Microbiology is the study of a diverse group of microscopic organisms, or microorganisms: bacteria, fungi, algae, protozoa, and viruses. Bacteria are prokaryotes; the other microorganisms are eukaryotes. Prokaryote cells lack a nuclear membrane and membrane-bound organelles. Recently, bacteria have been divided into eubacteria and archaebacteria, with the latter more closely related to eukaryote cells. Bacteria are mostly unicellular and range in size from tiny mycoplasmas, 200 nanometers (that is, 200 billionths of a meter, or less than 1/100,000 of an inch) in diameter, to the recently discovered Thiomargarita namibiensis, at one millimeter (or about 1/25 of an inch). E. coli cells are one to two micrometers in length (about five to ten times the diameter of the mycoplasmas). Fungi include yeasts, molds, and mushrooms. The bread, wine, and beer yeast, Saccharomyces cerevisiae, is ten micrometers (about 1/2,500 of an inch) in diameter. Algae are photosynthetic organisms, unicellular or multicellular. Protozoa are microscopic, unicellular, and usually motile. Viruses are not cellular organisms; they are intracellular parasites of animals, plants, or bacteria. They are composed of nucleic acid (DNA or RNA) enclosed in a protein coat. Viruses range from 18 to 450 nanometers (from less than one-millionth to almost 1/50,000 of an inch). Microorganisms, with the exception of viruses, can be observed with a compound light microscope (up to ,000 magnification). Electron microscopes (up to 100,000 magnification) are used to visualize viruses.

History of Microbiology Before Pasteur

Microorganisms were first visualized by Antoni van Leeuwenhoek (16321723), a Dutch cloth merchant and an expert lens grinder. His simple microscopes magnified up to three hundred diameters. In the eighteenth century, many people still believed that living organisms could arise spontaneously from organic matterthe doctrine of abiogenesis, or spontaneous generation.

Lazzaro Spallanzani (17291799), an Italian priest and physiologist, did an experiment that came close to proving that life (in this case, microorganisms) does not arise spontaneously from nonliving matter. He sealed flasks containing broth and then boiled them. No spontaneous generation or growth occurred in the flasks; however, the debate continued, as proponents of the doctrine said that air was needed for spontaneous generation. Opponents of this doctrine had a very difficult task trying to prove a negative, namely that something did not happen.

The ancient Egyptians and Romans were comfortable with the idea that organisms invisible to the naked eye could cause disease. During the Dark Ages and the medieval period of Western history, this idea virtually disappeared. In the sixteenth century, Girolamo Fracastoro (14831553) described disease passing from one person to another by "germs." Athanasius Kircher (16021680) furthered the "germ theory" by observing bacteria from plague victims.

History from Pasteur Onward

Louis Pasteur (18221895) was an intellectual giant who dominated science in the middle of the nineteenth century. In 1861, in the midst of a twenty-year study of microbial fermentation, Pasteur dealt the deathblow to the doctrine of spontaneous generation by demonstrating the presence of microorganisms in the air and then by showing that sterile liquid in a swan-necked flask remained sterile. Air could enter such a flask, but microorganisms could not. In 1875, Ferdinand Cohn (18281898) published the first classification of bacteria, and used the genus name, Bacillus, for a spore-forming bacterium. In 1875, Robert Koch (18431910), a German bacteriologist, proved that a spore-forming bacterium, Bacillus anthracis, caused anthrax. His experiments demonstrated four principles, now known as Koch's postulates, which are still the hallmark of disease etiology: (1) the microorganism must be present in every diseased animal studied, but not be isolated from healthy animals; (2) the microorganism must be isolated from the animal and cultivated; (3) an animal inoculated with the microorganism must develop the disease; (4) the same microorganism must be isolated from the diseased animal inoculated with the microorganism. Working independently on anthrax, Pasteur and his colleagues confirmed Koch's findings. Koch introduced three practices that allowed bacteriologists to obtain pure cultures simply: (1) a semisolid medium composed of nutrients solidified with gelatin, (2) platinum needles sterilized in a flame to pick up bacteria, (3) streaking of bacteria onto a gelatin surface to obtain single cells that would grow into colonies. In 1881, Fanny Hesse, the wife of German bacteriologist, Walther Hesse, suggested using a seaweed extract, agar, which she used to thicken jam, to solidify media in petri plates. Agar had neither of the disadvantages of gelatin: it was rarely degraded by microorganisms and it stayed solid at temperatures above 28C (about 82F). Agar is still the solidifying agent of choice. In 1882, Koch used the pure-culture techniques to isolate the bacterium that causes tuberculosis. In 1884, Charles Chamberland, a collaborator of Pasteur's, developed a porcelain filter that would retain all bacteria. When, in 1892, a young Russian scientist, Dmitri Iwanowski, transmitted tobacco mosaic disease to healthy plants using a porcelain-filtered extract, he postulated the presence of a toxin. In 1898, the Dutch microbiologist, Martinus Beijerinck, reproduced Iwanowski's results, but he postulated the existence of very small infectious agents, "filterable viruses." Thus began the field of virology, although visualization of viruses had to wait until the development of the electron microscope in the 1930s. Medical bacteriology progressed rapidly at the Pasteur Institute in Paris, where Pasteur presided, and the Koch Institute in Berlin, where Koch presided.

History of Food Preservation Microbiology

In 1810, Nicolas Appert (17501841) applied Spallanzani's results to develop a system of preserving food by sealing it in airtight cans and heating the cans. Without understanding that the heat treatment, or "appertization," was killing microorganisms in the canned food, Appert established the basis for the modern practice of canning. In 1852, Napoleon III asked Pasteur to study the problem of "wine diseases," particularly wine souring. In 1886, Pasteur proclaimed that the off-flavors in wine were caused by contaminating microorganisms. He suggested heating (pasteurizing) the grape juice to kill the spoilage bacteria. He discovered that some microorganisms could grow in the absence of oxygen. He used the term "anaerobic" to apply to microbial metabolism that occurs only in the absence of oxygen, and "aerobic" for metabolism that occurs under normal atmospheric conditions. Fermentation of grape juice by yeast is one kind of anaerobic metabolism. He also described the anaerobic degradation of protein, or putrefaction, by bacteria. Aerobic bacteria, namely the acetic-acid bacteria, were the cause of wine souring. Some of these bacteria metabolize ethanol to acetic acid; others metabolize the acetic acid to carbon dioxide and water. The process of pasteurization, a mild heat treatment of liquids, originated as a means of preserving the desired flavor of milk, fruit juices, beer, and wine. For example, Pasteur recommended that heating bottled wine for a short time at 122F (50C) would kill the lactic-acid and acetic-acid bacteria that can spoil wine. In traditional pasteurization, liquids are heated at about 145F (63C) for thirty minutes, then held at 50F (10C). Nowadays, flash or high-temperature, short-time (HTST) pasteurization is the preferred method (about 162F [72C] for fifteen seconds, followed by rapid cooling to 50F [10C]) because it has less effect on the flavor of the food being heated. Currently, milk is pasteurized to eliminate the bacteria responsible for tuberculosis, food poisoning, undulant fever, and Q fever. The treatment does not result in sterilization of milk, which can contain twenty thousand bacteria, such as lactobacilli, per ml post-pasteurization. More common in Europe than other parts of the world, is ultrahigh temperature (UHT) treatment (300F[148.9C] for one to two seconds), which sterilizes milk, allowing it to be stored without refrigeration for more than the limit of two to three weeks for pasteurized milk. Many brewing companies pasteurize their bottled or canned beer at 140F (60C) for a few minutes. Pasteurization is infrequently used, however, in modern winemaking, as it adversely affects the flavor.

Cohn and John Tyndall (18291893) both demonstrated that the endospores of Bacillus subtilis cells were far more resistant to heating than were vegetative bacteria. Tyndall developed a method of sterilizing liquids that contained bacterial spores: a medium was first incubated to allow the spores to germinate, then heated to kill most of the bacteria. This process, later termed "tyndallization," was repeated several times. This was a very important development in food science since the bacteria that form endospores include the food-borne pathogens, Clostridium botulinum, C. perfringens and C. difficile. Today, canned food is subjected to a temperaturetime treatment that ensures the death of heat-resistant bacterial endospores, particularly those of C. botulinum.

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