Microscopy and Its Founding Fathers

The development of microscopes started in the sixteenth century and evolved through time into a sophisticated tool used routinely in many branches of science. To this day all different types of microscopes continue to be improved and new ones are being developed.

Zaccharias and Hans Janssen, a father-and-son team of Dutch eyeglass makers (around the year 1590) found that optical images could be enlarged and viewed using different lenses. The first microscope they produced was a compound microscope consisting of a simple tube with lenses at each end. Depending on the size of the diaphragm, the part of the microscope that regulates the amount of light striking the specimen, the magnification of objects under view ranged from three times (or ×3) to ×9.

Antony van Leeuwenhoek (1632–1723), another native of Holland, is considered to be the father of microscopy and is believed to be the first to observe live bacteria and protozoans. He was fascinated by the power of lenses, which made it possible to observe what the naked human eye could not see. The microscope he used contained only one convex objective lens and is now called a simple microscope. His interest in science and his native curiosity led him to some of the most important observations of biology. Van Leeuwenhoek was able to see small life forms that he called “animalcules” (little animals). Throughout the years he observed bacteria, protozoans, blood cells, sperm cells, microscopic nematodes, rotifers, and more. Much of his inspiration came from Hooke’s Micrographia (see later in this chapter). He published his observations in 1678 in a letter to the Royal Society of London. As a result of his findings, van Leeuwenhoek is referred to as the “father of microbiology.” After some early skepticism, scientists in the late seventeenth century finally became convinced that microorganisms did, in fact, exist. Van Leeuwenhoek did not comment further on the origin of the microorganisms nor did he relate them to any diseases. The definitive relationship between microbes and disease was established later by Hooke, Pasteur, Koch, and others in what became known as the “germ theory of disease.”

Robert Hooke (1635–1703), an English scientist with remarkable engineering abilities and an interest in many aspects of science, greatly improved the design and capabilities of the compound light microscope. With his microscope he observed insects, sponges, bryozoans, foraminifera, bird feathers, and plant cells. He published his observations with magnificent drawings in the book Micrographia. He was requested by the Royal Society of London to confirm van Leeuwenhoek’s finding of animalcules and succeeded in doing so.

Table 1.1 lists some significant events in the history of microbiology.

Types of Microscopes

With advances in technology, continued development of microscopes for specific uses continues and many kinds of microscopes are now available to scientists. A brief overview and description of light and electron microscopes currently used in teaching, service, and research laboratories follows.

All light microscopes use visible light to illuminate, and optical lenses to observe, enlarged images of specimens. They are classified as either simple or compound. A simple light microscope, such as van Leeuwenhoek’s, has a single magnifying lens and a visible light source, and can magnify objects approximately ×266. A compound light microscope also uses visible light, usually provided by an electric source, but uses multiple lenses for magnification. The lens or lenses close to the eye are called ocular lenses and are located in the headpiece of the microscope. The lenses closer to the specimen are located in the body of the microscope and are referred to as objective lenses. Each lens has its own magnifying power, and the final magnification of a compound microscope is the product of the enlarging power of the ocular lens multiplied by the power of the objective lens. Most often the ocular lenses, either single (monocular) or in pairs (binocular), magnify by a power of 10 (×10). The objective lenses are mounted on a revolving nosepiece and usually magnify ×4 or ×5, ×10, ×40, and ×100. In general, compound microscopes can magnify an object up to 1000 times (i.e., an ocular lens with a magnification of ×10 times the objective lens with a power of ×100 = ×1000). The specimens for compound light microscopy can either be visualized as whole (i.e., bacteria and other microorganisms) or are specially prepared for viewing with a given type of microscope. After specific dehydration procedures larger specimens are cut into 1.0- to 10-µm sections. Both smear preparations, for single cells, and sections are usually stained for better visual images (see Chapter 4, Microbiological Laboratory Techniques). Photographs taken through a microscope are referred to as photomicrographs or micrographs.

Dissection microscopes and stereomicroscopes are low-power microscopes designed for observing larger objects such as insects, worms, plants, or any objects that may have to be dissected for further observation. These microscopes provide three-dimensional images to determine surface structures and specific locations on a specimen.

Bright-field Microscopes

The bright-field microscope, a type of compound microscope, can be used to examine small specimens and some of their details. The microscope exhibits a background brighter than the observed specimen and is dependent on altering the light path (refraction) only. For this reason most specimens require staining for optimal observation. Bright-field microscopes are most commonly used to observe sectioned and stained tissues, organs, and microorganisms (Figure 1.2).

Dark-field Microscopes

Unfixed, unstained specimens such as living organisms (i.e., bacteria) can be observed with a dark-field microscope. Because of its design, the background of the microscope slide is dark, whereas the organism itself is bright (Figure 1.3). Some bacteria that are difficult to stain, including spirochetes (see Chapter 6, Bacteria and Archaea), can best be observed by dark-field microscopy. Bacterial capsules are also good candidates to be observed with dark-field microscopes.

Phase-contrast Microscopes

Phase-contrast microscopy, first described in 1934 by Frits Zernike, is done with a contrast-enhancing optical instrument that can be used for a wide variety of applications. It produces high-contrast images of transparent specimens such as:

• Living plant and animals cells

• Microorganisms

• Thin tissue slices, and more (Figure 1.4)

This type of instrument is ideally suited for the observation of cytoplasmic streaming, motility, and the dynamic states of cell organelles. Cell division and phagocytosis are examples of processes well suited for phase-contrast microscopy. The development of video technology enabled the recording and demonstration of these processes.

Fluorescence Microscopes

If a specimen can emit light (fluoresce) of one color when illuminated by ultraviolet radiation then fluorescence microscopy may be the method of choice. Fluorescence microscopes are used to visualize specimens that contain natural fluorescent substances such as chlorophyll or those stained with a fluorescent dye such as fluorescein, auramine, or rhodamine B (Figure 1.5). Fluorescence microscopy is an important and widely used tool in the diagnosis of infectious disease and in studies in microbial ecology. Fluorescence techniques are applied to identify specific antibodies, which are proteins produced in response to antigens (see Chapter 20, The Immune System). By attaching a fluorescent dye to these protein molecules, labeled antibodies are created that can be visualized, monitored, and studied.

Transmission Electron Microscopes

In a transmission electron microscope the electron beam travels through an ultrathin sectioned specimen (approximately 100 nm in thickness) and provides a two-dimensional image of the cell or other object. The resolving power of a TEM is approximately 0.002 µm, which is 100 times greater than can be achieved with a light microscope. The usual magnification of a TEM ranges from 500,000 to 1,000,000. Pictures taken of images created with an electron microscope are called electron micrographs (Figure 1.6).

Scanning Electron Microscopes

A scanning electron microscope also provides images of high resolution, but in contrast to the TEM, the SEM does not require ultrathin sections. It scans the surface of an object, producing a three-dimensional image (Figure 1.7). Moreover, the SEM has a large depth of field that allows the surface areas of large samples to be in focus at the same time. The usual magnification that can be achieved with an SEM ranges from ×10 to ×100,000.

Spontaneous Generation

Before microorganisms were discovered and described by Antony van Leeuwenhoek, life was hypothesized to develop from nonliving matter (abiogenesis). Maggots, for example, were believed to arise spontaneously from rotting meat. The big question among scientists at that time was whether microbes were produced spontaneously by decay and fermentation, or whether they caused decay and fermentation—raising the basic dilemma of “which comes first, the chicken or the egg?”

One of the first serious attacks on this controversial spontaneous generation theory came in 1688 from Francesco Redi, an Italian physician. He believed that maggots developed from fly eggs. In his experiments he put meat into different flasks, kept some of them exposed to the atmosphere and flies, and the rest sealed. As he expected, maggots developed only in the open flasks where flies had access to the meat.

At about the same time, Antony van Leeuwenhoek’s invention of the first microscope allowed him to observe small organisms, which he called “animalcules.” He further showed that when hay was placed into a mixture of water and soil and allowed to incubate for a few days, new creatures in the hay infusion could be observed. Yet, these findings were interpreted by some scientists of the time as being additional proof of spontaneous generation.

This debate over the existence of spontaneous generation continued for centuries and in 1745 an English clergyman, John Needham, claimed victory for spontaneous generation with his experimental observations. He boiled chicken and corn broth, put it into a flask, and sealed it. After a few days cloudiness appeared in the broth, which is evidence of microbial growth. However, Lazzaro Spallanzani, an Italian priest, questioned Needham’s interpretation and suggested that microorganisms could have entered the flask from the air before the tube was sealed. In his own experiments he also placed chicken broth in a flask, sealed it, and then drew off the air before boiling the broth. As a result no microorganisms grew, which he interpreted as evidence that spontaneous generation did not exist. However, proponents of spontaneous generation held that Spallanzani only proved that spontaneous generation could not occur in the absence of air.

Louis Pasteur (1822–1895), a French chemist, finally ended the controversy. In 1861 he designed and conducted a definitive experiment in which he boiled meat broth in a flask and then heated the neck of the flask until it could be bent it into an S shape (swan-necked flask). Thus air could enter the flask but microorganisms trapped in the S-shaped neck were unable to reach the broth. There was no evidence of microbial growth. However, when he tilted the flask so that the broth could reach the organisms in the neck, the broth gradually became cloudy due to microbial growth (Figure 1.8).

In 1877 John Tyndall (1820–1893) demonstrated that microorganisms exist in dust. In a set of experiments similar to Pasteur’s, but in the absence of dust, the broths remained sterile even when exposed to air. In addition, Tyndall provided evidence of the existence of heat-resistant bacteria. Ferdinand Cohn (1828–1898), independent of Tyndall’s studies, also described heat-resistant bacterial endospores (see Chapter 3, Cell Structure and Function).

Pasteurization

In addition to his other findings Louis Pasteur invented the process we know as pasteurization (Box 1.1). The Emperor Napoleon III asked Pasteur to investigate the spoiling of wine that caused a negative impact in the production of wine, damaging the French wine industry. Pasteur discovered that wine spoilage (wine disease) is caused by microorganisms. He “pasteurized” the wine by heating it to 55° C for several minutes, which killed enough microorganisms to prevent the wine from spoiling. Pasteurization does not kill all microorganisms, but reduces the number of viable organisms so they are less likely to cause spoilage or disease. Sterilization (see Chapter 4, Microbiological Laboratory Techniques), on the other hand, kills all microorganisms including their endospores.

Germ Theory of Disease

During the time when the existence of spontaneous generation was being debated, several physicians began to suspect that microorganisms not only could cause spoilage and decay but might also play a role in infectious disease. Two physicians in different parts of the world, one in the United States and one in Vienna, significantly contributed to the concept that microorganisms had a role in infectious diseases. Oliver Wendell Holmes (1809–1894) in the United States showed that death following childbirth was often caused by material on the hands of midwives or physicians. Ignaz Semmelweis (1818–1865) in Vienna observed that women in the maternity ward became infected after being examined by physicians or students who came directly from the autopsy room or from examining infected patients. Semmelweis also noted that these students’ and physicians’ hands stunk from putrefaction and that they did not wash their hands before examining patients in the maternity wards. He also suspected that microorganisms were responsible for the odor. Consequently, he required everyone to wash their hands in a solution of chlorine before entering the maternity ward and examining patients. The result was a drastic decline in the death rate on the wards.

Joseph Lister (1827–1912) studied the observations of Semmelweis and hypothesized that airborne microbes might play a role in postsurgical infections as well. He discovered that applying carbolic acid to dressings and using an aerosol of carbolic acid on the surgical field significantly decreased the number of wound infections. He was the first physician to introduce the use of aseptic techniques.

The germ theory of disease proposed by Louis Pasteur and Robert Koch (1843–1910) is based on the existence of infectious microorganisms. Although Pasteur was convinced that microbes caused disease in humans he was never able to link a specific microbe with a particular disease. Koch’s investigations focused on anthrax, an infectious disease that seriously affects animal herds and humans brought in contact with the microorganism. Koch also discovered that anthrax produced endospores that persist after the death of the exposed animals. Moreover, he proved that these spores can survive and later develop into the active anthrax microbe and infect other animals. This established that a specific organism caused a specific disease. After many years of experimentation he developed what we now know as Koch’s postulates (Box 1.2), which set forth the conditions that should identify an organism as the specific cause of a specific disease.

Immunology begins with the work of Edward Jenner (1749–1823). Jenner observed that milkmaids who had contracted cowpox did not become infected with smallpox, a major killer during the eighteenth century. He applied material from cowpox lesions, containing the vaccinia virus, to small incisions or puncture wounds made in human arms. These human subjects did not develop smallpox but only occasionally showed a mild fever associated with the disease. With this procedure Jenner proved that immunity against smallpox could be achieved through vaccination.

Evolution

Evolution is an important concept that involves all living things including microorganisms. Evolution implies that living things gradually change over millions of years, resulting in structural and functional changes of organisms throughout generations. The millions of different species on earth and their successful adaptation to different habitats are indicators of the evolutionary process.

For the first three quarters of evolutionary history the only organisms on earth were microscopic and mostly unicellular. Fossils of prokaryotes go back to 3.5 to 4 billion years followed by eukaryotic life approximately 2.2 billion years ago. Eukaryotic cells are larger and more complex than prokaryotic cells and scientists believe that eukaryotes have evolved from prokaryotic symbiotic communities. It is estimated that there are about 5 to 100 million species of organisms living on earth today. The evolutionary relationship between organisms is the subject of phylogeny. Today, the phylogenetic relationship between organisms can be determined by nucleic acid and nucleotide sequencing. Results from ribosomal RNA (rRNA) sequencing identify three evolutionarily distinct cell lines that are classified as bacteria, archaea, and eukarya. These three distinct lineages, based on the origin of cell lines, are referred to as domains (Figure 1.9).

Prokaryotes versus Eukaryotes

Differentiation between prokaryotes (pro, before; karyon, nucleus) and eukaryotes (eu, good or with; karyon, nucleus) was greatly advanced as more sophisticated microscopes became available, allowing scientists to identify membrane-bound structures in unicellular organisms. Significantly, scientists discovered that some unicellular organisms contained membrane-bound organelles such as a nucleus, whereas others did not. The cells without membrane-bound organelles and therefore without a nucleus were classified as prokaryotes and the ones with membrane-bound organelles and a nucleus were designated eukaryotes. All bacterial cells are prokaryotic, whereas algae, fungi, and protozoans are eukaryotes (see Chapter 3, Cell Structure and Function).

Bacteria, Archaea, and Eukaryotes

Bacteria, archaea, and eukaryotes are examples of diversity and the root of microbial diversity is evolution. Within bacteria several evolutionary branches are present including some pathogenic (disease-causing) bacteria, occurring in soil and water, in animal digestive tracts and skin, as well as in many other environments. Also, different bacteria require different sources of energy such as light, organic chemicals, inorganic chemicals, or combinations thereof.

Archaea are a group of single-celled microorganisms, similar to bacteria because they are also prokaryotes, but evolutionarily different. They are characterized by their preference to live in extreme environments including hot springs, glaciers, or highly salty environments (see Chapter 6, Bacteria and Archaea).

Eukaryotic microorganisms include algae, fungi, and protozoans (see Chapter 8, Eukaryotic Microorganisms). Algae are a large and diverse group of simple organisms, ranging from unicellular to multicellular forms, containing chlorophyll that is needed for photosynthesis. They are common in aquatic bodies (both freshwater and saltwater) and in all types of soil, but have limited medical significance. Fungi include yeasts, molds, and mushrooms, which are not microbes. Their energy sources are organic compounds found in soil and water. Fungi play a major role in the breakdown of dead organic matter in a variety of environments. However, some fungi are pathogenic and can cause disease in humans and animals. Protozoans are colorless, mobile organisms feeding on other organisms for their energy source. In general, protozoans are free-living microorganisms and several of them are also pathogenic to humans and animals.

Viruses

Viruses are noncellular, submicroscopic particles (visible only by electron microscopy) and consist of nucleic acid surrounded by a protein coat. They are metabolically inert, incapable of carrying out biosynthetic functions, and they are dependent on host machinery for their multiplication. Viruses are classified according to the type of nucleic acid (RNA or DNA) they contain. Many viruses are pathogenic and can be classified on the basis of their host as animal viruses, plant viruses, and bacterial viruses (see Chapter 7, Viruses).

Prions

Prions (proteinaceous infectious particles) are not cellular organisms, nor are they viruses. Prions lack nucleic acids; they are normal proteins of animal tissues that can misfold during protein synthesis and become an infectious agent (see Chapter 7, Viruses).

Viroids

Viroids are plant pathogens that can cause serious economic problems. They are much smaller than viruses (15 to 100 nm) and exist of a small single-stranded, naked RNA circle. Viroids cannot produce proteins and do not have a protein coat (see Chapter 7, Viruses).

Taxonomy

The formal system of organizing, classifying, and naming of living organisms is called taxonomy. Taxonomy sorts organisms on the basis of mutual similarities into nonoverlapping groups called taxa. The goal of taxonomy is classification, nomenclature, and identification for clarification and ease of reference.

The current system of taxonomy began with a Swedish botanist, Carolus Linnaeus (1707–1778). He provided a system that standardized the naming and classification of organisms on the basis of common characteristics. Linnaeus’s system groups similar species into genera, genera sharing common features into families, similar families into orders, orders into classes, classes into phyla, and phyla into kingdoms (see Figure 1.9). In addition, in 1990, Carl Woese (1928–) and his colleagues introduced a three-domain system of taxonomy (archaea, bacteria and eukarya) based on genetic rather than morphological similarities (see Figure 1.9).

All these categories in the Linnaeus system are taxa, which are hierarchical, starting with the species and genera, followed by successive taxa, each with a broader description than the preceding one. All names in the taxa are Latin or Latinized.

Linnaeus assigned each species a descriptive name of its genus and a specific name for the species. This method of assigning the scientific name is called the binomial (two-name) system of nomenclature. A genus is the name of an organism and is often abbreviated by a single capital letter, whereas the species name is never abbreviated. For example, Escherichia coli can be abbreviated to E. coli. A genus typically contains several species, and a particular species can be further subdivided into different strains. For instance, there are several species in the genus Bacillus, such as B. subtilis or B. cereus.

The taxonomic resource for bacteria is the Bergey’s Manual of Systematic Bacteriology, the second edition of which is being published in five volumes. In addition to the number of keys for bacterial identification, newer versions of the manual also contain some molecular sequencing information for various bacterial groups. An outline of Bergey’s Manual can be found in Appendix A of this book.

Microbes in the Environment

Microorganisms in nature are often organized into complex communities of different organisms called biofilms. A biofilm consists of surface-associated microbial cells enclosed in an extracellular polymeric substance matrix. Antony van Leeuwenhoek observed and described layers of microorganisms on tooth surfaces, representing a microbial biofilm (see Chapter 3, Cell Structure and Function). However, detailed, high-magnification observations of microbial biofilms did not occur until electron microscopy became available. Biofilms are found on a variety of surfaces such as medical devices (Table 1.2), industrial water system piping, natural aquatic systems, foods, and also on living tissues. An established biofilm structure provides a perfect environment for the exchange of genetic material between different organisms in the biofilm community.

Microorganisms in Food Production

Many nonpathogenic microorganisms occur naturally in food, are beneficial, and are used as starter cultures to produce foods such as vinegar, sauerkraut, pickles, fermented milks, yogurt, cheese, and bread (Box 1.3). For example, vinegar is made from foods containing starch or smaller sugars. The production requires a two-way fermentation process that begins with apple juice or other raw materials, to which the yeast Saccharomyces cerevisiae is added to speed up the fermentation process. In the second stage, cultures of acetic acid bacteria such as Acetobacter aceti are added to this alcoholic liquid, converting the alcohol into acetic acid. In the United States most vinegar is produced from apples and is named apple cider vinegar.

Sauerkraut is another product made by fermentation of shredded cabbage in the presence of salt. The shredded cabbage is tightly packed in an anaerobic environment, where it becomes dehydrated. The addition of salt promotes the growth of lactic acid bacteria.

Dairy products such as butter, buttermilk, sour cream, cottage cheese, and yogurt are produced from fermented milk. Raw milk contains the fermentable sugar lactose in addition to several acid-producing microorganisms. The natural microflora of raw milk is often inefficient, uncontrollable, and can produce unpredictable results. Moreover, these organisms are destroyed during heat treatments needed to pasteurize milk. Therefore, starter cultures that can provide a more controlled and predictable fermentation process are added to produce the desired products. The function of the lactic acid starters is the production of lactic acid from lactose of the milk. Other starter cultures may provide flavor, aroma, and alcohol production.

Microorganisms in the Production of Alcoholic Beverages

Wine is produced by yeast fermentation of carbohydrates in freshly harvested grape juice, peaches, berries, pears, and other fruits or plants (even dandelions), and is marketed as such. In beer production barley or other grain is used as the source of fermentable carbohydrate. Beer and wine generally are limited to about 15% alcohol because yeast fails to ferment beyond this point. Beverages higher in alcohol content are produced through distillation and are called spirits or liquor.

Treatment of Water Supplies

Good health depends on a clean, drinkable water supply. It must be free of pathogens, toxins, odor, color, and bad taste to achieve these standards. The microbial content of drinking water should be constantly monitored to make sure the water is free of infectious agents. Most water purity assays are focused on the detection of fecal material, which would indicate the presence of pathogens. Wells, reservoirs, and other water resources can be analyzed for the presence of indicator bacteria that can be readily identified by routine laboratory procedures before selection of the appropriate water treatment.

Microbes and the Production of Pharmaceutical Agents

Louis Pasteur observed the inhibition of microorganisms by products formed by other microbes. This phenomenon is called antibiosis. The discovery of the penicillin-producing mold Penicillium by Alexander Fleming in the 1920s started the successful search for other antibiotic-producing microorganisms. Today, with the help of genetic engineering and recombinant DNA technology, not only antibiotics and semisynthetic antibiotics but also various hormones and other drugs are available on the market (see Chapter 22, Antimicrobial Drugs).

Microbes in Agriculture

Agricultural microbiology focuses on the relationships between microbes and domesticated plants and animals. Farmers use microbes and their products in a variety of ways, particularly for crop management via recombinant DNA biotechnology (see Chapter 25, Biotechnology). Plant microbiology involves the management of plant disease, soil fertility, and nutritional interactions. For example, the availability of nitrogen in the soil is essential for the growth of crops and bacteria involved in the nitrogen cycle of the soil are essential (see Soil Microbiology in Chapter 24, Microorganisms in the Environment and Environmental Safety). On the animal side, agricultural microbiology deals with the management and prevention of infectious diseases in farm animals as well as other associations animals have with microorganisms.

Bioremediation

Bioremediation is the process of using microorganisms to clean up toxic, hazardous, or unmanageable compounds by degrading them to harmless compounds. Some microbes performing these tasks have been genetically engineered to clean up specific wastes or pollutants. For example, genetically engineered petroleum-digesting bacteria assist in the cleanup of oil spills. Another form of bioremediation that has long been in use is the treatment of water and sewage. Reclamation procedures for treatment of polluted water to convert it to drinkable water are becoming more important worldwide because of the rapid dwindling of clean freshwater sources.

Microbes, Biomass, and Energy

By a process referred to as bioconversion, microorganisms can convert biomass such as organic matter and human, agricultural, and industrial wastewater into alternative fuels, including ethanol, methane, and hydrogen.

Ethanol produced during fermentation is one of the simplest alternative fuel sources to produce and can be mixed with gasoline to make gasohol. Although at present crops such as corn are used, it would be more economic to utilize crop wastes (i.e., corn stalks).

Some communities already use landfills as immense sources of methane, where methanogens anaerobically convert wastes into methane by the process of fermentation. Methane gas can be piped through natural gas lines and used as an energy source in common households.

The concept of using microbes to power fuel cells is not new; however, in 2003, scientists found a way to generate electricity by feeding bacteria common sugars and other carbohydrates. In this study bacteria were grown on graphite electrodes within a fuel cell. When the organisms were fed sugars, they generated electrons and transferred them to the graphite electrodes. This flow of electrons generated electricity that the battery could store. The generation of power by microbes has led many scientists to develop microbial fuel cells. In 2005, Pennsylvania State University environmental engineers and scientists at Ion Power Inc. (in New Castle, Delaware) published the first process that shows bacteria can retrieve four times the amount of hydrogen out of a biomass than would typically be generated by fermentation alone.

Microbial Forensics

Microbial forensics is a relatively new field applied to solving bioterrorism cases, medical negligence, and outbreaks of foodborne diseases. Use of microorganisms as weapons is not a new idea. They can be the weapon of choice in terrorists’ activities such as in the anthrax attacks of 2001. Forensic cases also have been reported about HIV-infected people intentionally infecting others as well. Microbes are also of interest in cases of medical negligence, in which medical personnel are implicated in postsurgical or other hospital-acquired infections due to inadequate or relaxed hygiene practices. In the case of outbreaks of foodborne diseases or intentional food contamination, it is critical to trace the infecting microbe to the source, either the company or person(s) of origin. Microbial forensics is becoming an essential part of data collection and their interpretation. Inquiries should stand up to the review of scientists and healthcare professionals as well as to the scrutiny of judges and juries.