Koch's Postulates

For his successful determination of the bacterium that causes tuberculosis, Mycobacterium tuberculosis, Koch was awarded the Nobel Prize in Physiology or Medicine in 1905. Koch formulated his famous set of criteria for establishing a causative link between an infectious agent and a disease (Fig. 1.17). These four criteria are known as Koch's postulates:
            1. The microbe is found in all cases of the disease but is absent from healthy individuals.
            2. The microbe is isolated from the diseased host and grown in pure culture.
            3. When the microbe is introduced into a healthy, susceptible host (or animal model), the same disease occurs.
            4. The same strain of microbe is obtained from the newly diseased host. When cultured, the strain shows the same characteristics as before.    

Koch's postulates continue to be used to determine whether a given strain of microbe causes a disease. Mod-em examples include Lyme disease, a tick-borne infection that has become widespread in New England and the Mid-Atlantic states; and hantaviral pneumonia, an emerging disease particularly prevalent among Native Americans in the Southwest. Nevertheless, the postulates remain only a guide; individual diseases and pathogens may confound one or more of the criteria. For example, tuberculosis bacteria are now known to cause symptoms in only 10% of the people infected. If Koch had been able to detect these silent bacilli, they would not have fulfilled his first criterion. In the case of AIDS, the concentration of HIV virus is so low that initially no virus could be detected in patients with fully active symptoms. It took the invention of the polymerase chain reaction (PCR), a method of producing any number of copies of DNA or RNA sequences, to detect the presence of HIV. Another difficulty with AIDS and many other human diseases is the absence of an animal host that exhibits the same disease. In the case of AIDS, even chimpanzees, our closest relatives, are not susceptible, although they exhibit a similar disease from a related pathogen, simian immunodeficiency virus (SIV). Experimentation on humans is prohibited, although in rare instances researchers have voluntarily exposed themselves to a proposed pathogen. For example, Australian researcher Barry Marshall ingested Ileiicobacter pylori to convince skeptical col-leagues that this organism could colonize the extremely acidic stomach. II. pylori turned out to be the causative agent of gastritis and stomach ulcers, conditions that had long been thought to be caused by stress rather than infection. For the discovery of H. pylori, Marshall and col-league Robin Warren won the 2005 Nobel Prize in Physiology or Medicine.

Growth of Microbes in Pure Culture

Unlike Pasteur, who was a university professor, Koch took up a medical practice in a small Polish-German town. To make space in his home for a laboratory to study anthrax and other deadly diseases, his wife curtained off part of his patients' examining room. Anthrax interested Koch because its epidemics in sheep and cattle caused economic hardship among local farmers. Today, anthrax is no longer a major problem for agriculture, as its transmission is prevented by effective environmental controls and vaccination. It has, however, gained notoriety as a bioterror agent because anthrax bacteria can survive for long periods in the dormant, desiccated form of an endospore. In 2001, anthrax spores sent through the mall contaminated post offices throughout the northeastern United States, as well as an office building of the United States Senate, causing several deaths. To investigate whether anthrax was a transmissible disease, Koch used blood from an anthrax-infected carcass to inoculate a rabbit. When the rabbit died, he used the rabbit's blood to inoculate a second rabbit, which then died in turn. The blood of the unfortunate animal had turned black with long, rod-shaped bacilli. Upon introduction of these bacilli into healthy animals, the animals became ill with anthrax. Thus, Koch demonstrated an important principle of epidemiology: the chain of infection, or transmission of a disease. In retrospect, his choke of anthrax was fortunate, for the microbes generate disease very quickly, multiply in the blood to an extraordinary concentration, and remain infective outside the body for long periods.
        Koch and his colleagues then applied their experimental logic and culture methods to a more challenging disease: tuberculosis. In Koch's day, tuberculosis caused one-seventh of reported deaths in Europe; today, tuberculosis bacteria continue to infect millions of people worldwide. Koch's approach to anthrax, however, was less applicable to tuberculosis, a disease that develops slowly after many years of dormancy. Furthermore, the causative bacteria, Mycobacterium tuberculosis, are small and difficult to distinguish from human tissue or from different bacteria of similar appearance associated with the human body. How could Koch prove that a particular bacterium caused a particular disease? What was needed was to isolate a pure culture of microorganisms, a culture grown from a single "parental" cell. This had been done by previous researchers using the laborious process of serial dilution of suspended bacteria until a culture tube contained only a single cell. Alternatively, inoculation of a solid surface such as a sliced potato could produce isolated colonies, distinct populations of bacteria, each grown from a single cell. For M. tuberculosis, Koch inoculated serum, which then formed a solid gel after heating. Later he refined the solid-substrate technique by adding gelatin to a defined liquid medium, which could then be chilled to form a solid medium in a glass dish. A covered version called the petri dish (also called a petri plate) was invented by a colleague, Julius Richard Petri (1852-1921). The petri dish consists of a round dish with vertical walls covered by an inverted dish of slightly larger diameter. Today, the petri dish, generally made of disposable plastic, remains an indispensable part of the microbiological laboratory. Another improvement in solid-substrate culture was the replacement of gelatin with materials that remain solid at higher temperatures, such as the gelling agent agar (a polymer of the sugar galactose). The use of agar was recommended by Angelina Hesse (1850-1934), a microscopic and illustrator, to her husband, Walther Hesse (1846-1911), a young medical colleague of Koch (Fig.1.16). Agar comes from red algae (seaweed), which is used by East Indian birds to build nests; it is the main ingredient in the delicacy "bird's nest soup." Dutch colonists used agar to make jellies and preserves, and a Dutch colonist from Java introduced it to Angelina Hesse. The Hesses used agar to develop the first effective growth medium for tuberculosis bacteria. (Pure culture is discussed further in Chapter 4.) Note that some kinds of microbes cannot be grown in pure culture without other organisms. For example, viruses can be cultured only in the presence of their host cells. The discovery of viruses is explored at the end of this section

Medical Microbiology

Over the centuries, thoughtful observers, such as Fracastoro and Bassi (see Table 1.2), noted a connection between microbes and disease. Ultimately, researchers developed the germ theory of disease, the theory that many diseases are caused by microbes. The first to establish a scientific basis for determining that a specific microbe causes a specific disease was If all life on Earth shares descent from a microbial ancestor, how did the first microbe arise? The earliest fossil evidence of cells in the geological record appears in sedimentary rock that formed as early as 3.8 billion years ago. Although the nature of the earliest reported fossils remains controversial, it is generally accepted that "microfossils" from over 2 billion years ago were formed by living cells. Moreover, the living cells that formed these microfossils looked remarkably similar to bacterial cells today, forming chains of simple rods or spheres (Fig. 1). The exact composition of the first environment for life is controversial. The components of the first living cells may have formed from spontaneous reactions sparked by ultraviolet absorption or electrical discharge. American chemists Stanley Miller (1930-2007) and Harold C. Urey (1893-1981) argued that the environment of early Earth contained mainly reduced compounds compounds that have a strong tendency to donate electrons, such as ferrous iron, methane, and ammonia. More recent evidence has modified this view, but it is agreed that the strong electron acceptor oxygen gas (02) was absent until the first photosynthetic microbes produced it. Today, all our cells are composed of highly reduced molecules that are readily oxidized (accept electrons from 02). This seem ingly hazardous composition may reflect our cellular origin in the chemically reduced environment of early Earth. In 1953, Miller attempted to simulate the highly reduced conditions of early Earth to test whether ultraviolet absorption or electrical discharge could cause reactions producing the fundamental components of life (Fig. 2A). Miller boiled a solution of water containing hydrogen gas, methane, and ammonia and applied an electrical discharge (comparable to a lightning strike). The electrical discharge excites electrons in the molecules and causes them to react. Astonishingly, the reaction produced a number of amino acids, including glycine, alanine, and aspartic acid. A similar experiment in 1961 by Spanish-American researcher Juan Or (1923-2004) (Fig. 2B) combined hydrogen cyanide and ammonia under electrical discharge to obtain adenine, a fundamental component of DNA and of the energy carrier adenosine triphosphate (ATP). How could early cells have survived the heat and chemically toxic environment of early Earth? Clues may be found in the survival of archaea that thrive under habitat conditions that we consider extreme, such as solutions of boiling sulfuric acid. The specially adapted structures of such microbes may resemble those of the earliest life-forms.

Spontaneous Generation: Do Microbes Have Parents?

The observation of microscopic organisms led priests and philosophers to wonder where they came from. In the eighteenth century, scientists and church leaders intensely debated the question of spontaneous generation, the theory that living creatures such as maggots could arise spontaneously, without parental organisms. Chemists of the day tended to support spontaneous generation, at it appeared similar to the changes in matter that could occur when chemicals were mixed. Christian church leaders, however, supported the biblical view that all organisms have "parents" going back to the first week of creation. The Italian priest Francesco Redi (1626-1697) showed that maggots in decaying meat were the offspring of flies. Meat kept in a sealed container, excluding flies, did not produce maggots. Thus, Redi's experiment argued against spontaneous generation for macroscopic organisms. The meat still putrefied, however, producing microbes that seemed to arise "without parents." To disprove spontaneous generation of microbes, another Italian priest, Lazzaro Spallanzani (1729-1799), showed that a sealed flask of meat broth sterilized by boiling failed to grow microbes. Spallanzani also noticed that microbes often appeared in pairs. Were these two parental microbes coupling to produce offspring, or did one microbe become two? By long and tenacious observation, Spallanzani watched a single microbe grow in size until it split in two. Thus, he demonstrated cell fission, the process by which cells arise by the splitting of preexisting cells. Even Spallanzani's experiments, however, did not put the matter to rest. Proponents of spontaneous generation argued that the microbes in the priest's flask lacked access to oxygen and therefore could not grow. The pursuit of this question was left to future microbiologists, including the famous French microbiologist Louis Pasteur (1822-1895) (Fig. 1.14A). In addressing spontaneous generation and related questions, Pasteur and his contemporaries laid the foundations for modern microbiology.
     Louis Pasteur reveals the biochemical basis of microbial growth. Pasteur began his scientific career as a chemist and wrote his doctoral thesis on the structure of organic crystals. He discovered the fundamental chemical property of chirality, the fact that some organic molecules exist in two forms that differ only by mirror symmetry. In other words, the two structures are mirror images of one another, like the right and left hands. Pasteur found that when microbes were cultured on a nutrient substance containing both mirror forms, only one mirror form was consumed. He concluded that the metabolic preference for one mirror form was a fundamental property of life. Subsequent research has confirmed that most biological molecules, such as DNA and proteins, occur in only one of their mirror forms. As a chemist, Pasteur was asked to help with a widespread problem encountered by French manufacturers of wine and beer. The production of alcoholic beverages is now known to occur by fermentation, a process by which microbes gain energy by converting sugars into alcohol. In the time of Pasteur, however, the conversion of grapes or grain to alcohol was believed to be a spontaneous chemical process. No one could explain why some fermentation mixtures produced vinegar (acetic acid) instead of alcohol. Pasteur discovered that fermentation is actually caused by living yeast, a single-celled fungus. In the absence of oxygen, yeast produces alcohol as a terminal waste product. But when the yeast culture is contaminated with bacteria, the bacteria outgrow the yeast and produce acetic acid instead of alcohol. (Fermentative metabolism is discussed further in Chapter 13.) Pasteur's work on fermentation led him to test a key claim made by proponents of spontaneous generation. The proponents claimed that Spallanzani's failure to find spontaneous appearance of microbes was due to lack of oxygen. From his studies of yeast fermentation, Pasteur knew that some microbial species do not require oxygen for growth. So he devised an unsealed flask with a long, bent "swan neck" that admitted air but kept the boiled contents free of microbes (Fig. 1.148). The famous swan-necked flasks remained free of microbial growth for many years; but when a flask was tilted to enable contact of broth with microbe containing dust, growth occurred immediately. Thus, Pasteur disproved that lack of oxygen was the reason for the failure of spontaneous generation in Spallanzani's flasks. But even Pasteur's work did not prove that microbial growth requires preexisting microbes. The Irish scientist John Tyndall (1820-1893) attempted the same experiment as Pasteur, but sometimes found the opposite result. Tyndall found that the broth sometimes gave rise to microbes, no matter how long it was sterilized by boiling. The microbes appear because some kinds of organic matter, particularly hay infusion, arecontaminated with a heat resistant form of bacteria called endospores (or spores). The spore form can be eliminated only by repeated cycles of boiling and resting, in which the spores germinate to the growing, vegetative form that is killed at 100°C. It was later discovered that endospores could be killed by boiling under pressure, as in a pressure cooker, which generates higher temperatures than can be obtained at atmospheric pressure. The steam pressure device called the autoclave became a standard method for the sterilization of materials required for the controlled study of microbes. (Microbial control and antisepsis are discussed further in Chapter 5.) Although spontaneous generation has been discredited as a continual source of microbes, at some point in the past the first living organisms must have originated from nonliving materials. The origin of life is explored in Special Topic 1.1, and discussed further in Chapter 17.

Microscopes Reveal the Microbial World

 The seventeenth century was a time of growing inquiry and excitement about the "natural magic" of science and patterns of our world, such as the laws of gravitation and motion formulated by Isaac Newton (1642-1727). Robert Boyle (1627-1691) performed the first controlled experiments on the chemical conversion of matter. Physicians attempted new treatments for disease involving the application of "stone and minerals" (that is, the application of chemicals), what today we would call chemotherapy. Minds were open to consider the astounding possibility that our surroundings, indeed our very bodies, were inhabited by tiny living beings.

  Robert Hooke observes the microscopic world.

 The first micros copist to publish a systematic study of the world as seen under a microscope was Robert Hooke (1635-1703). As curator of experiments for the Royal Society of London, Hooke built the first compound microscopea magnifying instrument containing two or more lenses that multiply their magnification in series. With his microscope, Hooke observed biological mate-rials such as nematode "vinegar eels," mites, and mold filaments, illustrations of which he published in Micrographia (1665), the first publication that illustrated objects observed under a microscope (Fig. 1.12). Hooke was the first to observe distinct units of living material, which he called "cells." Hooke first named the units cells because the shape of hollow cell walls in a slice of cork reminded him of the shape of monks' cells in a monastery. But his crude lenses achieved at best 30-fold power (30 x), so he never observed single celled organisms.

Antonie van Leeuwenhoek observes bacteria with a single lens.

Hooke's Micrographia inspired other microscopists, including Antonie van Leeuwenhoek (1632-1723), who became the first individual to observe single-celled microbes (Fig. 1.13A). As a young man, Leeuwenhoek lived in the Dutch city of Delft, where he worked as a cloth draper, a profession that introduced him to magnifying glasses. (The magnifying glasses were used to inspect the quality of the cloth, enabling the worker to count the number of threads.) Later in life, he took up the hobby of grinding ever stronger lenses to see into the world of the unseen. Leeuwenhoek ground lenses stronger than Hooke's, which he used to build single-lens magnifiers, complete with sample holder and focus adjustment (Fig. 1.13B). First he observed insects, including lice and fleas; then the relatively large single cells of protists and algae; then ultimately bacteria. news on Microscopes Reveal the Microbial World One day he applied his microscope to observe matter extracted from between his teeth. He wrote, "To my great surprise [I] perceived that the aforesaid matter contained very many small living Animals, which moved themselves very extravagantly." Over the rest of his life, Leeuwenhoek recorded page after page on the movement of microbes, reporting their size and shape so accurately that in many cases we can determine the species he observed (Fig. 1.13C).
          He performed experiments, comparing, for example, the appearance of "small animals" from his teeth before and after drinking hot coffee. The disappearance of microbes from his teeth after drinking a hot beverage suggested that heat killed microbes a profoundly important principle for the study and control of microbes ever since. Ironically, Leeuwenhoek is believed to have died of a disease contracted from sheep whose bacteria he observed. Historians have often wondered why it took so many centuries for Leeuwenhoek and his successors to determine the link between microbes and dis-ease. Although observers such as Agostino Bassi de Lodi (1773-1856) noted isolated cases of microbes associated with pathology (see Table 1.2), the very ubiquity of microbes most of them actually harmless may have obscured their more deadly roles. In addition, it was hard to distinguish between microbes and the single-celled components of the human body, such as blood cells and sperm. It was not until the nineteenth century that human tissues could be distinguished from microbial cells by the application of differential chemical stains (discussed in Chapter 2).