory of disease. For example, he proved that the anthrax bacillus (B. anthracis), which had been discovered earlier by other scientists, was truly the causative agent of anthrax. He accomplished this using a series of scientific steps that he and his colleagues had developed; these steps later became known as Koch’s Postulates.
  • Koch discovered that B. anthracis produces spores, capable of resisting adverse conditions.
  • Koch developed methods of fixing, staining, and photographing bacteria.
  • Koch developed methods of cultivating bacteria on solid media. One of Koch’s colleagues, R.J. Petri, invented a flat glass dish (now known as a Petri dish) in which to culture bacteria on solid media. It was Frau Hesse—the wife of another of Koch’s colleagues—who suggested the use of agar (a polysaccharide obtained from seaweed) as a solidifying agent. These methods enabled Koch to obtain pure cultures of bacteria. The term pure culture refers to a condition in which only one type of organism is growing on a solid culture medium or in a liquid culture medium in the laboratory; no other types of organisms are present. Petri dishes containing agar are still used to culture bacteria and fungi in laboratories.
  • Koch discovered the bacterium (M. tuberculosis) that causes tuberculosis and the bacterium (Vibrio cholerae) that causes cholera.
  • Koch’s work on tuberculin (a protein derived from M. tuberculosis) ultimately led to the development of a skin test valuable in diagnosing tuberculosis.

Koch’s Postulates 

During the mid- to late-1800s, Koch and his colleagues established an experimental procedure to prove that a specific microbe is the cause of a specific infectious disease. This scientific procedure, published in 1884, became known as Koch’s Postulates (Fig. 4).
Fig. 4: Koch’s Postulates: proof of the germ theory of disease.
Fig. 4: Koch’s Postulates: proof of the germ theory of disease.

Koch’s Postulates (paraphrased):
  1. A particular microbe must be found in all cases of the disease and must not be present in healthy animals or humans.
  2. The microbe must be isolated from the diseased animal or human and grown in pure culture in the laboratory.
  3. The same disease must be produced when microbes from the pure culture are inoculated into healthy susceptible laboratory animals.
  4. The same microbe must be recovered from the experimentally infected animals and grown again in pure culture.
After completing these steps, the microbe is said to have fulfilled Koch’s Postulates and has been proven to be the cause of that particular infectious disease. Koch’s Postulates not only helped to prove the germ theory of disease, but also gave a tremendous boost to the development of microbiology by stressing laboratory culture and identification of microbes.

Exceptions to Koch’s Postulates 

Circumstances do exist in which Koch’s Postulates cannot be fulfilled. Examples of such circumstances are as follows:
  • To fulfill Koch’s Postulates, it is necessary to grow (culture) the pathogen in the laboratory (in vitro = the term in vitro refers to something that occurs outside the living body, whereas the term in vivo refers to something that occurs within the living body. In vitro often refers to something that occurs in the laboratory) in or on artificial culture media. However, certain pathogens will not grow on artificial media. Such pathogens include viruses, rickettsias (a category of bacteria), chlamydias (another category of bacteria), and the bacteria that cause leprosy and syphilis. Viruses, rickettsias, and chlamydias are called obligate intracellular pathogens (or obligate intracellular parasites) because they can survive and multiply only within living host cells. Such organisms can be grown in cell cultures (cultures of living human or animal cells of various types), embryonated chicken eggs, or certain animals (referred to as laboratory animals). In the laboratory, the leprosy bacterium (Mycobacterium leprae) is propagated in armadillos, and the spirochetes of syphilis (Treponema pallidum) grow well in the testes of rabbits and chimpanzees. Microbes having complex and demanding nutritional requirements are said to be fastidious (meaning fussy). Although certain fastidious organisms can be grown in the laboratory by adding special mixtures of vitamins, amino acids, and other nutrients to the culture media, others cannot be grown in the laboratory because no one has discovered what ingredient(s) to add to the medium to enable them to grow.
  • To fulfill Koch’s Postulates, it is necessary to infect laboratory animals with the pathogen being studied. However, many pathogens are species-specific, meaning that they infect only one species of animal. For example, some pathogens that infect humans will infect only humans. Thus, it is not always possible to find a laboratory animal that can be infected with a pathogen that causes human disease. Because human volunteers are difficult to obtain and ethical considerations limit their use, the researcher may only be able to observe the changes caused by the pathogen in human cells that can be grown in the laboratory (called cell cultures).
  • Some diseases, called synergistic infections or poly-microbial infections, are caused not by one particular microbe, but by the combined effects of two or more different microbes. Examples of such infections include acute necrotizing ulcerative gingivitis (ANUG; also known as “trench mouth”) and bacterial vaginosis. It is very difficult to reproduce such synergistic infections in the laboratory.
  • Another difficulty that is sometimes encountered while attempting to fulfill Koch’s Postulates is that certain pathogens become altered when grown in  vitro. Some become less pathogenic, whereas others become nonpathogenic. Thus, they will no longer infect  animals after being cultured on artificial media.
It is also important to keep in mind that not all diseases are caused by microbes. Many diseases, such as rickets and scurvy, result from dietary deficiencies. Some diseases are inherited because of an abnormality in the chromosomes, as in sickle cell anemia. Others, such as diabetes, result from malfunction of a body organ or system. Still others, such as cancer of the lungs and skin, are influenced by environmental factors. However, all infectious diseases are caused by microbes, as are all microbial intoxications. All infectious diseases and microbial intoxications are caused by microbes.