Home > Encyclopedia of Food & Culture > Microbiology

MICROBIOLOGY. 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 (1632–1723), 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 matter—the doctrine of abiogenesis, or spontaneous generation.

Lazzaro Spallanzani (1729–1799), 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 (1483–1553) described disease passing from one person to another by "germs." Athanasius Kircher (1602–1680) furthered the "germ theory" by observing bacteria from plague victims.

History from Pasteur Onward

Louis Pasteur (1822–1895) 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 (1828–1898) published the first classification of bacteria, and used the genus name, Bacillus, for a spore-forming bacterium. In 1875, Robert Koch (1843–1910), 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 28°C (about 82°F). 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 (1750–1841) 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 122°F (50°C) would kill the lactic-acid and acetic-acid bacteria that can spoil wine. In traditional pasteurization, liquids are heated at about 145°F (63°C) for thirty minutes, then held at 50°F (10°C). Nowadays, flash or high-temperature, short-time (HTST) pasteurization is the preferred method (about 162°F [72°C] for fifteen seconds, followed by rapid cooling to 50°F [10°C]) 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 (300°F[148.9°C] 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 140°F (60°C) for a few minutes. Pasteurization is infrequently used, however, in modern winemaking, as it adversely affects the flavor.

Cohn and John Tyndall (1829–1893) 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 temperature–time treatment that ensures the death of heat-resistant bacterial endospores, particularly those of C. botulinum.

For hundreds of years, substances that inhibit microbial growth have been added to foods in an attempt to prevent spoilage. One of the oldest practices is the salting of meat and fish as a means of preservation. Growth of most bacteria is inhibited by the high osmotic strength generated by the salt. In a relatively dry climate, salted meat can last up to twelve months. In 1958, the United States government determined that no chemical could be added to food or beverages without having been tested for safety. Three important antifungal preservatives for acidic foods (foods with a pH of 4.6 or less) such as canned drinks, salad dressings, cheese, and wines, are benzoic acid, sorbic acid, and propionic acid (or their salts). Sodium nitrate has been used in meat in China and the Middle East since 1200 B.C.E. A bacterial conversion of nitrate to nitrite results in a reaction with the heme pigment, giving the pink color of ham. Nitrite is antibacterial and prevents the germination of C. botulinum and other anaerobic bacteria in meats like ham, bacon, and frankfurters. Sulfur dioxide in some form, for example as produced by sodium metabisulfite, is used to control yeast and bacteria in wines and bacteria in brewing. Sulfur dioxide or bisulfite is an unusual chemical in that it is also an extremely effective antioxidant. Fermentation is another method of preservation. A commonly held dictum is that pathogenic bacteria do not grow at pH levels below 4.5. Fermented foods are inoculated with microorganisms, which reduce the pH of the food by producing acid during their growth. Acids such as acetic or citric acid are also added to decrease the pH of foods. Heat treatments are more effective at killing microorganisms at lower pH. It appears that low pH does not ensure safety from pathogens: in 1993, E. coli 0157:H7 in fresh-pressed apple juice caused an outbreak of diarrhea and hemolytic uremic syndrome. Yersinia spp. may also be able to survive in low pH foods.

Irradiation is a process that destroys microbial pathogens in food. Gamma rays from cobalt 60 or cesium 137, X rays (five million electron volts [5 MeV] maximum), and electrons (10 MeV maximum) are approved sources in the U.S. Irradiation was first used in the U.S. to ensure safe food for astronauts. Subsequently, the Food and Drug Administration (FDA) approved irradiation for wheat, wheat flour, and potatoes. Currently, irradiation is used mostly for spices, but also to disinfect cured meats, to kill Trichinella spiralis in pork, to control salmonella on chicken carcasses, and to reduce microbial load on fresh fruits and vegetables. There is some public resistance to irradiated foods, as the thought is that the food becomes radioactive. It does not.

Control of Microorganisms in Food

The contemporary food microbiologist has the challenges of a growing number of food pathogens and food spoilage. For example, eighty percent of commercial chickens in the U.S. are contaminated with Campylobacter jejuni. The food microbiologist may be involved in food manufacturing and processing, in retail food, in research in a university or government organization, such as the Agricultural Research Service of the United States Department of Agriculture (USDA), FDA, Centers for Disease Control and Prevention, and National Institutes of Health (NIH); or in food-plant inspection or the USDA's Food Safety Inspection Service (FSIS). In food plants, the food microbiologist is often a food technologist with a thorough training in chemistry as well as microbiology, who establishes a laboratory quality assurance manual (LQAM), a training program, and a statistical quality-control program. The food microbiologist must be versed in good manufacturing practices (GMP's), standard operating procedures (SOP's), sanitation, Hazard Analysis and Critical Control Points (HACCP's), rapid methods for the isolation and identification of microorganisms, as well as assays for toxins. HACCP's are designed to ensure food safety, extending beyond microbiological hazards, to chemical (for example, those from mycotoxins and pesticides) and physical (for example, from glass breakage) dangers. To generate an HACCP, the hazards in the plant's processes must be identified, the risk involved at each Critical Control Point must be established, and the critical levels of pathogens at each step in the process must be determined. The process must be monitored, and the monitoring verified. HACCP's are rapidly being required by government industries for more and more food processors. The USDA now mandates that all meat and poultry processors that are federally inspected have an HACCP in operation. The FDA now requires HACCP's for fruit-juice producers. Sanitation methods and monitoring are an extremely important part of any HACCP and a chief duty of a plant's food microbiologist. Surfaces in food-processing plants, meat carcasses, fruits, and vegetables must be kept pathogen-free. The use of simple rapid ATP detection systems (ATP—adenosine triphosphate—degrades quickly and is only found in living cells) allows a food microbiologist to involve plant workers in the sanitation effort. Workers swab sanitized surfaces, process the swab, and read the printout that has been calibrated to tell them the level of cellular contamination. They can resanitize surfaces until the results are acceptable.

In food-production and retail-food plants and in the home, good hygiene, especially hand washing, is the most effective way to eliminate the transmission of these pathogens. Another important practice is proper refrigeration of foods. Finally, proper cooking of raw meats, fish, and eggs by the consumer will destroy any remaining pathogens.

The explosion in genetic and immunological research in the 1980s resulted in many antibody-based and DNA-based methods for the identification of bacteria and toxins. These methods are rapid, reliable, sensitive, and becoming simpler daily. The time-consuming step in the assays is the necessity of initially growing or in some way enriching for the pathogen of interest. There are DNA-based assays for all the major food-borne pathogens. These assays use either DNA probes (usually of 16S rRNA genes, since there are relatively so many copies of these genes in cells) or PCR (a short DNA sequence is amplified in a thermocycler). There are also antibody-based assays for the major food-borne pathogens and toxins. These assays depend upon an antibody produced to some component of a bacterial cell or toxin. The most commonly used antibody-based assay is an ELISA, an enzyme-linked immunosorbent assay. It is described as a "sandwich assay." The test substance is added to a solid support to which the antibody to a particular pathogen is bound. The cell or toxin binds to the antibody. A secondary antibody, which is conjugated to an enzyme, binds to the primary antibody. Addition of the enzyme's substrate results in activity that can be detected. As the field of diagnostics speeds on, the food microbiologist must devote time to a continuing evaluation of newly emerging technologies aimed at reducing or eliminating pathogens as well as microorganisms that adversely affect the quality of food.

Use of Microorganisms for Various Helpful Ends

Before ancient people had any idea of microorganisms, they were using them to ferment foods. Bacteria, yeast, and molds are now used extensively to preserve foods and improve their aroma and flavor. Beer is probably the oldest fermentation product consumed by humans. Its history has been traced back to the Sumerians in 7500 B.C.E. The basic component of beer is a grain or cereal, for example, malted barley, rice, corn, or millet. The major food source in cereals is starch. Barley is germinated to produce starch-degrading enzymes, or amylases. This mixture, "malt," is used to process the starch in barley or other cereals; starch must be broken down into sugar for fermentation to take place. Strains of Saccharomyces cerevisiae are used for lager-style beers, and strains of S. uvarum for ales. Wine, also an ancient beverage, is made by inoculating fruit juice, usually grape juice, with strains of S. cerevisiae, fermenting the high level of sugar in the juice. Many kinds of lactic-acid bacteria (LAB) are also found during yeast fermentation: Lactobacillus spp., Leuconostoc spp., and Pediococcus spp. Winemakers often inoculate their wines with commercial LAB cultures to reduce overly high acidity of juice (grape juice, for example, has a pH of 3.0 to 3.8) and, through their metabolism, to add flavors or "complexity" to the wine.

The bacteria that Pasteur identified as wine spoilers, the acetic-acid bacteria, are used to make vinegar. Wine or cider is inoculated with Acetobacter spp., which produce acetic acid by oxidizing ethanol.

Basic bread is made by adding water and salt to wheat flour. Yeast is added to "leaven" bread. The Egyptians obtained yeast from beer vats to leaven their bread. The Greeks and Romans used yeast from wine vats. Now, strains of S. cerevisiae are used to ferment the sugars in bread dough. LAB are also used to give special flavors to some breads: for example, Lactobacillus sanfrancisco for sourdough and Lactobacillus plantarum for rye bread.

LAB are most important in dairy products. In 1878, Joseph Lister (1827–1912) isolated, in pure culture, a bacterium that caused milk souring. LAB are used to curdle milk for cheese production, and to ripen certain cheeses (Propionibacterium spp., for instance, for Swiss cheese). LAB are also used in the production of yogurt, buttermilk, and kefir, an alcoholic fermented-milk product. Molds, mainly of the penicillia family, are used to break down the fats in cheese and add distinctive flavors, for example, Penicillium roqueforti in Roquefort cheese.

Plant material is fermented to make pickled vegetables, sauerkraut, Spanish-style olives, and soy sauce. LAB, for example, are used to ferment cabbage into sauerkraut. Several genera of bacteria, including LAB, and fungi are used to ferment olives, which are inedible before this processing.

Many fermented products are made in the Far East, often from soybean meal. A koji, or mixed culture of bacteria, yeast, and molds, is used to inoculate the food. For soy sauce, for example, soybean meal is inoculated with a koji containing Aspergillus oryzae and LAB such as Lactobacillus delbrueckii. Other common fermented-soybean foods are tempeh, miso, and sufu (a traditional Chinese cheeselike product). The acids these microorganisms produce, chiefly acetic, butyric, and lactic, prevent the growth of most other microorganisms. The fermentations not only favorably modify flavors and textures, but also have preservative action.

A bacterium, Xanthomonas campestris, produces a polymer, xanthan gum, which is used as a thickener in such foods as salad dressings, cottage cheese, yogurt, ice cream, and frostings.

Harmful Microorganisms

Bacteria, viruses, and protozoa that cause gastroenteritis are transmitted by the fecal–oral route—that is, by the consumption of food or water fecally contaminated by infected persons. The major bacteria that cause gastroenteritis are Salmonella spp, E. coli, Campylobacter jejuni, Vibrio parahaemolyticus, and Yersinia enterocolotica. Ingesting toxins produced by bacteria that have grown in food can also result in gastroenteritis. Bacteria that cause gastroenteritis by producing toxins are Staphylococcus aureus, Clostridium perfringens, and Bacillus cereus. Rotavirus and the Norwalk virus group are the two major viruses causing gastroenteritis.

Milk is pasteurized to eliminate Mycobacterium tuberculosis, M. bovis, Salmonella spp., Listeria spp., enteric viruses, Brucella spp., Coxiella burnetii, and Campylobacter jejuni.

Food-borne bacteria can cause serious diseases: Salmonella typhi causes typhoid fever; Shigella spp. cause bacillary dysentery; E. coli strains can cause dysentery; M. tuberculosis and M. bovis cause tuberculosis; Vibrio cholerae causes cholera; Brucella spp. cause undulant fever; and Coxiella burnetii causes Q fever. Listeria monocytogenes causes listeriosis in predisposed populations, for example, immune-compromised individuals. Hepatitis is caused by the Hepatitis A virus and the recently discovered Hepatitis E virus, which is common in Africa and India and other Asian countries, but not in Western countries. In meat and poultry products, Salmonella, E. coli, Campylobacter jejuni, Listeria, and Clostridium perfringens are the major pathogens. Listeria monocytogenes is the major cheese pathogen. Unlike other food-borne pathogens, its temperature growth range (from 31 to 122°F [–0.4 to 50°C]) allows it to grow under refrigeration conditions. Hepatitis viruses and Yersinia enterocolitica are major oyster pathogens. Some of the pathogens found on fish are of marine origin, for example, Vibrio vulnificus, V. parahaemolyticus, and V. cholerae, and others are from sewage, for example Salmonella spp. and Campylobacter spp. Nuts and grains can become contaminated with mycotoxins, aflatoxins produced by Aspergillus flavus, being the most dangerous.

Prions are particles, smaller than viruses, and mostly composed of protein. It has recently been proposed that prions are the cause of four diseases: Creutzfeldt-Jacob disease and kuru in humans, bovine spongiform encephalopathy (BSE or mad cow disease) in cows, and scrapie in sheep. It is now thought that several dozen people have gotten a human form of BSE by eating the meat of infected cattle. This disease has been named new variant Creutzfeldt-Jakob disease, or nvCJD.

See also Beer; Cheese; Dairy Products; Fermentation; Fermented Beverages other than Wine or Beer; Government Agencies, U.S.; Microorganisms; Packaging and Canning; Pasteur, Louis; Safety, Food; Wine.

Susan Rodriguez Roy Thornton