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Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 PART I Introduction to Microbiology Chapter 1 The History and Scope of Microbiology Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation Chapter 3 Procaryotic Cell Structure and Function Chapter 4 Eucaryotic Cell Structure and Function CHAPTER 1 The History and Scope of Microbiology Louis Pasteur, one of the greatest scientists of the nineteenth century, maintained that “Science knows no country, because knowledge belongs to humanity, and is a torch which illuminates the world.” Outline 1.1 The Discovery of Microorganisms 2 1.2 The Conflict over Spontaneous Generation 2 1.3 The Role of Microorganisms in Disease 7 Recognition of the Relationship between Microorganisms and Disease 7 The Development of Techniques for Studying Microbial Pathogens 8 Immunological Studies 9 1.4 Industrial Microbiology and Microbial Ecology 10 1.5 Members of the Microbial World 11 1.6 The Scope and Relevance of Microbiology 11 1.7 The Future of Microbiology 13 Concepts 1. Microbiology is the study of organisms that are usually too small to be seen by the unaided eye; it employs techniques—such as sterilization and the use of culture media—that are required to isolate and grow these microorganisms. 2. Microorganisms are not spontaneously generated from inanimate matter but arise from other microorganisms. 3. Many diseases result from viral, bacterial, fungal, or protozoan infections. Koch’s postulates may be used to establish a causal link between the suspected microorganism and a disease. 4. The development of microbiology as a scientific discipline has depended on the availability of the microscope and the ability to isolate and grow pure cultures of microorganisms. 5. Microorganisms are responsible for many of the changes observed in organic and inorganic matter (e.g., fermentation and the carbon, nitrogen, and sulfur cycles that occur in nature). 6. Microorganisms have two fundamentally different types of cells—procaryotic and eucaryotic—and are distributed among several kingdoms or domains. 7. Microbiology is a large discipline, which has a great impact on other areas of biology and general human welfare

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 Dans les champs de l’observation, le hasard ne favorise que les esprits préparés. (In the field of observation, chance favors only prepared minds.) —Louis Pasteur One can’t overemphasize the importance of microbiology. Society benefits from microorganisms in many ways. They are necessary for the production of bread, cheese, beer, antibiotics, vaccines, vitamins, enzymes, and many other important products. Indeed, modern biotechnology rests upon a microbiological foundation. Microorganisms are indispensable components of our ecosystem. They make possible the cycles of carbon, oxygen, nitrogen, and sulfur that take place in terrestrial and aquatic systems. They also are a source of nutrients at the base of all ecological food chains and webs. Of course microorganisms also have harmed humans and disrupted society over the millennia. Microbial diseases undoubtedly played a major role in historical events such as the decline of the Roman Empire and the conquest of the New World. In 1347 plague or black death (see chapter 39) struck Europe with brutal force. By 1351, only four years later, the plague had killed 1/3 of the population (about 25 million people). Over the next 80 years, the disease struck again and again, eventually wiping out 75% of the European population. Some historians believe that this disaster changed European culture and prepared the way for the Renaissance. Today the struggle by microbiologists and others against killers like AIDS and malaria continues. The biology of AIDS and its impact (pp. 878–84) In this introductory chapter the historical development of the science of microbiology is described, and its relationship to medicine and other areas of biology is considered. The nature of the microbial world is then surveyed to provide a general idea of the organisms and agents that microbiologists study. Finally, the scope, relevance, and future of modern microbiology are discussed. Microbiology often has been defined as the study of organisms and agents too small to be seen clearly by the unaided eye—that is, the study of microorganisms. Because objects less than about one millimeter in diameter cannot be seen clearly and must be examined with a microscope, microbiology is concerned primarily with organisms and agents this small and smaller. Its subjects are viruses, bacteria, many algae and fungi, and protozoa (see table 34.1). Yet other members of these groups, particularly some of the algae and fungi, are larger and quite visible. For example, bread molds and filamentous algae are studied by microbiologists, yet are visible to the naked eye. Two bacteria that are visible without a microscope, Thiomargarita and Epulopiscium, also have been discovered (see p. 45). The difficulty in setting the boundaries of microbiology led Roger Stanier to suggest that the field be defined not only in terms of the size of its subjects but also in terms 2 Chapter 1The History and Scope of Microbiology of its techniques. A microbiologist usually first isolates a specific microorganism from a population and then cultures it. Thus microbiology employs techniques—such as sterilization and the use of culture media—that are necessary for successful isolation and growth of microorganisms. The development of microbiology as a science is described in the following sections. Table 1.1 presents a summary of some of the major events in this process and their relationship to other historical landmarks. 1.1 The Discovery of Microorganisms Even before microorganisms were seen, some investigators suspected their existence and responsibility for disease. Among others, the Roman philosopher Lucretius (about 98–55 B.C.) and the physician Girolamo Fracastoro (1478–1553) suggested that disease was caused by invisible living creatures. The earliest microscopic observations appear to have been made between 1625 and 1630 on bees and weevils by the Italian Francesco Stelluti, using a microscope probably supplied by Galileo. However, the first person to observe and describe microorganisms accurately was the amateur microscopist Antony van Leeuwenhoek (1632–1723) of Delft, Holland (figure 1.1a). Leeuwenhoek earned his living as a draper and haberdasher (a dealer in men’s clothing and accessories), but spent much of his spare time constructing simple microscopes composed of double convex glass lenses held between two silver plates (figure 1.1b). His microscopes could magnify around 50 to 300 times, and he may have illuminated his liquid specimens by placing them between two pieces of glass and shining light on them at a 45° angle to the specimen plane. This would have provided a form of dark-field illumination (see chapter 2) and made bacteria clearly visible (figure 1.1c). Beginning in 1673 Leeuwenhoek sent detailed letters describing his discoveries to the Royal Society of London. It is clear from his descriptions that he saw both bacteria and protozoa. 1.2 The Conflict over Spontaneous Generation From earliest times, people had believed in spontaneous generation—that living organisms could develop from nonliving matter. Even the great Aristotle (384–322 B.C.) thought some of the simpler invertebrates could arise by spontaneous generation. This view finally was challenged by the Italian physician Francesco Redi (1626–1697), who carried out a series of experiments on decaying meat and its ability to produce maggots spontaneously. Redi placed meat in three containers. One was uncovered, a second was covered with paper, and the third was covered with a fine gauze that would exclude flies. Flies laid their eggs on the uncovered meat and maggots developed. The other two pieces of meat did not produce maggots spontaneously. However, flies were attracted to the gauze-covered container and laid their eggs on the gauze; these eggs produced maggots

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 1.2 The Conflict over Spontaneous Generation 3 Table 1.1 Some Important Events in the Development of Microbiology Date Microbiological History Other Historical Events 1546 Fracastoro suggests that invisible organisms cause disease Publication of Copernicus’s work on the heliocentric solar system (1543) 1590–1608 Jansen develops first useful compound microscope Shakespeare’s Hamlet (1600–1601) 1676 Leeuwenhoek discovers “animalcules” J. S. Bach and Handel born (1685) 1688 Redi publishes work on spontaneous generation of maggots Isaac Newton publishes the Principia (1687) Linnaeus’s Systema Naturae (1735) Mozart born (1756) 1765–1776 Spallanzani attacks spontaneous generation 1786 Müller produces first classification of bacteria French Revolution (1789) 1798 Jenner introduces cowpox vaccination for smallpox Beethoven’s first symphony (1800) The battle of Waterloo and the defeat of Napoleon (1815) Faraday demonstrates the principle of an electric motor (1821) 1838–1839 Schwann and Schleiden, the Cell Theory England issues first postage stamp (1840) 1835–1844 Bassi discovers that silkworm disease is caused by a fungus and proposes that many diseases are microbial in origin Marx’s Communist Manifesto (1848) 1847–1850 Semmelweis shows that childbed fever is transmitted by Velocity of light first measured by Fizeau (1849) physicians and introduces the use of antiseptics to prevent the disease Clausius states the first and second laws of thermodynamics (1850) 1849 Snow studies the epidemiology of a cholera epidemic Graham distinguishes between colloids and crystalloids in London Melville’s Moby Dick (1851) Otis installs first safe elevator (1854) Bunsen introduces the use of the gas burner (1855) 1857 Pasteur shows that lactic acid fermentation is due to a microorganism 1858 Virchow states that all cells come from cells Darwin’s On the Origin of Species (1859) 1861 Pasteur shows that microorganisms do not arise by American Civil War (1861–1865) spontaneous generation Mendel publishes his genetics experiments (1865) Cross-Atlantic cable laid (1865) 1867 Lister publishes his work on antiseptic surgery Dostoevski’s Crime and Punishment (1866) 1869 Miescher discovers nucleic acids Franco-German War (1870–1871) 1876–1877 Koch demonstrates that anthrax is caused by Bell invents telephone (1876) Bacillus anthracis Edison’s first light bulb (1879) 1880 Laveran discovers Plasmodium, the cause of malaria 1881 Koch cultures bacteria on gelatin Ives produces first color photograph (1881) Pasteur develops anthrax vaccine 1882 Koch discovers tubercle bacillus, Mycobacterium tuberculosis First central electric power station constructed by Edison (1882) 1884 Koch’s postulates first published Mark Twain’s The Adventures of Huckleberry Finn (1884) Metchnikoff describes phagocytosis Autoclave developed Gram stain developed 1885 Pasteur develops rabies vaccine First motor vehicles developed by Daimler (1885–1886) Escherich discovers Escherichia coli, a cause of diarrhea 1886 Fraenkel discovers Streptococcus pneumoniae, a cause of pneumonia 1887 Petri dish (plate) developed by Richard Petri 1887–1890 Winogradsky studies sulfur and nitrifying bacteria Hertz discovers radio waves (1888) 1889 Beijerinck isolates root nodule bacteria Eastman makes box camera (1888) 1890 Von Behring prepares antitoxins for diphtheria and tetanus 1892 Ivanowsky provides evidence for virus causation of tobacco mosaic disease First zipper patented (1895) 1894 Kitasato and Yersin discover Yersinia pestis, the cause of plague 1895 Bordet discovers complement Röntgen discovers X rays (1895) 1896 Van Ermengem discovers Clostridium botulinum, the cause of botulism 1897 Buchner prepares extract of yeast that ferments Thomson discovers the electron (1897) Ross shows that malaria parasite is carried by the mosquito Spanish-American War (1898) 1899 Beijerinck proves that a virus particle causes the tobacco mosaic disease 1900 Reed proves that yellow fever is transmitted by the mosquito Planck develops the quantum theory (1900) 1902 Landsteiner discovers blood groups First electric typewriter (1901)

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 4 Chapter 1The History and Scope of Microbiology Table 1.1 Continued Date Microbiological History Other Historical Events 1903 Wright and others discover antibodies in the blood of First powered aircraft (1903) immunized animals 1905 Schaudinn and Hoffmann show Treponema pallidum Einstein’s special theory of relativity (1905) causes syphilis 1906 Wassermann develops complement fixation test for syphilis 1909 Ricketts shows that Rocky Mountain spotted fever is transmitted First model T Ford (1908) by ticks and caused by a microbe (Rickettsia rickettsii) Peary and Hensen reach North Pole (1909) 1910 Ehrlich develops chemotherapeutic agent for syphilis Rutherford presents his theory of the atom (1911) 1911 Rous discovers a virus that causes cancer in chickens Picasso and cubism (1912) World War I begins (1914) 1915–1917 D’Herelle and Twort discover bacterial viruses Einstein’s general theory of relativity (1916) Russian Revolution (1917) 1921 Fleming discovers lysozyme 1923 First edition of Bergey’s Manual Lindberg’s transatlantic flight (1927) 1928 Griffith discovers bacterial transformation 1929 Fleming discovers penicillin Stock market crash (1929) 1931 Van Niel shows that photosynthetic bacteria use reduced compounds as electron donors without producing oxygen 1933 Ruska develops first transmission electron microscope Hitler becomes chancellor of Germany (1933) 1935 Stanley crystallizes the tobacco mosaic virus Domagk discovers sulfa drugs 1937 Chatton divides living organisms into procaryotes Krebs discovers the citric acid cycle (1937) and eucaryotes World War II begins (1939) 1941 Beadle and Tatum, one-gene-one-enzyme hypothesis 1944 Avery shows that DNA carries information during The insecticide DDT introduced (1944) transformation Waksman discovers streptomycin Atomic bombs dropped on Hiroshima and Nagasaki (1945) 1946 Lederberg and Tatum describe bacterial conjugation United Nations formed (1945) First electronic computer (1946) 1949 Enders, Weller, and Robbins grow poliovirus in human tissue cultures 1950 Lwoff induces lysogenic bacteriophages Korean War begins (1950) 1952 Hershey and Chase show that bacteriophages inject DNA First hydrogen bomb exploded (1952) into host cells Stalin dies (1952) Zinder and Lederberg discover generalized transduction First commercial transistorized product (1952) 1953 Phase-contrast microscope developed U.S. Supreme Court rules against segregated schools (1954) Medawar discovers immune tolerance Watson and Crick propose the double helix structure for DNA 1955 Jacob and Wollman discover the F factor is a plasmid Montgomery bus boycott (1955) Jerne and Burnet propose the clonal selection theory Sputnik launched by Soviet Union (1957) 1959 Yalow develops the radioimmunoassay technique Birth control pill (1960) 1961 Jacob and Monod propose the operon model of gene regulation First humans in space (1961) 1961–1966 Nirenberg, Khorana, and others elucidate the genetic code Cuban missile crisis (1962) Nuclear test ban treaty (1963) 1962 Porter proposes the basic structure for immunoglobulin G Civil Rights March on Washington (1963) First quinolone antimicrobial (nalidixic acid) synthesized President Kennedy assassinated (1963) Arab-Israeli War (1967) Martin Luther King assassination (1968) Neil Armstrong walks on the moon (1969) 1970 Discovery of restriction endonucleases by Arber and Smith Discovery of reverse transcriptase in retroviruses by Temin and Baltimore 1973 Ames develops a bacterial assay for the detection of mutagens Salt I Treaty (1972) Cohen, Boyer, Chang, and Helling use plasmid vectors to clone Vietnam War ends (1973) genes in bacteria 1975 Kohler and Milstein develop technique for the production of President Nixon resigns because of Watergate cover-up (1974) monoclonal antibodies Lyme disease discovered 1977 Recognition of archaea as a distinct microbial group by Panama Canal Treaty (1977) Woese and Fox

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 1.2 The Conflict over Spontaneous Generation 5 Table 1.1 Continued Date Microbiological History Other Historical Events Gilbert and Sanger develop techniques for DNA sequencing 1979 Insulin synthesized using recombinant DNA techniques Hostages seized in Iran (1978) Smallpox declared officially eliminated Three Mile Island disaster (1979) 1980 Development of the scanning tunneling microscope Home computers marketed (1980) 1982 Recombinant hepatitis B vaccine developed AIDS first recognized (1981) 1982–1983 Discovery of catalytic RNA by Cech and Altman First artificial heart implanted (1982) 1983–1984 The human immunodeficiency virus isolated and identified Meter redefined in terms of distance light travels (1983) by Gallo and Montagnier The polymerase chain reaction developed by Mullis 1986 First vaccine (hepatitis B vaccine) produced by genetic Gorbachev becomes Communist party general secretary (1985) engineering approved for human use Berlin Wall falls (1989) 1990 First human gene-therapy testing begun Persian Gulf War with Iraq begins (1990) Soviet Union collapse; Boris Yeltsin comes to power (1991) 1992 First human trials of antisense therapy 1995 Chickenpox vaccine approved for U.S. use Haemophilus influenzae genome sequenced 1996 Methanococcus jannaschii genome sequenced Water found on the moon (1998) Yeast genome sequenced 1997 Discovery of Thiomargarita namibiensis, the largest known bacterium Escherichia coli genome sequenced 2000 Discovery that Vibrio cholerae has two separate chromosomes Figure 1.1 Antony van Leeuwenhoek. Leeuwenhoek (1632–1723) and his microscopes. (a) Leeuwenhoek holding a microscope. (b) A drawing of one of the microscopes showing the lens, a; mounting pin, b; and focusing screws, c and d. (c) Leeuwenhoek’s drawings of bacteria from the human mouth. (b) Source: C. E. Dobell, Antony van Leeuwenhoek and His Little Animals (1932), Russell and Russell, 1958. d d c c b b a a (a) (b) (c)

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 Thus the generation of maggots by decaying meat resulted from the presence of fly eggs, and meat did not spontaneously generate maggots as previously believed. Similar experiments by others helped discredit the theory for larger organisms. Leeuwenhoek’s discovery of microorganisms renewed the controversy. Some proposed that microorganisms arose by spontaneous generation even though larger organisms did not. They pointed out that boiled extracts of hay or meat would give rise to microorganisms after sitting for a while. In 1748 the English priest John Needham (1713–1781) reported the results of his experiments on spontaneous generation. Needham boiled mutton broth and then tightly stoppered the flasks. Eventually many of the flasks became cloudy and contained microorganisms. He thought organic matter contained a vital force that could confer the properties of life on nonliving matter. A few years later the Italian priest and naturalist Lazzaro Spallanzani (1729–1799) improved on Needham’s experimental design by first sealing glass flasks that contained water and seeds. If the sealed flasks were placed in boiling water for 3/4 of an hour, no growth took place as long as the flasks remained sealed. He proposed that air carried germs to the culture medium, but also commented that the external air might be required for growth of animals already in the medium. The supporters of spontaneous generation maintained that heating the air in sealed flasks destroyed its ability to support life. Several investigators attempted to counter such arguments. Theodore Schwann (1810–1882) allowed air to enter a flask containing a sterile nutrient solution after the air had passed through a red-hot tube. The flask remained sterile. Subsequently Georg Friedrich Schroder and Theodor von Dusch allowed air to enter a flask of heat-sterilized medium after it had passed through sterile cotton wool. No growth occurred in the medium even though the air had not been heated. Despite these experiments the French naturalist Felix Pouchet claimed in 1859 to have carried out experiments conclusively proving that microbial growth could occur without air contamination. This claim provoked Louis Pasteur (1822–1895) to settle the matter once and for all. Pasteur (figure 1.2) first filtered air through cotton and found that objects resembling plant spores had been trapped. If a piece of the cotton was placed in sterile medium after air had been filtered through it, microbial growth appeared. Next he placed nutrient solutions in flasks, heated their necks in a flame, and drew them out into a variety of curves, while keeping the ends of the necks open to the atmosphere (figure 1.3). Pasteur then boiled the solutions for a few minutes and allowed them to cool. No growth took place even though the contents of the flasks were exposed to the air. Pasteur pointed out that no growth occurred because dust and germs had been trapped on the 6 Chapter 1The History and Scope of Microbiology Figure 1.2 Louis Pasteur. Pasteur (1822–1895) working in his laboratory. Figure 1.3 The Spontaneous Generation Experiment. Pasteur’s swan neck flasks used in his experiments on the spontaneous generation of microorganisms. Source: Annales Sciences Naturelle, 4th Series, Vol. 16, pp.1–98, Pasteur, L., 1861, “Mémoire sur les Corpuscules Organisés Qui Existent Dans L’Atmosphère: Examen de la Doctrine des Générations Spontanées

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 walls of the curved necks. If the necks were broken, growth commenced immediately. Pasteur had not only resolved the controversy by 1861 but also had shown how to keep solutions sterile. The English physicist John Tyndall (1820–1893) dealt a final blow to spontaneous generation in 1877 by demonstrating that dust did indeed carry germs and that if dust was absent, broth remained sterile even if directly exposed to air. During the course of his studies, Tyndall provided evidence for the existence of exceptionally heat-resistant forms of bacteria. Working independently, the German botanist Ferdinand Cohn (1828–1898) discovered the existence of heat-resistant bacterial endospores (see chapter 3). 1. Describe the field of microbiology in terms of the size of its subject material and the nature of its techniques. 2. How did Pasteur and Tyndall finally settle the spontaneous generation controversy? 1.3 The Role of Microorganisms in Disease The importance of microorganisms in disease was not immediately obvious to people, and it took many years for scientists to establish the connection between microorganisms and illness. Recognition of the role of microorganisms depended greatly upon the development of new techniques for their study. Once it became clear that disease could be caused by microbial infections, microbiologists began to examine the way in which hosts defended themselves against microorganisms and to ask how disease might be prevented. The field of immunology was born. Recognition of the Relationship between Microorganisms and Disease Although Fracastoro and a few others had suggested that invisible organisms produced disease, most believed that disease was due to causes such as supernatural forces, poisonous vapors called miasmas, and imbalances between the four humors thought to be present in the body. The idea that an imbalance between the four humors (blood, phlegm, yellow bile [choler], and black bile [melancholy]) led to disease had been widely accepted since the time of the Greek physician Galen (129–199). Support for the germ theory of disease began to accumulate in the early nineteenth century. Agostino Bassi (1773–1856) first showed a microorganism could cause disease when he demonstrated in 1835 that a silkworm disease was due to a fungal infection. He also suggested that many diseases were due to microbial infections. In 1845 M. J. Berkeley proved that the great Potato Blight of Ireland was caused by a fungus. Following his successes with the study of fermentation, Pasteur was asked by the French government to investigate the pébrine disease of silkworms that was disrupting the silk industry. After several years of work, he showed that the disease was due to a protozoan parasite. The disease was controlled by raising caterpillars from eggs produced by healthy moths. Indirect evidence that microorganisms were agents of human disease came from the work of the English surgeon Joseph Lister (1827–1912) on the prevention of wound infections. Lister impressed with Pasteur’s studies on the involvement of microorganisms in fermentation and putrefaction, developed a system of antiseptic surgery designed to prevent microorganisms from entering wounds. Instruments were heat sterilized, and phenol was used on surgical dressings and at times sprayed over the surgical area. The approach was remarkably successful and transformed surgery after Lister published his findings in 1867. It also provided strong indirect evidence for the role of microorganisms in disease because phenol, which killed bacteria, also prevented wound infections. The first direct demonstration of the role of bacteria in causing disease came from the study of anthrax (see chapter 39) by the German physician Robert Koch (1843–1910). Koch (figure 1.4) used the criteria proposed by his former teacher, Jacob Henle (1809–1885), to establish the relationship between Bacillus anthracis and anthrax, and published his findings in 1876 (Box 1.1 briefly discusses the scientific method). Koch injected healthy mice with material from diseased animals, and the mice became ill. After transferring anthrax by inoculation through a series of 20 mice, he incubated a piece of spleen containing the anthrax bacillus in beef serum. The bacilli grew, reproduced, and produced spores. When the isolated bacilli or spores were injected into mice, anthrax developed. His criteria for proving the causal relationship between a microorganism and a specific disease are known as Koch’s postulates and can be summarized as follows: 1. The microorganism must be present in every case of the disease but absent from healthy organisms. 1.3 The Role of Microorganisms in Disease 7 Figure 1.4 Robert Koch. Koch (1843–1910) examining a specimen in his laboratory

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 2. The suspected microorganism 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. 4. The same microorganism must be isolated again from the diseased host. Although Koch used the general approach described in the postulates during his anthrax studies, he did not outline them fully until his 1884 publication on the cause of tuberculosis (Box 1.2). Koch’s proof that Bacillus anthracis caused anthrax was independently confirmed by Pasteur and his coworkers. They discovered that after burial of dead animals, anthrax spores survived and were brought to the surface by earthworms. Healthy animals then ingested the spores and became ill. The Development of Techniques for Studying Microbial Pathogens During Koch’s studies on bacterial diseases, it became necessary to isolate suspected bacterial pathogens. At first he cultured bacteria on the sterile surfaces of cut, boiled potatoes. This was unsatisfactory because bacteria would not always grow well on potatoes. He then tried to solidify regular liquid media by adding gelatin. Separate bacterial colonies developed after the surface had been streaked with a bacterial sample. The sample could also be mixed with liquefied gelatin medium. When the gelatin medium hardened, individual bacteria produced separate colonies. Despite its advantages gelatin was not an ideal solidifying agent because it was digested by many bacteria and melted when the temperature rose above 28°C. A better alternative was provided by Fannie 8 Chapter 1The History and Scope of Microbiology Although biologists employ a variety of approaches in conducting research, microbiologists and other experimentally oriented biologists often use the general approach known as the scientific method. They first gather observations of the process to be studied and then develop a tentative hypothesis—an educated guess—to explain the observations (see Box figure). This step often is inductive and creative because there is no detailed, automatic technique for generating hypotheses. Next they decide what information is required to test the hypothesis and collect this information through observation or carefully designed experiments. After the information has been collected, they decide whether the hypothesis has been supported or falsified. If it has failed to pass the test, the hypothesis is rejected, and a new explanation or hypothesis is constructed. If the hypothesis passes the test, it is subjected to more severe testing. The procedure often is made more efficient by constructing and testing alternative hypotheses and then refining the hypothesis that survives testing. This general approach is often called the hypothetico-deductive method. One deduces predictions from the currently accepted hypothesis and tests them. In deduction the conclusion about specific cases follows logically from a general premise (“if . ..,then . . .” reasoning). Induction is the opposite. A general conclusion is reached after considering many specific examples. Both types of reasoning are used by scientists. When carrying out an experiment, it is essential to use a control group as well as an experimental group. The control group is treated precisely the same as the experimental group except that the experimental manipulation is not performed on it. In this way one can be sure that any changes in the experimental group are due to the experimental manipulation rather than to some other factor not taken into account. If a hypothesis continues to survive testing, it may be accepted as a valid theory. A theory is a set of propositions and concepts that provides a reliable, systematic, and rigorous account of an aspect of nature. It is important to note that hypotheses and theories are never absolutely proven. Scientists simply gain more and more confidence in their accuracy as they continue to survive testing, fit with new observations and experiments, and satisfactorily explain the observed phenomena. Box 1.1 The Scientific Method The Hypothetico-Deductive Method. This approach is most often used in scientific research. Problem Develop hypothesis Select information needed to test hypothesis Collect information by observation or experiment Analyze information Falsification Hypothesis rejected Construct new hypothesis Hypothesis supported Expose to more tests Eventual falsification Develop new hypothesis incorporating strong points of old hypothesis

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 Eilshemius Hesse, the wife of Walther Hesse, one of Koch’s assistants (figure 1.5). She suggested the use of agar as a solidifying agent—she had been using it successfully to make jellies for some time. Agar was not attacked by most bacteria and did not melt until reaching a temperature of 100°C. One of Koch’s assistants, Richard Petri, developed the petri dish (plate), a container for solid culture media. These developments made possible the isolation of pure cultures that contained only one type of bacterium, and directly stimulated progress in all areas of bacteriology. Isolation of bacteria and pure culture techniques (pp. 106–10). Koch also developed media suitable for growing bacteria isolated from the body. Because of their similarity to body fluids, meat extracts and protein digests were used as nutrient sources. The result was the development of nutrient broth and nutrient agar, media that are still in wide use today. By 1882 Koch had used these techniques to isolate the bacillus that caused tuberculosis. There followed a golden age of about 30 to 40 years in which most of the major bacterial pathogens were isolated (table 1.1). The discovery of viruses and their role in disease was made possible when Charles Chamberland (1851–1908), one of Pasteur’s associates, constructed a porcelain bacterial filter in 1884. The first viral pathogen to be studied was the tobacco mosaic disease virus (see chapter 16). The development of virology (pp. 362–63). Immunological Studies In this period progress also was made in determining how animals resisted disease and in developing techniques for protecting humans and livestock against pathogens. During studies on chicken cholera, Pasteur and Roux discovered that incubating their cultures for long intervals between transfers would attenuate the bacteria, which meant they had lost their ability to cause the disease. If the chickens were injected with these attenuated cultures, they remained healthy but developed the ability to resist the disease. He called the attenuated culture a vaccine [Latin vacca, cow] in honor of Edward Jenner because, many years earlier, Jenner had used vaccination with material from cowpox lesions to protect people against smallpox (see section 16.1). Shortly after this, Pasteur and Chamberland developed an attenuated anthrax vaccine in two ways: by treating cultures with potassium bichromate and by incubating the bacteria at 42 to 43°C. Vaccines and immunizations (pp. 764–68). 1.3 The Role of Microorganisms in Disease 9 Although the criteria that Koch developed for proving a causal relationship between and a microorganism and a specific disease have been of immense importance in medical microbiology, it is not always possible to apply them in studying human diseases. For example, some pathogens cannot be grown in pure culture outside the host; because other pathogens grow only in humans, their study would require experimentation on people. The identification, isolation, and cloning of genes responsible for pathogen virulence (see p. 794) have made possible a new molecular form of Koch’s postulates that resolves some of these difficulties. The emphasis is on the virulence genes present in the infectious agent rather than on the agent itself. The molecular postulates can be briefly summarized as follows: 1. The virulence trait under study should be associated much more with pathogenic strains of the species than with nonpathogenic strains. Box 1.2 Molecular Koch’s Postulates 2. Inactivation of the gene or genes associated with the suspected virulence trait should substantially decrease pathogenicity. 3. Replacement of the mutated gene with the normal wild-type gene should fully restore pathogenicity. 4. The gene should be expressed at some point during the infection and disease process. 5. Antibodies or immune system cells directed against the gene products should protect the host. The molecular approach cannot always be applied because of problems such as the lack of an appropriate animal system. It also is difficult to employ the molecular postulates when the pathogen is not well characterized genetically. Figure 1.5 Fannie Eilshemius (1850–1934) and Walther Hesse (1846–1911). Fannie Hesse first proposed using agar in culture media

Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 1. The History and Scope of Microbiology © The McGraw−Hill Companies, 2002 Pasteur next prepared rabies vaccine by a different approach. The pathogen was attenuated by growing it in an abnormal host, the rabbit. After infected rabbits had died, their brains and spinal cords were removed and dried. During the course of these studies, Joseph Meister, a nine-year-old boy who had been bitten by a rabid dog, was brought to Pasteur. Since the boy’s death was certain in the absence of treatment, Pasteur agreed to try vaccination. Joseph was injected 13 times over the next 10 days with increasingly virulent preparations of the attenuated virus. He survived. In gratitude for Pasteur’s development of vaccines, people from around the world contributed to the construction of the Pasteur Institute in Paris, France. One of the initial tasks of the Institute was vaccine production. After the discovery that the diphtheria bacillus produced a toxin, Emil von Behring (1854–1917) and Shibasaburo Kitasato (1852–1931) injected inactivated toxin into rabbits, inducing them to produce an antitoxin, a substance in the blood that would inactivate the 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). It became clear that blood cells were also important in immunity (cellular immunity) when Elie Metchnikoff (1845–1916) discovered that some blood leukocytes could engulf disease-causing bacteria (figure 1.6). He called these cells phagocytes and the process phagocytosis [Greek phagein, eating]. 1. Discuss the contributions of Lister, Pasteur, and Koch to the germ theory of disease and to the treatment or prevention of diseases. 2. What other contributions did Koch make to microbiology? 3. Describe Koch’s postulates. What are the molecular Koch’s postulates and why are they important? 4. How did von Behring and Metchnikoff contribute to the development of immunology? 1.4 Industrial Microbiology and Microbial Ecology Although Theodore Schwann and others had proposed in 1837 that yeast cells were responsible for the conversion of sugars to alcohol, a process they called alcoholic fermentation, the leading chemists of the time believed microorganisms were not involved. They were convinced that fermentation was due to a chemical instability that degraded the sugars to alcohol. Pasteur did not agree. It appears that early in his career Pasteur became interested in fermentation because of his research on the stereochemistry of molecules. He believed that fermentations were carried out by living organisms and produced asymmetric products such as amyl alcohol that had optical activity. There was an intimate connection between molecular asymmetry, optical activity, and life. Then in 1856 M. Bigo, an industrialist in Lille, France, where Pasteur worked, requested Pasteur’s assistance. His business produced ethanol from the fermentation of beet sugars, and the alcohol yields had recently declined and the product had become sour. Pasteur discovered that the fermentation was failing because the yeast normally responsible for alcohol formation had been replaced by microorganisms producing lactic acid rather than ethanol. In solving this practical problem, Pasteur demonstrated that all fermentations were due to the activities of specific yeasts and bacteria, and he published several papers on fermentation between 1857 and 1860. His success led to a study of wine diseases and the development of pasteurization (see chapter 7) to preserve wine during storage. Pasteur’s studies on fermentation continued for almost 20 years. One of his most important discoveries was that some fermentative microorganisms were anaerobic and could live only in the absence of oxygen, whereas others were able to live either aerobically or anaerobically. Fermentation (pp. 179–81); The effect of oxygen on microorganisms (pp. 127–29). A few of the early microbiologists chose to investigate the ecological role of microorganisms. In particular they studied microbial involvement in the carbon, nitrogen, and sulfur cycles taking place in soil and aquatic habitats. Two of the pioneers in this endeavor were Sergei N. Winogradsky (1856–1953) and Martinus W. Beijerinck (1851–1931). Biogeochemical cycles (pp. 611–18). 10 Chapter 1The History and Scope of Microbiology Figure 1.6 Elie Metchnikoff. Metchnikoff (1845–1916) shown here at work in his laboratory