Scope of Microbiology

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Scope of Microbiology

WHY YOU NEED TO KNOW

To see where we’re going from where we are, we must know where we’ve been. Measles, whooping cough, mumps, polio, cholera, influenza, rheumatic fever, pneumonia, diphtheria, tuberculosis, typhoid fever, meningitis, leprosy, syphilis, gonorrhea, tetanus, anthrax, the common cold, chicken pox, smallpox, rabies, encephalitis, malaria, dysentery, etc., etc., etc., numerous epidemics, pandemics, and too many others to list have been with humankind from approximately 4000 bce. to the present. Humankind has lived with diseases and been able to survive with varying degrees of success. Knowledge of the nature of disease has been slow and difficult to obtain. Rational use of this knowledge to alleviate the distress caused by disease has been even slower. Technological advances were and are needed to cope with the problems. For example, cholera—a killer of epidemic proportions—was linked to a public water source by John Snow, an English physician in the mid-1800s. He removed the pump handle, preventing access to the water, and the epidemic ended—ingenious, but serendipitous and of limited use.

The advancement of microbiology began with Robert Hooke’s (1635–1703) observations utilizing the then new compound microscope that could magnify objects ×20 to ×30. Later, Antony van Leeuwenhoek (1632–1723), using his skills at lens grinding and the use of light, improved the resolving power of the microscope to ×200. His were among the first observations of bacteria and, arguably, the beginning of microbiology. Bacteria were eventually recognized as causative agents of disease. These observations and others led Pasteur and Koch in the late 1800s to develop the germ theory of disease, an understanding that boosted disease prevention and treatment significantly. For example, Robert Koch in 1883 microscopically identified the causative pathogen for cholera (Vibrio cholerae), which he had grown on a plate of agar. After acceptance of the germ theory of disease, new pathogens were reported on the average of every year and a half. Technological advancements in light microscopy and the development of electron microscopy permitted visualization of pathogens or their shadows, allowing assessment of the effectiveness of treatment. Although it was deduced that the causative agent for the influenza pandemic of 1918 was a filterable agent, it was the advent of electron microscopy that allowed visualization of the virus in a rare lung sample from a victim. More recent advances in the biotechnology of genetic analysis have provided information on the nature of the functions of hemagglutinin and neuraminidase, viral coat proteins of the influenza virus. It is hoped that this information will direct investigations for methods of protection from another potential influenza pandemic.

Microbiology not only affords the detailed study of recognized pathogenic microorganisms but is invaluable in the identification of new pathogens in emerging diseases. Such studies are essential in monitoring the presence of microbes everywhere from water supplies to door knobs. Results from these studies are used to direct personal as well as public health and hygiene practices and policies. Technological advances in microscopy and biotechnology have provided a basis for the scientific discipline of microbiology and have stimulated the development of new concepts for research and vice versa. Meanwhile, hospital microorganisms such as Staphylococcus aureus, enterococci, and Pseudomonas aeruginosa are becoming resistant to the old tried and true antibiotics. Cephalosporins in many cases are the last line of defense. Clearly, much work lies ahead if we wish to bias the balance in favor of survival.

Origins of Microbiology and Microscopy

Microbiology is the study of microorganisms, using a variety of techniques for purposes of visualization, identification, and study of their function. The science of microbiology originated with the invention and development of the microscope. Microscopy allowed humans to magnify objects and microorganisms not detectable by the naked eye. Technological advances then have led to the improvement of microscopes, which became an essential investigative tool for biology in general and for the study of cells, tissues, and microorganisms (Figure 1.1) in particular.

FIGURE 1.1 A, Comparison of cell sizes. This drawing shows relative size comparisons of bacteria, eukaryotic cells, and viruses. Although a few bacteria are actually larger than some eukaryotic cells, the vast majority are much smaller. B, Cheek cell with resident bacteria. This photo shows a human cheek cell with resident bacteria, which are stained and appear as small dark purple spheres or rods. Note that the bacteria are even much smaller than the nucleus in the center of the cell. (Approximate magnification: ×1000.)

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.

TABLE 1.1

Significant Events in Microbiology

Name	Year	Event
Zaccharias Janssen	1590	Invention of the first compound microscope
Robert Hooke	1660	Explores living and nonliving matter with a compound microscope
Francesco Redi	1668	Experiments to disprove spontaneous generation
Antony van Leeuwenhoek	1676	Observes bacteria and protozoan “animalcules” with simple microscope
Francesco Redi	1688	Published experiments on spontaneous generation of maggots
Lazzaro Spallanzani	1776	Conducts further experiments to disprove spontaneous generation
Edward Jenner	1796	Introduction of smallpox vaccination
Ignaz Semmelweis	1847–1850	First use of antiseptics to reduce hand-borne disease
Louis Pasteur	1857	Proves that fermentation is caused by microorganisms; introduces pasteurization
Louis Pasteur	1861	Completes experiments that show without doubt that spontaneous generation does not occur
Joseph Lister	1867	Antiseptic surgery—begins the trend toward modern aseptic techniques
Robert Koch	1876–1877	Studies anthrax in cattle and implicates Bacillus anthracis as causative agent
Louis Pasteur	1881	Develops anthrax vaccine for animals
Robert Koch	1882	Identifies causative agent of tuberculosis
Robert Koch	1884	Describes his postulates

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).

FIGURE 1.2 Bright-field microscopy.
Bright-field microscopes vary somewhat in appearance based on brands, special features, or attached equipment, but they all have the same basic parts as labeled in the photo.

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.

FIGURE 1.3 Dark-field microscopy.
The dark-field microscope illuminates from the side rather than directly through the specimen. These cheek cells are in a living state and did not require any staining. (Approximate magnification: ×400.)

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)

FIGURE 1.4 Phase-contrast microscopy.
The phase-contrast microscope enhances the contrast of the image by shifting the phase of the light. These cheek cells are in a living state and did not require any staining. (Approximate magnification: ×400.)

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.

FIGURE 1.5 Fluorescence microscopy.
The fluorescence microscope uses an ultraviolet lamp to illuminate specimens stained with fluorescent stain. This is a fluorescence microscopy photo of a modified strain of Pseudomonas. (Approximate magnification: ×1000.) Courtesy Dr. Larry Halverson, Iowa State University.

Confocal Microscopes

In 1955 Marvin Minsky invented confocal microscopy, which was patented in 1957. Confocal microscopes have several advantages over the conventional optical microscopes, such as creating sharper images of specimens that would appear blurred with the use of a conventional compound microscope. In a confocal imaging system a single point of light focuses sequentially across a specimen, avoiding most of the unwanted scattered light that usually obscures an image when the entire specimen is illuminated at one time. An image is built by visualizing different planes of the layers of the specimen as the stage of the microscope is moved up and down.

Electron Microscopes

Electron microscopes (EMs) are sophisticated instruments of the twentieth century that use a beam of electrons rather than light as the source of energy to visualize specimens. Magnetic fields instead of optical lenses are used to focus the electron beam. This allows much better resolution of the image than is possible with the light microscope. Specimens for EM studies require more extensive preparation, expensive laboratory equipment, and specially trained personnel than are required for the preparation of specimens used for light microscopy.

Bacteria can be visualized by light microscopy, but their detailed structure or specifics of their attachment to hosts are best seen by electron microscopy. Although most viruses are not visible by light microscopy, their effects on cells and tissues are. Investigations of specimen surfaces use scanning electron microscopy (SEM), whereas studies of the interior of cells and tissues use transmission electron microscopy (TEM).

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).

FIGURE 1.6 Transmission electron microscopy.
This is a TEM image of a neuron.

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.

FIGURE 1.7 Scanning electron microscopy.
Photo of an SEM image of the bacteria Campylobacter passing through the pores of a filter. (Approximate magnification: ×5000.) Courtesy Dr. James Dickson, Iowa State University.

Spontaneous Generation

Before microorganisms were discovered and described by Antony van Leeuwenhoek, life was hypothesized to develop from nonliving matter (abiogenesis).

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