Chapter 4 - Microscopy, Staining and Classification PDF

4 Microscopy, Staining, and Classification Before You Begin 1. Consider bacteria, fungi, protozoa, and viruses. Which of these is generally smallest in size? 2. What is the main difference between the cell walls of Gram-positive and Gram-negative bacteria? 3. Among endospores, fimbriae, flagella, an...

4 Microscopy, Staining, and Classification Before You Begin 1. Consider bacteria, fungi, protozoa, and viruses. Which of these is generally smallest in size? 2. What is the main difference between the cell walls of Gram-positive and Gram-negative bacteria? 3. Among endospores, fimbriae, flagella, and inclusions, which allow bacteria to survive very harsh conditions? MICRO IN THE CLINIC End of the Camping Trip? MARISOL IS A SOPHOMORE studying biology at the University of Minnesota. This has been a hard year—starting upperlevel courses, volunteering off campus, and playing on the women’s softball team. While she loves all of it, Marisol is really looking forward to the two-week camping trip that she and her girlfriends are taking right after finals week. Finals come and go, and Marisol and her friends head to the Boundary Waters Wilderness area in northern Minnesota. The first week of the trip is amazing—great weather, canoeing, hiking, hanging out with friends. At the start of the second week, Marisol wakes up feeling really achy and tired. They’ve been paddling canoes for the last seven days, so she assumes that it’s just muscle fatigue. However, the next day she still feels even more tired and achy and she thinks that she has a slight fever. The group decides to take a day off from canoeing and just hang out at their camp, giving Marisol a chance to rest. One of Marisol’s friends notices something on the back of Marisol’s right leg. It looks like a bruise—it has a blue center, and the edges are a little red. Since they’ve been hiking, camping, hauling gear, and climbing in and out of canoes, her friend disregards it. The morning after the rest day, Marisol says that she feels a little bit better, but she still has the fever and aches. Her friends think they should paddle out and cut the trip short; Marisol thinks she’s OK to continue on the trip. 1. What do you think? 2. Are Marisol’s symptoms severe enough to warrant an early end to the trip? Scanning electron micrograph of two strains of bacteria, isolated from a housefly’s feet, growing together. M04_BAUM2302_06_SE_C04.indd 96 Turn to the end of the chapter (p. 120) to find out. 08/06/19 8:00 AM Microscopy Scientists can see the marvelous microbial world through advances in microscopy. With the invention of new laboratory techniques and the construction of new instruments, biologists are still discovering wonders in the microbial world. In this chapter, we will examine some of the techniques microbiologists use to enter that world. We begin with a discussion of metric units as they relate to measuring the size of microbes. We then examine the instruments and staining techniques used in microbiology. Finally, we consider the classification schemes used to categorize the inhabitants Play Microscopy and Staining: Overview of the microbial @ Mastering Microbiology wonderland. Units of Measurement L E A R N I N G O U TCO M E S 4.1 4.2 Identify the two primary metric units used to measure the diameters of microbes. List the metric units of length in order, from meter to nanometer. Microorganisms are small. This may seem an obvious statement, but it is one that should not be taken for granted. Exactly how small are they? How can we measure the width and length of microbes? Typically, a unit of measurement is smaller than the object being measured. For example, we measure a person’s height in feet or inches, not in miles. Likewise, the diameter of a dime is measured in fractions of an inch, not in feet. So, measuring the size of a microbe requires units that are smaller than even the smallest interval on a ruler marked with English units (typically 1/16 inch). Even smaller units, such as 1/64 inch or 1/128 inch, become quite cumbersome and very difficult to use when we are dealing with microorganisms. So that they can work with units that are simpler and in standard use the world over, scientists use metric units of TABLE 4.1 measurement. Unlike the English system, the metric system is a decimal system, so each unit is one-tenth the size of the next largest unit. Even extremely small metric units are much easier to use than the fractions involved in the English system. The unit of length in the metric system is the meter (m), which is slightly longer than a yard. One-tenth of a meter is a decimeter (dm), and one-hundredth of a meter is a centimeter (cm), which is equivalent to about a third of an inch. One-tenth of a centimeter is a millimeter (mm), which is the thickness of a dime. A millimeter is still too large to measure the size of most microorganisms, but in the metric system we continue to divide by multiples of 10 until we have a unit appropriate for use. Thus, one-thousandth of a millimeter is a micrometer (mm), which is small enough to be useful in measuring the size of cells. One-thousandth of a micrometer is a nanometer (nm), a unit used to measure the smallest cellular organelles and viruses. A nanometer is one-billionth of a meter. TABLE 4.1 presents these metric units and some English equivalents. (Refer to Chapter 3, Figure 3.4, for a visual size comparison of a typical eukaryotic cell, prokaryotic cell, and virus.) TELL ME WHY Why do scientists use metric rather than English units? Microscopy L E A R N I N G O U TCO M E Define microscopy. 4.3 Microscopy1 refers to the use of light or electrons to magnify objects. The science of microbiology began when Antoni van Leeuwenhoek (lā′ven-hŭk, 1632–1723) used primitive microscopes to observe and report the existence of microorganisms. Since that time, scientists and engineers have developed a variety of light and electron microscopes. Metric Units of Length Metric Unit (abbreviation) Meaning of Prefix Metric Equivalent U.S. Equivalent Meter (m) —a 1m 39.37 in (about a yard) Length of pork tapeworm, Taenia solium (e.g., 1.8–8.0 m) Decimeter (dm) 1/10 0.1 m = 10 -1 m 3.94 in —b Centimeter (cm) 1/100 0.01 m = 10 -2 m 0.39 in; 1 in = 2.54 cm Diameter of a mushroom cap (e.g., 12 cm) Millimeter (mm) 1/1000 0.001 m = 10 — Diameter of a bacterial colony (e.g., 2.3 mm); length of a tick (e.g., 5.7 mm) Micrometer (mm) 1/1,000,000 0.000001 m = 10 -6 m — Diameter of white blood cells (e.g.,5925 mm) Nanometer (nm) 1/1,000,000,000 0.000000001 m = 10 -9 m — Diameter of a poliovirus (e.g., 25 nm) -3 97 m Representative Microbiological Application of the Unit a The meter is the standard metric unit of length. Decimeters are rarely used. b 1 From Greek micro, meaning “small,” and skopein, meaning “to view.” 98 CHAPTER 4 Microscopy, Staining, and Classification MICRO CHECK 1. What metric unit is one-thousandth of a millimeter and is a useful unit to use when measuring whole cells? 2. Other than light, what is used to magnify objects in microscopy? Electrons are negatively charged particles that orbit the nuclei of atoms. Besides being particulate, moving electrons also act as waves, with wavelengths dependent on the voltage of an electron beam. For example, the wavelength of electrons at 10,000 volts (V) is 0.01 nm; that of electrons at 1,000,000 V is 0.001 nm. As we will see, using radiation of smaller wavelengths results in enhanced microscopy. Magnification General Principles of Microscopy L E A R N I N G O U TCO M E S 4.4 4.5 4.6 4.7 Explain the relevance of electromagnetic radiation to microscopy. Define magnification. List and explain two factors that determine resolving power. Discuss the relationship between contrast and staining in microscopy. General principles involved in both light and electron microscopy include the wavelength of radiation, the magnification of an image, the resolving power of the instrument, and contrast in the specimen. Wavelength of Radiation Visible light is one part of a spectrum of electromagnetic radiation that includes X rays, microwaves, and radio waves (FIGURE 4.1). Note that beams of radiation may be referred to as either rays or waves. These various forms of radiation differ in wavelength— the distance between two corresponding parts of a wave. The human eye discriminates among different wavelengths of visible light and sends patterns of nerve impulses to the brain, which interprets the impulses as different colors. For example, we see wavelengths of 400 nm as violet and of 650 nm as red. White light, composed of many colors (wavelengths), has an average wavelength of 550 nm. Magnification is an apparent increase in the size of an object. It is indicated by a number and *, which is read “times.” For example, 16,000* is 16,000 times. Magnification results when a beam of radiation refracts (bends) as it passes through a lens. Curved glass lenses refract light, and magnetic fields act as lenses to refract electron beams. Let’s consider the magnifying power of a glass lens that is convex on both sides. A lens refracts light because the lens is optically dense compared to the surrounding medium (such as air); that is, light travels more slowly through the lens than through air. Think of a car moving at an angle from a paved road onto a dirt shoulder. As the right front tire leaves the pavement, it has less traction and slows down. Since the other wheels continue at their original speed, the car veers toward the dirt, and the line of travel bends to the right. Likewise, the leading edge of a light beam slows as it enters glass, and the beam bends (FIGURE 4.2a). Light also bends as it leaves the glass and reenters the air. Because of its curvature, a lens refracts light rays that pass through its periphery more than light rays that pass through its center, so that the lens focuses light rays on a focal point. Importantly for the purpose of microscopy, light rays spread Light Air 400 nm Glass 700 nm (a) Focal point Visible light Gamma rays 10–12 m X UV rays light 10–8 m Infrared Microwave Radio waves and Television Increasing wavelength 10–4 m 100 m Crest Specimen 103 m One wavelength Trough Increasing resolving power ▲ FIGURE 4.1 The electromagnetic spectrum. Visible light is made up of a narrow band of wavelengths of radiation. Visible light and ultraviolet (UV) light are used in microscopy. (b) Convex lens Inverted, reversed, and enlarged image ▲ FIGURE 4.2 Light refraction and image magnification by a convex glass lens. (a) Light passing through a lens refracts (bends) because light rays slow down as they enter the glass. Light at the leading edge of a beam that strikes the glass at an angle slows first. (b) A convex lens focuses light on a focal point. The image is enlarged and inverted as light rays pass the focal point and spread apart. Microscopy apart as they travel past the focal point and produce an enlarged, inverted image (FIGURE 4.2b). The degree to which the image is enlarged depends on the thickness of the lens, its curvature, and the speed of light through its substance. Microscopists could combine lenses to obtain an image magnified millions of times, but the image would be so faint and blurry that it would be useless. Such magnification is said to be empty magnification. The properties that determine the clarity of an image, which in turn determines the useful magnification of a microscope, are resolution and contrast. the ability to distinguish two objects that are close to one another. Modern microscopes have fivefold better resolution than van Leeuwenhoek’s; they can distinguish objects as close together as 0.2 mm. FIGURE 4.3 illustrates the size of various objects that can be resolved by the unaided human eye and by various types of microscopes. Why do modern microscopes have better resolution than van Leeuwenhoek’s microscopes? A principle of microscopy is that resolution is dependent on (1) the wavelength of the electromagnetic radiation and (2) the numerical aperture of the lens, which refers to the ability of a lens to gather light. Resolution may be calculated using the following formula: Resolution Resolution, also called resolving power, is the ability to distinguish two points that are close together. An optometrist’s eye chart is a test of resolution at a distance of 20 feet (6.1 m). Van Leeuwenhoek’s microscopes had a resolving power of about 1 mm; that is, he could distinguish objects if they were more than about 1 mm apart, whereas objects closer together than 1 mm appeared as a single object. The better the resolution, the better Diameter of DNA Ribosomes Atoms H Proteins 0.61 * wavelength numerical aperture The resolution of today’s microscopes is greater than that of van Leeuwenhoek’s microscopes because modern microscopes use shorter wavelength radiation, such as blue light or electron beams, and because they have lenses with larger numerical apertures. Flea Large protozoan (Euglena) Chloroplasts Chicken egg N C O H Amino acids 0.01 nm resolution = Typical bacteria and archaea Viruses 0.1 nm Mitochondrion 10 nm 1 nm 100 nm 1 µm 10 µm Human red blood cell 100 µm 1 mm 10 mm 100 mm Scanning tunneling microscope (STM) 0.01 nm–10 nm Transmission electron microscope (TEM) 0.078 nm–100 µm Unaided human eye 200 µm– Scanning electron microscope (SEM) 0.4 nm–1 mm Atomic force microscope (AFM) 1 nm–10 nm Compound light microscope (LM) 200 nm–10 mm ▲ FIGURE 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of various types of microscopes. 99 1m 100 CHAPTER 4 Microscopy, Staining, and Classification Contrast Contrast refers to differences in intensity between two objects or between an object and its background. Contrast is important in determining resolution. For example, although you can easily distinguish two golf balls lying side by side on a putting green 15 m away, at that distance it is much more difficult to distinguish them if they are lying on a white towel. Most microorganisms are colorless and have very little contrast whether one uses light or electrons. One way to increase the contrast between microorganisms and their background is to stain them. Stains and staining techniques are covered later in the chapter. As we will see, the use of light that is in phase—that is, in which all of the waves’ crests and troughs are aligned—can also enhance contrast. MICRO CHECK 3. What term best describes the ability to distinguish two separate points that are close together when observing objects through a microscope? Light Microscopy L E A R N I N G O U TCO M E S Describe the difference between simple and compound microscopes. 4.9 Compare and contrast bright-field microscopy, dark-field microscopy, and phase microscopy. 4.10 Compare and contrast fluorescence and confocal microscopes. 4.8 Several classes of microscopes use various types of light to examine microscopic specimens. The most common microscopes are bright-field microscopes, in which the background (or field) is illuminated. In dark-field microscopes, the specimen is made to appear light against a dark background. Phase microscopes use the alignment or misalignment of light waves to achieve the desired contrast between a living specimen and its background. Fluorescence microscopes use invisible ultraviolet light to cause specimens to radiate visible light, a phenomenon called fluorescence. Microscopes that use lasers to illuminate fluorescent chemicals in a thin plane of a specimen are called confocal microscopes. Next, we examine each of these kinds of light microscope in turn. Bright-Field Microscopes There are two basic types of bright-field microscopes: simple microscopes and compound microscopes. Simple Microscopes Van Leeuwenhoek first reported his observations of microorganisms using a simple microscope in 1674. A simple microscope, which contains a single magnifying lens, is more similar to a magnifying glass than to a modern microscope (see Figure 1.2). Though van Leeuwenhoek did not invent the microscope, he was the finest lens maker of his day and produced microscopes of exceptional quality. They were capable of approximately 300* magnification and achieved excellent clarity, far surpassing other microscopes of his time. Compound Microscopes Simple microscopes have been replaced in modern laboratories by compound microscopes. A compound microscope uses a series of lenses for magnification (FIGURE 4.4a). Many scientists, including Galileo Galilei (1564–1642), made compound microscopes as early as 1590, but it was not until about 1830 that scientists developed compound microscopes that exceeded the clarity and magnification of van Leeuwenhoek’s simple microscope. In a basic compound microscope, magnification is achieved as light rays pass through a specimen and into an objective lens, which is the lens immediately above the object being magnified (FIGURE 4.4b). An objective lens is really a series of lenses that not only create a magnified image but also are engineered to reduce aberrations in the shape and color of the image. Most light microscopes used in biology have three or four objective lenses mounted on a revolving nosepiece. The objective lenses on a typical microscope are scanning objective lens (4*), low-power objective lens (10*), high-power lens or high dry objective lens (40*), and oil immersion objective lens (100*). An oil immersion lens increases not only magnification but also resolution. As we have seen, light refracts as it travels from air into glass and also from glass into air; therefore, some of the light passing out of a glass slide is bent so much that it bypasses the lens (FIGURE 4.5a). Placing immersion oil (historically, cedarwood oil; today, more commonly a synthetic oil) between the slide and an oil immersion objective lens enables the lens to capture this light because light travels through immersion oil at the same speed as through glass. Because light is traveling at a uniform speed through the slide, the immersion oil, and the glass lens, it does not refract (FIGURE 4.5b). Immersion oil increases the numerical aperture, which increases resolution because more light rays are gathered into the lens to produce the image. Obviously, the space between the slide and the lens can be filled with oil only if the distance between the lens and the specimen, called the working distance, is small. An objective lens bends the light rays, which then pass up through one or two ocular lenses, which are the lenses closest to the eyes. Microscopes with a single ocular lens are monocular, and those with two are binocular. Ocular lenses magnify the image created by the objective lens, typically another 10*. The total magnification of a compound microscope is determined by multiplying the magnification of the objective lens by the magnification of the ocular lens. Thus, total magnification using a 10* ocular lens and a 10* low-power objective lens is 100*. Using the same ocular and a 100* oil immersion objective produces 1000* magnification. Some light microscopes, using higher-magnification oil immersion objective lenses and ocular lenses, can achieve 2000* magnification, but this is the limit of useful magnification for light microscopes because their resolution is restricted by the wavelength of visible light. Modern compound microscopes also have a condenser lens (or lenses), which directs light through the specimen, as well as one or more mirrors or prisms that deflect the path of the light rays from an objective lens to the ocular lens (see Figure 4.4b). Some microscopes have mirrors or prisms that direct light to a Microscopy 101 Line of vision Ocular lens Remagnifies the image formed by the objective lens Body Transmits the image from the objective lens to the ocular lens using prisms Arm Ocular lens Objective lenses Primary lenses that magnify the specimen Body Path of light Prism Objective lenses Stage Holds the microscope slide in position Specimen Condenser Focuses light through specimen Diaphragm Controls the amount of light entering the condenser Illuminator Light source Condenser lenses Illuminator Coarse focusing knob Moves the stage up and down to focus the image Fine focusing knob Slightly moves the stage up and down for focusing Base (a) (b) ▲ FIGURE 4.4 A bright-field compound light microscope. (a) The parts of a compound microscope, which uses a series of lenses to produce an image at up to 2000* magnification. (b) The path of light in a compound microscope; light travels from bottom to top. Why can’t light microscopes produce clear images that are magnified 10,000 *? Figure 4.4 Although it is possible for a light microscope to produce an image that is magnified 10,000 *, magnification above 2000 * is empty magnification because pairs of objects in the specimen are too close together to resolve with even the shortest-wavelength (blue) light. camera through a special tube. A photograph of such a microscopic image is called a light micrograph (LM); micrograph refers to any microscopic image. Dark-Field Microscopes Pale objects are often better observed with dark-field microscopes. These microscopes prevent light from directly entering the objective lens. Instead, light rays are reflected inside the condenser, so that they pass into the slide at such an oblique angle that they completely miss the objective lens. Only light rays scattered by a specimen can enter the objective lens and be seen, so the specimen appears light against a dark background—the field. Dark-field microscopy increases contrast and enables observation of more details than are visible in bright-field microscopy. Dark-field microscopes are especially useful for examining small or colorless cells. Phase Microscopes Scientists use phase microscopes to examine living microorganisms or specimens that would be damaged or altered by attaching them to slides or staining them. Basically, phase microscopes treat one set of light rays differently from another set of light rays. When a phase microscope’s lenses bring the two sets of rays together, contrast is created. There are two types of phase microscopes: phase-contrast and differential interference contrast microscopes. Phase-Contrast Microscopes The simplest phase microscopes, phase-contrast microscopes, produce sharply defined images in which fine structures can be seen in living cells. These microscopes are particularly useful for observing cilia and flagella. Differential Interference Contrast Microscopes Differential interference contrast microscopes (also called Nomarski2 microscopes) significantly increase contrast and give an image a dramatic three-dimensional and shadowed appearance, almost as though light were striking the specimen from one side. This technique can also produce unnatural colors, which enhance contrast. 2 After the French physicist Georges Nomarski, who invented the differential interference contrast microscope. 102 CHAPTER 4 Microscopy, Staining, and Classification b FIGURE 4.6 Four Nucleus Microscope objective Lenses Refracted light rays lost to lens Bacterium Glass cover slip Slide Specimen (a) Bright field LM (b) Dark field LM (c) Phase contrast LM (d) Differential interference contrast (Nomarski) LM 20 μm Light source (a) Without immersion oil Microscope objective Lenses More light enters lens Immersion oil Glass cover slip 20 μm Slide Light source (b) With immersion oil ▲ FIGURE 4.5 The effect of immersion oil on resolution. (a) Without immersion oil, light is refracted as it moves from the glass cover slip into the air. Part of the scattered light misses the objective lens. (b) With immersion oil. Because light travels through the oil at the same speed as it does through glass, no light is refracted as it leaves the specimen and more light enters the lens, which increases resolution. 20 μm FIGURE 4.6 illustrates the differences that can be observed in a single specimen when viewed using four different types of light microscopy. Fluorescence Microscopes Molecules that absorb energy from invisible radiation (such as ultraviolet light) and then radiate the energy back as a longer, visible wavelength are said to be fluorescent. Fluorescence microscopes use an ultraviolet (UV) light source to fluoresce objects. UV light increases resolution because it has a shorter wavelength than visible light, and contrast is improved because fluorescing structures are visible against a nonfluorescing, black background. 20 μm kinds of light microscopy. All four photos show the same human cheek cell and bacteria. (a) Brightfield microscopy reveals some internal structures. (b) Darkfield microscopy increases contrast between some internal structures and between the edges of the cell and the surrounding medium. (c) Phase-contrast microscopy provides greater resolution of internal structures. (d) Differential interference contrast (Nomarski) microscopy produces a threedimensional effect. 103 Microscopy Some cells—for example, the pathogen Pseudomonas aeruginosa (soo-dō-mō′nas ā-roo-ji-nō′să)—and some cellular molecules (such as chlorophyll in photosynthetic organisms) are naturally fluorescent. Other cells and cellular structures can be stained with fluorescent dyes. When these dyes are bombarded with ultraviolet light, they emit visible light and show up as bright orange, green, yellow, or other colors (see Figure 3.34b). Some fluorescent dyes are specific for certain cells. For example, the dye fluorescein isothiocyanate attaches to cells of Bacillus anthracis (ba-sil′ŭs an-thrā′sis), the causative agent of anthrax, and appears apple green when viewed in a fluorescence microscope. Another fluorescent dye, auramine O, stains Mycobacterium tuberculosis (mī′kō-bak-tēr′ē-ŭm too-ber-kyū-lō′sis) (FIGURE 4.7). Fluorescence microscopy is also used in a process called immunofluorescence. First, fluorescent dyes are chemically linked to Y-shaped immune system proteins called antibodies (FIGURE 4.8a). Antibodies will bind specifically to complementary-shaped antigens, which are portions of molecules that are present, for example, on the surface of microbial cells. When viewed under UV light, a microbial specimen that has bound dye-tagged antibodies becomes visible (FIGURE 4.8b). In addition to identifying pathogens, including those that cause syphilis, rabies, and Lyme disease, scientists use immunofluorescence to locate and make visible a variety of proteins of interest. Antibodies Bacterium Fluorescent dye Antibodies carrying dye Cell-surface antigens Bacterial cell with bound antibodies carrying dye (a) Confocal Microscopes Confocal3 microscopes also use fluorescent dyes or fluorescent antibodies, but these microscopes use ultraviolet lasers to illuminate the fluorescent chemicals in only a single plane that is no thicker than 1.0 mm; the rest of the specimen remains dark and out of focus. Visible light emitted by the dyes passes through a pinhole aperture that helps eliminate blurring that can occur with other types of microscopes and increases (b) LM 2 μm ▲ FIGURE 4.8 Immunofluorescence. (a) After a fluorescent dye is covalently linked to an antibody, the dye-antibody combination binds to the antibody’s target, making the target visible under fluorescent microscopy. (b) Immunofluorescent staining of Yersinia pestis, the causative agent of bubonic plague. The bacteria are brightly colored against a dark background. (a) LM 10 μm (b) LM 10 μm ▲ FIGURE 4.7 Fluorescence microscopy. Fluorescent chemicals absorb invisible short-wavelength radiation and emit visible (longerwavelength) radiation. (a) When viewed under normal illumination, Mycobacterium tuberculosis cells stained with the fluorescent dye auramine O are invisible amid the mucus and debris in a sputum smear. (b) When the same smear is viewed under UV light, the bacteria fluoresce and are clearly visible. 3 From coinciding focal points of (laser) light. resolution by up to 40%. Each image from a confocal microscope is thus an “optical slice” through the specimen, as if it had been thinly cut. Once individual images are digitized, a computer is used to construct a three-dimensional representation, which can be rotated and viewed from any direction (FIGURE 4.9 on the next page). Confocal microscopes have been particularly useful for examining the relationships among various organisms within complex microbial communities called biofilms. Regular light microscopy cannot produce clear images of structures within a living biofilm, and removing surface layers from a biofilm Play Light Microscopy would change the dynamics of @ Mastering Microbiology a biofilm community. 104 CHAPTER 4 Microscopy, Staining, and Classification Polysaccharide matrix Sand grain Generally, researchers use electron microscopes to magnify objects 10,000* to 100,000*, although millions of times magnification with good resolution is possible. Electron microscopes provide detailed views of the smallest bacteria, viruses, internal cellular structures, and even molecules and large atoms. Cellular structures that can be seen only by using electron microscopy are referred to as a cell’s ultrastructure. Ultrastructural details cannot be made visible by light microscopy because they are too small to be resolved. There are two general types of electron microscopes: transmission electron microscopes and scanning electron microscopes. Bacteria Transmission Electron Microscopes CM 5 µm ▲ FIGURE 4.9 Confocal microscopy. A laser is used to stimulate fluorescent dyes to produce single “optical slices.” A computer then constructs a three-dimensional representation—here, a marine biofilm. MICRO CHECK 4. What type of microscope is essentially a magnifying glass? 5. What type of light microscope uses a series of lenses that together contribute to the total magnification of a specimen? 6. Which style of microscope, used in virtually every school science lab, uses a white light source below the specimen to illuminate the specimen? Electron Microscopy L E A R N I N G O U TCO M E 4.11 Contrast transmission electron microscopes with scanning electron microscopes in terms of how they work, the images they produce, and the advantages of each. Even with the most expensive phase microscope using the best oil immersion lens with the highest numerical aperture, resolution is still limited by the wavelength of visible light. Because the shortest visible radiation (violet) has a wavelength of about 400 nm, structures closer together than about 200 nm cannot be distinguished using even the best light microscope. By contrast, electrons traveling as waves can have wavelengths of 0.001 nm, which is one hundred-thousandth the wavelength of visible light. The resolving power of electron microscopes is therefore much greater than that of light microscopes, and with greater resolving power comes the possibility of greater magnification. A transmission electron microscope (TEM) generates a beam of electrons that ultimately produces an image on a fluorescent screen (FIGURE 4.10a). The path of electrons is similar to the path of light in a light microscope. From their source, the electrons pass through the specimen, through magnetic fields (instead of glass lenses) that manipulate and focus the beam, and then onto a fluorescent screen that changes some of their energy into visible light (FIGURE 4.10b). Dense areas of the specimen block electrons, resulting in a dark area on the screen. In regions where the specimen is less dense, the screen fluoresces more brightly. As with light microscopy, contrast and resolution can be enhanced through the use of electron-dense stains, which are discussed later. The brightness of each region of the screen corresponds to the number of electrons striking it. Therefore, the image on the screen is composed of light and dark areas. The screen can be folded out of the way to enable the electrons to strike a photographic film, located in the base of the microscope. Prints made from the film are called transmission electron micrographs or TEM images (FIGURE 4.10c). Photographers often colorize such images to emphasize certain features. Matter, including air, absorbs electrons, so the specimen must be very thin, and the column of a transmission electron microscope must be a vacuum. When dealing with thicker specimens, technicians dehydrate them, embed them in plastic, and cut them to a thickness of about 100 nm with a diamond or glass knife mounted in a slicing machine called an ultramicrotome so that the electron beam can pass through them. Because the vacuum and slicing of the specimens are required, transmission electron microscopes cannot be used to study living organisms. Scanning Electron Microscopes A scanning electron microscope (SEM) also uses magnetic fields within a vacuum tube to manipulate a beam of electrons; however, rather than passing electrons through a specimen, a SEM rapidly focuses the electrons back and forth across a specimen’s surface, which has previously been coated with a metal such as platinum or gold. The electrons knock other electrons off the surface of the coated specimen, and these scattered electrons pass through a detector and a photomultiplier, producing an amplified signal that is displayed on a monitor. Typically, scanning microscopes are used to magnify up to 10,000* with a resolution of about 20 nm. One advantage of scanning microscopy over transmission microscopy is that whole specimens can be observed because sectioning is not required. Scanning electron micrographs can be beautifully realistic and appear three-dimensional (FIGURE 4.11). 105 Microscopy Light microscope (upside down) Column of transmission electron microscope Lamp Electron gun Condenser lens Condenser lens (magnet) Specimen Specimen Objective lens Objective lens (magnet) Eyepiece Projector lens (magnet) Final image seen by eye Final image on fluorescent screen (a) (b) (c) TEM μm ▲ FIGURE 4.10 A transmission electron microscope (TEM). (a) The path of electrons through a TEM, as compared to the path of light through a light microscope (at left, drawn upside down to facilitate the comparison). (b) TEMs are much larger than light microscopes. (c) Transmission electron image of a bacterium, Bacillus subtilis. A transmission electron micrograph reveals much internal detail not visible by light microscopy. Why must air be evacuated from the column of an electron microscope? Figure 4.10 Air would absorb electrons, so there would be no radiation to produce an image. Two disadvantages of a scanning electron microscope are that it magnifies only the external surface of a specimen and that, like TEM, it requires a vacuum Play Electron Microscopy and thus can examine only @ Mastering Microbiology dead organisms. Probe Microscopy (a) Arachnoidiscus L E A R N I N G O U TCO M E SEM μm 4.12 (b) Aspergillus SEM 0 μm Describe two variations of probe microscopes. A recent advance in microscopy utilizes minuscule, pointed electronic probes to magnify more than 100,000,000*. There are two variations of probe microscopes: scanning tunneling microscopes and atomic force microscopes. Scanning Tunneling Microscopes (c) Paramecium, SEM 20 μm a unicellular protozoan on top of filamentous bacteria. (d) Streptococcus SEM ▲ FIGURE 4.11 Scanning electron microscope (SEM) images. 2 μm (a) Arachnoidiscus, a marine diatom (alga). (b) Aspergillus, a fungus. (c) Paramecium, a unicellular “animal” on top of rod-shaped bacteria. (d) Streptococcus, a bacterium. A scanning tunneling microscope (STM) passes a metallic probe, sharpened to end in a single atom, back and forth across and slightly above the surface of a specimen. Rather than scattering a beam of electrons into a detector, as in scanning electron microscopy, a scanning tunneling microscope measures the flow of electrons to and from the probe and the specimen’s surface. The amount of electron flow, called a tunneling current, is directly proportional to the distance from the probe to the specimen’s surface. A scanning tunneling microscope can measure distances as small as 0.01 nm and reveal three-dimensional details on the surface of a specimen at the atomic level (FIGURE 4.12a). A requirement for scanning tunneling microscopy is that the specimen be electrically conductive. 106 CHAPTER 4 Microscopy, Staining, and Classification DNA Enzyme and its background. Electron microscopy requires that specimens be treated with stains or coatings to enhance contrast. In this section, we examine how scientists prepare specimens for staining and how stains work, and we consider seven kinds of stains used for light microscopy. We conclude with a look at staining for electron microscopy. Preparing Specimens for Staining L E A R N I N G O U TCO M E (a) STM 1 nm (b) AFM 15 nm 4.13 ▲ FIGURE 4.12 Probe microscopy. (a) Scanning tunneling micro- scopes reveal surface detail; in this case, three turns of a DNA double helix. (b) Plasmid DNA being digested by an enzyme, viewed through atomic force microscopy. Atomic Force Microscopes An atomic force microscope (AFM) also uses a pointed probe, but it traverses the tip of the probe lightly on the surface of the specimen rather than at a distance. This might be likened to the way a person reads Braille. Deflection of a laser beam aimed at the probe’s tip measures vertical movements, which when translated by a computer reveals the three-dimensional atomic topography. Unlike tunneling microscopes, atomic force microscopes can magnify specimens that do not conduct electrons. They can also magnify living specimens because neither an electron beam nor a vacuum is required (FIGURE 4.12b). Researchers have used atomic force microscopes to magnify the surfaces of bacteria, viruses, proteins, and amino acids. Recent studies using them have examined single living bacteria in three dimensions as they grow and divide. TABLE 4.2 on p. 107 summarizes the features of the various types of microscopes. MICRO CHECK 7. What sort of electron microscope is used in order to see the internal details of cells? TELL ME WHY Why is magnification high and color absent in an unretouched electron micrograph? Staining Earlier we discussed the difficulty of resolving two distant white golf balls viewed against a white background. If the balls were painted black, they could be distinguished more readily from the background and from one another. This illustrates why staining increases contrast and resolution. Most microorganisms are colorless and difficult to view with bright-field microscopes. Microscopists use stains to make microorganisms and their parts more visible because stains increase contrast between structures and between a specimen Explain the purposes of a smear, heat fixation, and chemical fixation in the preparation of a specimen for microscopic viewing. Many investigations of microorganisms, especially those seeking to identify pathogens, begin with light microscopic observation of stained specimens. Staining simply means coloring specimens with stains, which are also called dyes. Before microbiologists stain microorganisms, they must place them on and then firmly attach them to a microscope slide. Typically, this involves making a smear and fixing it to the slide (FIGURE 4.13). If the organisms are growing in a liquid, the microscopist spreads a small drop of the broth across the surface of the slide. If the organisms are growing on a solid surface, such as an agar plate, they are mixed into a small drop of water on the slide. Either way, the thin film of organisms on the slide is called a smear. The smear is air-dried completely and then attached or fixed to the surface of the slide. In heat fixation, developed more than a hundred years ago by Robert Koch, the slide is gently heated by passing the slide, smear up, through a flame. Alternatively, chemical fixation involves applying a chemical such as methyl alcohol to the smear for one minute. Desiccation (drying) and fixation kill the microorganisms, attach them firmly to the slide, and generally preserve their shape and size. It is important to smear and fix specimens properly so that they are not lost during staining. Specimens prepared for electron microscopy must be dry because water vapor from a wet specimen would stop an electron beam. As we have seen, transmission electron microscopy requires that the desiccated sample also be sliced very thin, generally before staining. Specimens for scanning electron microscopy are coated, not stained. MICRO CHECK 8. What is the process by which microbes are air dried and “attached” to a microscope slide using flame? Principles of Staining L E A R N I N G O U TCO M E 4.14 Describe the uses of acidic and basic dyes, mentioning ionic bonding and pH. Dyes used as microbiological stains for light microscopy are usually salts. A salt is composed of a positively charged cation and a negatively charged anion. At least one of the two ions in Staining TABLE 4.2 107 Comparison of Types of Microscopes Type of Microscope Typical Image Description of Image Special Features Typical Uses Light Microscopes Useful magnification 1* to 2000*; resolution to 200 nm Use visible light; shorter, blue wavelengths provide better resolution Bright-field Colored or clear specimen against bright background Simple to use; relatively inexpensive; stained specimens often required To observe killed stained specimens and naturally colored live ones; also used to count microorganisms Dark-field Bright specimen against dark background Uses a special filter in the condenser that prevents light from directly passing through a specimen; only light scattered by the specimen is visible To observe living, colorless, unstained organisms Phase-contrast Specimen has light and dark areas Uses a special condenser that splits a polarized light beam into two beams, one of which passes through the specimen, and one of which bypasses the specimen; the beams are then rejoined before entering the oculars; contrast in the image results from the interactions of the two beams To observe internal structures of living microbes Differential interference contrast (Nomarski) Image appears three-dimensional Uses two separate beams instead of a split beam; false color and a threedimensional effect result from interactions of light beams and lenses; no staining required To observe internal structures of living microbes Fluorescence Brightly colored fluorescent structures against dark background An ultraviolet light source causes fluorescent natural chemicals or dyes to emit visible light To localize specific chemicals or structures; used as an accurate and quick diagnostic tool for detection of pathogens Confocal Single plane of structures or cells that have been specifically stained with fluorescent dyes Uses a laser to fluoresce only one plane of the specimen at a time Detailed observation of structures of cells within communities Electron Microscopes Typical magnification 1000* to 100,000*; resolution to 0.001 nm Use electrons traveling as waves with short wavelengths; require specimens to be in a vacuum, so cannot be used to examine living microbes Transmission Monotone, two-dimensional, highly magnified images; may be color enhanced Produces two-dimensional image of ultrastructure of cells To observe internal ultrastructural detail of cells and observation of viruses and small bacteria Scanning Monotone, three-dimensional, surface images; may be color enhanced Produces three-dimensional view of the surface of microbes and cellular structures To observe the surface details of structures Probe Microscopes Magnification greater than 100,000,000* with resolving power greater than that of electron microscopes Uses microscopic probes that move over the surface of a specimen Scanning tunneling Individual molecules and atoms visible Measures the flow of electrical current between the tip of a probe and the specimen to produce an image of the surface at atomic level To observe the surface of objects; provide extremely fine detail, high magnification, and great resolution Atomic force Individual molecules and atoms visible Measures the deflection of a laser beam aimed at the tip of a probe that travels across the surface of the specimen To observe living specimens at the molecular and atomic levels 108 CHAPTER 4 Microscopy, Staining, and Classification Simple stains are composed of a single basic dye, such as crystal violet, safranin, or methylene blue. They are “simple” because they involve no more than soaking the smear in the dye for 30–60 seconds and then rinsing off the slide with water. (A properly fixed specimen will remain attached to the slide despite this treatment.) After carefully blotting the slide dry, the microbiologist observes the smear under the microscope. Simple stains are used to determine size, shape, and arrangement of cells (FIGURE 4.14). Spread culture in thin film over slide Air-dry Differential Stains Pass slide through flame to fix it Most stains used in microbiology are differential stains, which use more than one dye so that different cells, chemicals, or structures can be distinguished when microscopically examined. Common differential stains are the Gram stain, the acid-fast stain, the endospore stain, Gomori methenamine silver stain, and hematoxylin and eosin stain. Gram Stain ▲ FIGURE 4.13 Preparing a specimen for staining. Microorganisms are spread in liquid across the surface of a slide using a circular motion. After drying in the air, the smear is passed through the flame of a Bunsen burner to fix the cells to the glass. Alternatively, chemical fixation can be used. Why must a smear be fixed to the slide? Figure 4.13 Fixation causes the specimen to adhere to the glass so that it does not easily wash off during staining. the molecular makeup of dyes is colored; this colored portion of a dye is known as the chromophore. Chromophores bind to chemicals via covalent, ionic, or hydrogen bonds. For example, methylene blue chloride is composed of a cationic chromophore, methylene blue, and a chloride anion. Because methylene blue is positively charged, it ionically bonds to negatively charged molecules in cells, including DNA and many proteins. In contrast, anionic dyes, for example, eosin, bind to positively charged molecules, such as some amino acids. Anionic chromophores are also called acidic dyes because they stain alkaline structures and work best in acidic (low pH) environments. Positively charged, cationic chromophores are called basic dyes because they combine with and stain acidic structures; further, they work best under basic (higher pH) conditions. In microbiology, basic dyes are used more commonly than acidic dyes because most cells are negatively charged. Acidic dyes are used in negative staining, which is discussed shortly. Some stains do not form bonds with cellular chemicals but rather function because of their solubility characteristics. For example, Sudan black selectively stains membranes because it is lipid soluble and accumulates in phospholipid bilayers. Simple Stains L E A R N I N G O U TCO M E 4.15 Describe simple stains, four kinds of differential stains, and two kinds of special stains. In 1884, the Danish scientist Hans Christian Gram (1853–1938) developed the most frequently used differential stain, which now bears his name. The Gram stain differentiates between two large groups of microorganisms: purple-staining Gram-positive cells and pink-staining Gram-negative cells. These cells differ significantly in the chemical and physical structures of their cell walls (see Figure 3.16). Typically, a Gram stain is the first step a medical laboratory scientist performs to identify bacterial pathogens. Let’s examine the Gram staining procedure as it was originally developed and as it is typically performed today. For the purposes of our discussion, we’ll assume that a smear has been made on a slide and heat fixed and that the smear contains both Gram-positive and Gram-negative colorless bacteria. (a) LM 0 μm (b) LM 0 μm ▲ FIGURE 4.14 Simple stains. Simply and quickly performed, simple stains increase contrast and allow determination of size, shape, and arrangement of cells. (a) Unstained Escherichia coli and Staphylococcus aureus. (b) Same mixture stained with crystal violet. Note that all cells, no matter their type, stain almost the same color with a simple stain because only one dye is used. 109 Staining The classical Gram staining procedure involves the following four steps (FIGURE 4.15): 1 2 3 4 Flood the smear with the basic dye crystal violet for 1 minute, and then rinse with water. Crystal violet, which is called the primary stain, colors all cells. Flood the smear with an iodine solution for 1 minute, and then rinse with water. Iodine is a mordant, a substance that binds to a dye and makes it less soluble. After this step, all cells remain purple. Rinse the smear with a solution of ethanol and acetone for 10–30 seconds, and then rinse with water. This solution, which acts as a decolorizing agent, breaks down the thin cell wall of Gram-negative cells, allowing the stain and mordant to be washed away; these Gram-negative cells are now colorless. Gram-positive cells, with their thicker cell walls, remain purple. Flood the smear with safranin for 1 minute, and then rinse with water. This red counterstain provides a contrasting color to the primary stain. Although all types of cells may absorb safranin, the resulting pink color is masked by the darker purple dye already in Grampositive cells. After this step, Gram-negative cells now appear pink, whereas Gram-positive cells remain purple. After the final step, the slide is blotted dry in preparation for light microscopy. The Gram procedure works best with young cells. Older Gram-positive cells bleach more easily than younger cells and can therefore appear pink, which makes them appear to be Gram-negative cells. Therefore, smears for Gram staining should come from freshly grown bacteria. Microscopists have developed minor variations on Gram’s original procedure. For example, 95% ethanol may be used to decolorize instead of Gram’s ethanol-acetone mixture. In a threestep variation, safranin dissolved in ethanol simultaneously decolorizes and counterstains. Acid-Fast Stain The acid-fast stain is another important differential stain because it stains cells of the genera Mycobacterium and Nocardia (nō-kar′dē-ă), which cause many human diseases, including tuberculosis, leprosy, and other lung and skin infections. Cells of these bacteria have large amounts of waxy lipid in their cell walls, so they do not readily stain with the water-soluble dyes used in Gram staining. Microbiological laboratories can use a variation of the acidfast stain developed by Franz Ziehl (1857–1926) and Friedrich Neelsen (1854–1894) in 1883. Their procedure is as follows: 1. Cover the smear with a small piece of tissue paper to retain the dye during the procedure. 2. Flood the slide with the red primary stain, carbolfuchsin, for several minutes while warming it over steaming water. In this procedure, heat is used to drive the stain through the waxy wall and into the cell, where it remains trapped. 3. Remove the tissue paper, cool the slide, and then decolorize the smear by rinsing it with a solution of hydrochloric acid (pH 6 1.0) and alcohol. The bleaching action of 1 Slide is flooded with crystal violet for 1 min, then rinsed with water. Result: All cells are stained purple. LM 5 μm 2 Slide is flooded with iodine for 1 min, then rinsed with water. Result: Iodine acts as a mordant; all cells remain purple. LM 5 μm 3 Slide is rinsed with solution of ethanol and acetone for 10–30 sec, then rinsed with water. Result: Smear is decolorized; Gram-positive cells remain purple, but Gram-negative cells are now colorless. LM 5 μm 4 Slide is flooded with safranin for 1 min, then rinsed with water and blotted dry. Result: Gram-positive cells remain purple, Gram-negative cells are pink. LM 5 μm ▲ FIGURE 4.15 The Gram staining procedure. A specimen is smeared and fixed to a slide. The classical procedure consists of four steps. Gram-positive cells (in this case, Bacillus cereus) remain purple throughout the procedure; Gram-negative cells (here, Escherichia coli) end up pink. acid-alcohol removes color from both non-acid-fast cells and the background. Acid-fast cells retain their red color because the acid cannot penetrate the waxy wall. The name of the procedure is derived from this step; that is, the cells are colorfast in acid. 4. Counterstain with methylene blue, which stains only bleached, non-acid-fast cells. 110 CHAPTER 4 Microscopy, Staining, and Classification The Ziehl-Neelsen acid-fast staining procedure results in pink acid-fast cells, which can be differentiated from blue non-acid-fast cells, including human cells (FIGURE 4.16). The presence of acid-fast bacilli (AFBs) in sputum is indicative of mycobacterial infection. Endospore Stain Histological Stains Laboratory technicians use two popular stains to stain histological specimens, that is, tissue samples. Gomori4 methenamine silver (GMS) stain is commonly used to screen for the presence of fungi and the locations of carbohydrates in tissues. Hematoxylin and eosin (HE) stain, which involves applying the basic dye hematoxylin and the acidic dye eosin, is used to delineate many features of histological specimens, such as the presence of cancer cells. LM ▲ FIGURE 4.17 Schaeffer-Fulton endospore stain of Bacillus anthracis. The nearly impermeable spore wall retains the green dye during decolorization. Vegetative cells, which lack spores, pick up the counterstain and appear red. Why don’t the spores stain red as well? Special Stains Special stains are simple stains designed to reveal specific microbial structures. Three types of special stains are negative stains, flagellar stains, and fluorescent stains (which we already discussed in the section on fluorescence microscopy). Negative (Capsule) Stain 5 μm as these rod-shaped Mycobacterium bovis cells stain pink or red. Nonacid-fast cells—in this case, Staphylococcus—stain blue. Why isn’t the Gram stain utilized to stain Mycobacterium? Figure 4.16 Cell walls of Mycobacterium are composed of waxy materials that repel the water-based dyes of the Gram stain. Named for George Gomori, a noted Hungarian American histologist. 5 μm Most dyes used to stain bacterial cells, such as crystal violet, methylene blue, malachite green, and safranin, are basic dyes. These dyes stain cells by attaching to negatively charged molecules within them. Acidic dyes, by contrast, are repulsed by the negative charges on the surface of cells and, therefore, do not stain them. Such stains are called negative stains because they stain the background and leave cells colorless. Eosin and nigrosin are examples of acidic dyes used for negative staining. Negative stains are used primarily to reveal the presence of negatively charged bacterial capsules. Therefore, they are also called capsule stains. Encapsulated cells appear to have a halo surrounding them (FIGURE 4.18). ▲ FIGURE 4.16 Ziehl-Neelsen acid-fast stain. Acid-fast cells such 4 LM Figure 4.17 Heat from steam is used to drive the green primary stain into the endospores. Counterstaining is performed at room temperature, and the thick, impermeable walls of the endospores resist the counterstain. Some bacteria—notably those of the genera Bacillus and Clostridium (klos-trid′ē-ŭm), which contain species that cause such diseases as anthrax, gangrene, and tetanus—produce endospores. These dormant, highly resistant cells form inside the cytoplasm of the bacteria and can survive environmental extremes such as desiccation, heat, and harmful chemicals. Endospores cannot be stained by normal staining procedures because their walls are practically impermeable at room temperature. The Schaeffer-Fulton endospore stain uses heat to drive the primary stain, malachite green, into the endospore. After cooling, the slide is decolorized with water and counterstained with safranin. This staining procedure results in green-stained endospores and red-colored vegetative cells (FIGURE 4.17). Flagellar Stain Bacterial flagella are extremely thin and thus normally invisible with light microscopy, but their presence, number, and arrangement are important in identifying some species, including some pathogens. Flagellar stains, such as pararosaniline and carbolfuchsin, in combination with mordants—chemicals that combine with a dye and make it less soluble and therefore affix it in a material—are applied in a series of steps. Flagellar stains bind to flagella, increasing their diameter and colorizing them, which increases contrast and makes the flagella visible (FIGURE 4.19). 111 Staining Flagella Bacterium Capsule Background stain LM μm ▲ FIGURE 4.18 Negative (capsule) stain of Klebsiella pneumoniae. Notice that the acidic dye stains the background and does not penetrate the capsule. LM ▲ FIGURE 4.19 Flagellar stain of Proteus vulgaris. Various bacteria have different numbers and arrangements of flagella, features that might be important in identifying some species. How can the flagellar arrangement shown here be described? Staining for Electron Microscopy L E A R N I N G O U TCO M E 4.16 Explain how stains used for electron microscopy differ from those used for light microscopy. TABLE 4.3 5 μm Figure 4.19 Peritrichous Stains used for light microscopy are summarized in TABLE 4.3. Beneficial Microbes: Glowing Viruses (on p. 113) illustrates a unique kind of stain that uses fluorescent dye attached to viruses to stain specific strains of bacteria. Laboratory technicians increase contrast and resolution for transmission electron microscopy by using stains, just as they do for light microscopy. However, stains used for transmission electron microscopy are not colored dyes but instead chemicals Some Stains Used for Light Microscopy Type of Stain Examples Results Simple Stains (use a single dye) Crystal violet Uniform purple stain Methylene blue Uniform blue stain Differential Stains (use two or more dyes to differentiate between cells or structures) Gram stain Gram-positive cells are purple; Gram-negative cells are pink Differentiates Gram-positive and Gram-negative bacteria, which is typically the first step in their identification Ziehl-Neelsen acid-fast stain Pink to red acid-fast cells and blue non-acid-fast cells Distinguishes the genera Mycobacterium and Nocardia from other bacteria Schaeffer-Fulton endospore stain Green endospores and pink to red vegetative cells Highlights the presence of endospores produced by species in the genera Bacillus and Clostridium Negative stain for capsules Background is dark; cells unstained or stained with simple stain Reveals bacterial capsules Flagellar stain Bacterial flagella become visible Allows determination of number and location of bacterial flagella Special Stains Typical Image Representative Uses Reveals size, morphology, and arrangement of cells 112 CHAPTER 4 Microscopy, Staining, and Classification containing atoms of heavy metals, such as lead, osmium, tungsten, and uranium, which absorb electrons. Electron-dense stains may bind to molecules within specimens, or they may stain the background. The latter type of negative staining is used to provide contrast for extremely small specimens, such as viruses and molecules. Stains for electron microscopy can be general in that they stain most objects to some degree, or they may be highly specific. For example, osmium tetroxide (OsO4) has an affinity for lipids and is thus used to enhance the contrast of membranes. Electron-dense stains can also be linked to antibodies to provide an even greater degree of staining specificity because Play Staining antibodies bind only to their @ Mastering Microbiology specific target molecules. MICRO CHECK 9. What is the name of the process that uses a single dye such as crystal violet or methylene blue to color a microscope specimen? 10. Which stain is the most frequently used differential stain in modern microbiology labs, differentiating microorganisms into two distinct groups? 11. What type of chemical used in the staining process does not add color itself but binds to a dye, making the dye less soluble and more likely to be attached to cellular materials? 12. Atoms of what class of elements are used to absorb electrons in electron-dense stains? TELL ME WHY Why is a Gram-negative bacterium colorless but a Grampositive bacterium purple after it is rinsed with decolorizer? Classification and Identification of Microorganisms L E A R N I N G O U TCO M E 4.17 Discuss the purposes of taxonomy. Biologists classify organisms for several reasons: to bring a sense of order and organization to the variety and diversity of living things, to enhance communication, to make predictions about the structure and function of similar organisms, and to uncover and understand potential evolutionary connections. They sort organisms on the basis of mutual similarities into nonoverlapping groups called taxa.5 Taxonomy6 is the science of classifying and naming organisms. Taxonomy consists of classification, which is the assigning of organisms to taxa based on similarities; nomenclature, 5 From Greek taxis, meaning “order.” 6 From taxis and Greek nomos, meaning “rule.” which is concerned with the rules of naming organisms; and identification, which is the practical science of determining that an isolated individual or population belongs to a particular taxon. In this text, we concentrate on classification and identification. Because all members of any given taxon share certain common features, taxonomy enables scientists both to organize large amounts of information about organisms and to make predictions based on knowledge of similar organisms. For example, if one member of a taxon is important in recycling nitrogen in the environment, it is possible that others in the group will play a similar ecological role. Similarly, a clinician might suggest a treatment against one pathogen based on what has been effective against other pathogens in the same taxon. Identification of organisms is an essential part of taxonomy because it enables scientists to communicate effectively and be confident that they are discussing the same organism. Further, identification is often essential for treating groups of diseases, such as meningitis and pneumonia, which can be caused by pathogens as different as fungi, bacteria, and viruses. In this section, we examine the historical basis of taxonomy, consider modern advances in this field, and briefly consider various taxonomic methods. This chapter also presents a general overview of the taxonomy of prokaryotes (which are considered in greater detail in Chapter 11); of animals, protozoa, fungi, and algae (Chapter 12); and of viruses, viroids, and prions (Chapter 13). Linnaeus and Taxonomic Categories L E A R N I N G O U TCO M E S Discuss the difficulties in defining species of microorganisms. 4.19 List the hierarchy of taxa from general to specific. 4.20 Define binomial nomenclature. 4.21 Describe a few modifications of the Linnaean system of taxonomy. 4.18 Our current system of taxonomy began in 1753 with the publication of Species Plantarum by the Swedish botanist Carolus Linnaeus (1707–1778). Until his time, the names of organisms were often strings of descriptive terms that varied from country to country and from one scientist to another. Linnaeus provided a system that standardized the naming and classification of organisms based on characteristics they have in common. He grouped similar organisms that can successfully interbreed into categories called species. The definition of species as “a group of organisms that interbreed to produce viable offspring” works relatively well for sexually reproducing organisms, but it is not satisfactory for asexual organisms such as most microorganisms. As a result, some scientists define a microbial species as a collection of strains or serotypes—populations of cells that arose from a single cell—that share many stable properties, differ from other strains, and evolve as a group. Alternatively, biologists define a microbial species as cells that share at least 97% common genetic sequences. Not surprisingly, these definitions sometimes result in disagreements and inconsistencies in the classification of microbial life. Some researchers question whether unique microbial species exist at all. Classification and Identification of Microorganisms 113 BENEFICIAL MICROBES Glowing Viruses A bacteriophage is a virus that inserts its DNA into a bacterium. Commonly called a phage, it adheres only to a select bacterial strain for which each phage type has a specific adhesion factor. Many phages are so specialized for their particular bacterial strain that scientists have used phages to identify and classify bacteria. Such identification is called phage LM 20 μm typing. Fluorescent phages Scientists at San Diego State University light up bacteria. have taken phage specificity a step further. They successfully linked a fluorescent dye to the DNA of phages of the bacterium Salmonella and used the phages to detect and identify Salmonella species. Such fluorescent phages rapidly and accurately detect specific strains of Salmonella in mixed bacterial cultures. In Linnaeus’s system, which forms the basis of modern taxonomy, similar species are grouped into genera7 and similar genera into still larger taxonomic categories. That is, genera sharing common features are grouped together to form families; similar families are grouped into orders; orders are grouped into classes; classes into phyla;8 and phyla into kingdoms (FIGURE 4.20). All these categories, except strains (serotypes), and including species and genera, are taxa, which are hierarchical; that is, each successive taxon has a broader description than the preceding one, and each taxon includes all the taxa beneath it. For all groups except species of viruses, the rules of nomenclature require that every taxon have a Latin or Latinized name, in part because the language of science during Linnaeus’s time was Latin. The name Chondrus crispus (kon′drŭs krisp′ŭs) describes the exact same algal species all over the world despite the fact that in England its common name is Irish moss, in Ireland it is carragheen, in North America it is curly moss, and, in reality, it isn’t a moss at all. When new microscopic, genetic, or biochemical techniques identify new or more detailed characteristics of organisms, a taxon may be split into two or more taxa. Alternatively, several taxa may be lumped together into a single taxon. For example, the genus “Diplococcus” (dip′lō-kok′ŭs) has been united (synonymized) with the genus Streptococcus (strep-tō-kok′ŭs), so the name Diplococcus pneumoniae (nū-mō′nē-ī) is now Streptococcus pneumoniae to reflect this synonymy. Linnaeus assigned each species a descriptive name consisting of its genus name and a specific epithet. The genus name is always a noun, and it is written first and capitalized. The specific epithet contains only lowercase letters and is usually an adjective. Both names, together called a binomial, are either Fluorescent phages have advantages over fluorescent antibodies: Unlike antibodies, phages are not metabolized by bacteria. Phages are also more stable over time and are not as sensitive to vagaries in temperature, pH, and ionic strength. Further, fluorescent dyes within phages have a long shelf life; the phage coat protects the dye, which is attached to the DNA within the phage. There are numerous uses for test kits using fluorescent phages. Environmental scientists could use them to detect bacterial contamination of streams and lakes, food processors could identify potentially fatal Escherichia coli strain O157:H7 in meat and vegetables, or homeland security agents could positively establish the presence or absence of bacteria used for biological warfare. Antibody-based kits frequently failed to accurately detect Bacillus anthracis used in the 2001 terrorist attacks. Fluorescent phage field kits should be much more robust and precise. printed in italics or underlined. Because the Linnaean system assigns two names to every organism, it is said to use binomial9 nomenclature. Consider the following examples of binomial names of species: Enterococcus faecalis (en′ter-ō-kok′ŭs fē-kă′lis)10 is a fecal bacterium, whereas Enterococcus faecium (fē-sē′ŭm) in the same genus is a different species because of certain differing characteristics. Therefore, it has a different specific epithet. In some cases, binomials honor people. Examples include Pasteurella haemolytica (pas-ter-el′ă hē-mō-lit′i-kă), a bacterium named after the microbiologist Louis Pasteur; Escherichia coli (esh-ĕ-rik′ē-ă kō′lī), a bacterium named after the physician Theodor Escherich (1857–1911); and Izziella abbottiae (iz-ē-el′lă ab′ottē-ī), a marine alga named after the phycologist and taxonomist Isabella Abbott (1919–2010). Even though binomials are often descriptive of an organism, sometimes they can be misleading. For instance, Haemophilus influenzae (hē-mof′i-lŭs in-flu-en′zī) does not cause influenza. Most scientists still use the Linnaean system today, though significant modifications have been adopted. For example, scientists sometimes use additional categories, such as tribes, sections, subfamilies, and serotypes.11 Further, Linnaeus divided all organisms into only two kingdoms (Plantae and Animalia), and he did not know of the existence of viruses. As scientists learned more about organisms, they adopted taxonomic schemes to reflect advances in knowledge. For example, a widely accepted taxonomic approach of the last century was based on five kingdoms: Animalia, Plantae, Fungi, Protista, and Prokaryotae. Scientists created the kingdom Protista for eukaryotic organisms that did not fit neatly with true plants, animals, or fungi. This 9 From Greek bi, meaning “two,” and nomos, meaning “rule.” 7 Plural of Latin genus, meaning “race” or “birth.” 8 Plural of phylum, from Greek phyllon, meaning “tribe.” The term phylum is used for animals and bacteria; the corresponding taxon in mycology and botany is called a division. 10 From Greek enteron, meaning “intestine;” kokkos, meaning “berry”; and Latin faeces. 11 Serotypes, which are also called varieties, strains, or subspecies, differ only slightly from each other. 114 CHAPTER 4 Microscopy, Staining, and Classification b FIGURE 4.20 Levels in a taxonomic scheme. Domain Bacteria Archaea Eukarya Animalia Plantae Fungi Illustrated here are the taxonomic categories for the deer tick, Ixodes scapularis, the primary vector of Lyme disease. Notice that higher taxonomic categories are more inclusive than lower ones. To simplify the diagram, only selected classification possibilities are shown. Kingdom Phylum Chordata (vertebrates) Arthropoda (joint-legged animals) Platyhelminthes (tapeworms) Nematoda (unsegmented roundworms) Class Insecta Crustacea Arachnida Order Scorpionida Acariformes (mites) Parasitiformes (mites and ticks) Family Ixodidae (hard ticks) Argasidae (soft ticks) Dermacentor Ixodes Rhipicephalus I. scapularis (deer tick) I. pacificus (black-eyed tick) I. ricinus (castor bean tick) Genus Species Araneida Classification and Identification of Microorganisms scheme grouped together such obviously different eukaryotes as massive brown seaweeds (kelps) and unicellular microbes, such as Euglena (yū-glēn′ă). Because this five-kingdom approach does not fully address the taxonomy of prokaryotes or the many differences among protists, scientists have proposed other taxonomic schemes that have from 5 to more than 50 kingdoms. Sometimes students are upset that taxonomists do not agree about all the taxonomic categories or the species they contain, but it must be realized that classification of organisms reflects the state of our current knowledge and theories. Taxonomists change their schemes to accommodate new information, and not every expert agrees with every proposed modification. Another significant development in taxonomy is a shift in its basic goal—from Linnaeus’s goal of classifying and naming organisms as a means of cataloging them to the more modern goal of understanding the relationships among groups of organisms. Linnaeus based his taxonomic scheme primarily on organisms’ structural similarities, and whereas such terms as family may suggest the existence of some common lineage, he and his contemporaries thought of species as divinely created. However, when Charles Darwin (1809–1882) propounded his theory of the evolution of species by natural selection (a century after Linnaeus published his pivotal work on taxonomy), taxonomists came to consider that common ancestry explains the similarities among organisms in the various taxa. Today, most taxonomists agree that a major goal of modern taxonomy is to reflect a phylogenetic12 hierarchy—that is, that the ways in which organisms are grouped should reflect their evolution from common ancestors. Taxonomists’ efforts to classify organisms according to their ancestry have resulted in reduced emphasis on comparisons of physical and chemical traits, and in greater emphasis on comparisons of their genetic material. Such work led to the addition of a new, most inclusive taxon: the domain. 115 of bacteria. Repeated testing showed that methanogens, as they are called, were not like other prokaryotic or eukaryotic organisms. They were something new to science, a third branch of life. Woese and Fox had discovered that there are three basic types of ribosome, leading them to propose a new classification scheme in which a new taxon, called a domain, contains the Linnaean taxa of kingdoms. The three domains identified by Woese and Fox are Eukarya, Bacteria, and Archaea. Domain Eukarya includes all eukaryotic cells, all of which contain eukaryotic rRNA sequences. Domains Bacteria and Archaea include all prokaryotic cells. They contain bacterial and archaeal rRNA sequences, respectively, which differ significantly from one another and from those in eukaryotic cells. In addition to differences in rRNA sequences, cells of the three domains differ in many other characteristics, including the lipids in their cell membranes, transfer RNA (tRNA) molecules, and sensitivity to antibiotics. (Chapters 11 and 12, which cover prokaryotes and eukaryotes respectively, discuss the taxonomy of organisms within the three domains.) Ribosomal nucleotide sequences further suggest that there may be more than 50 kingdoms of Bacteria and five or more kingdoms of Archaea. Further, scientists examining substances such as human saliva, water, soil, and rock regularly discover novel nucleotide sequences that have been released from cells. When these sequences are compared to known sequences stored in a computer database, they cannot be associated with any previously identified organism. This suggests that many curious new forms of microbial life have never been grown in a laboratory and still await discovery. Ribosomal nucleotide sequences have also given microbiologists a new way to define prokaryotic species. Some scientists propose that prokaryotes whose rRNA sequence differs from that of other prokaryotes by more than 3% be classified as a distinct species. Although this definition has the advantage of being precise, not all taxonomists agree with it. Domains L E A R N I N G O U TCO M E 4.22 List and describe the three domains proposed by Woese and Fox. Carl Woese (1928–2012) and George Fox (1945–) labored for years to understand the taxonomic relationships among prokaryotes. Morphology (shape) and biochemical tests did not provide enough information to classify organisms fully, so for over a decade Woese and Fox painstakingly sequenced the DNA nucleotides coding for the smaller subunits of ribosomal RNA (rRNA) in an effort to unravel the relationships among these organisms. Because rRNA molecules are present in all cells and are crucial to protein synthesis, changes in the nucleotide sequences coding for them are presumably very rare. In 1976, they sequenced DNA from an odd group of prokaryotes that produce methane gas as a metabolic waste. Woese and Fox were surprised that the DNA sequences coding for the rRNA of these cells did not contain nucleotide sequences characteristic 12 From Greek phyllon, meaning “tribe,” and Latin genus, meaning “birth” (i.e., origin of a group). MICRO CHECK 13. What taxonomic group appears between kingdom and class? 14. What historical figure developed our current system of taxonomy, by which we assign names to organisms? 15. What were Woese and Fox studying that led them to propose the new taxon domain? Taxonomic and Identifying Characteristics L E A R N I N G O U TCO M E 4.23 Describe six procedures taxonomists use to identify and classify microorganisms. Criteria and laboratory techniques used for classifying and identifying microorganisms are quite numerous and include macroscopic and microscopic examination of physical characteristics, 116 CHAPTER 4 Microscopy, Staining, and Classification differential staining characteristics, growth characteristics, microorganisms’ interactions with antibodies, microorganisms’ susceptibilities to viruses, nucleic acid analysis, biochemical tests, and organisms’ environmental requirements, including the temperature and pH ranges of their various types of habitats. Clearly, then, microbial taxonomy is too broad a subject to cover in one chapter, and thus the details of the criteria for the classification of major groups are provided in subsequent chapters. It is important to note that even though scientists may use a given technique to either classify or identify microorganisms, the criteria used to identify a particular organism are not always the same as those that were used to classify it. For example, even though medical laboratory scientists distinguish the genus Escherichia from other bacterial genera by its inability to utilize citric acid (citrate) as a sole carbon source, this characteristic is not vital in the classification of Escherichia. Bergey’s Manual of Determinative Bacteriology, first published in 1923 and now in its ninth edition (1994), contains information used for the laboratory identification of prokaryotes. Bergey’s Manual of Systematic Bacteriology (second edition, 2001, 2005, 2009, 2010) is a similar reference work that is used for classification based on ribosomal RNA sequences, which taxonomists use to describe relationships among organisms. Each of these manuals is known as “Bergey’s Manual.” Linnaeus did not know of the existence of viruses and thus did not include them in his original taxonomic hierarchy, nor are viruses assigned to any of the five kingdoms or Woese and Fox’s three domains because viruses are acellular and generally lack rRNA. Virologists do classify viruses into families and genera, but higher taxa are poorly defined for viruses. (Chapter 13 further discusses viral taxonomy.) With this background, let’s turn now to some brief discussions of five types of information that microbiologists commonly use to distinguish among microorganisms: physical characteristics, biochemical tests, serological tests, phage typing, and analysis of nucleic acids. In addition, MALDI-TOF mass spectrometry is used in medical laboratories. rod-shaped prokaryotes, and thus visible characteristics alone are not sufficient to classify prokaryotes. Instead, taxonomists rely primarily on genetic differences as revealed by metabolic dissimilarities and, more and more frequently, on DNA sequences that code for subunits of rRNA. Biochemical Tests Microbiologists distinguish many prokaryotes on the basis of differences in their ability to utilize or produce certain chemicals. Biochemical tests include procedures that determine an organism’s ability to ferment various carbohydrates; utilize various substrates, such as specific amino acids, starch, citrate, and gelatin; or produce waste products, such as hydrogen sulfide (H2S) gas (FIGURE 4.21). Differences in fatty acid composition of bacteria are also used to distinguish among bacteria. Obviously, biochemical tests can be used to identify only those microbes that can be grown under laboratory conditions. Laboratory scientists utilize biochemical tests to identify pathogens, allowing physicians to prescribe appropriate treatments. Many tests require that the microorganisms be cultured (grown) for 12–24 hours, though this time can be greatly reduced by the use of rapid identification tools. Such tools exist for many groups of medically important pathogens, such as Gram-negative bacteria in the family Enterobacteriaceae, Gram-positive bacteria, yeasts, and filamentous fungi. Automated systems for identifying pathogens use the results of a whole battery of biochemical tests performed in a plastic plate containing numerous small wells (FIGURE 4.22). A color change in a well indicates Inverted tubes to trap gas Gas bubble Physical Characteristics Many physical characteristics are used to identify microorganisms. Scientists can usually identify protozoa, fungi, algae, and parasitic worms based solely on their morphology (shape). Medical laboratory scientists can also use the physical appearance of a bacterial colony13 to help identify microorganisms. As we have discussed, stains are used to view the size and shape of individual bacterial cells and to show the presence or absence of identifying features such as endospores and flagella. Linnaeus categorized prokaryotic cells into two genera based on two prevalent shapes. He classified spherical prokaryotes in the genus “Coccus,”14 and he placed rod-shaped cells in the genus Bacillus.15 However, subsequent studies have revealed vast differences among many of the thousands of spherical and 13 A group of bacteria that has arisen from a single cell grown on a solid laboratory medium. 14 From Greek kokkos, meaning “berry.” This genus name has been supplanted by many genera, including Staphylococcus, Micrococcus, and Streptococcus. 15 From Latin bacillum. Acid with gas Acid with no gas (a) Inert Hydrogen No sulfide hydrogen produced sulfide (b) ▲ FIGURE 4.21 Two biochemical tests for identifying bacteria. (a) A carbohydrate utilization test. At left is a tube in which the bacteria have metabolized a particular carbohydrate to produce acid (which changes the color of a pH indicator, phenol red, to yellow) and gas, as indicated by the bubble. At center is a tube with another bacterium that metabolized the carbohydrate to produce acid but no gas. At right is a tube inoculated with bacteria that are “inert” with respect to this test. (b) A hydrogen sulfide (H2S) test. Bacteria that produce H2S are identified by the black precipitate formed by the reaction of the H2S with iron present in the medium. Classification and Identification of Microorganisms 117 Wells Negative result Positive result Negative result Positive result (a) ▲ FIGURE 4.22 One tool for the rapid identification of bacteria, the automated MicroScan system. A MicroScan panel is a plate containing numerous wells, each the site of a particular biochemical test. The instrument ascertains the identity of the organism by reading the pattern of colors in the wells after the biochemical tests have been performed. (b) the presence of a particular metabolic reaction, and the machine reads the pattern of colors in the plate to ascertain the identity of the pathogen. Serological Tests In the narrowest sense, serology is the study of serum, the liquid portion of blood after the clotting factors have been removed and an important site of antibodies. In its most practical application, serology is the study of antigen-antibody reactions in laboratory settings. Antibodies are immune system proteins that bind very specifically to target antigens (Chapter 16). In this section, we briefly consider the use of serological testing to identify microorganisms. Many microorganisms are antigenic; that is, within a host organism they trigger an immune response that results in the production of antibodies. Suppose, for example, that a scientist injects a sample of Borrelia burgdorferi (bō-rē′lē-ă burg-dōr′fer-ē), the bacterium that causes Lyme disease, into a rabbit. The bacterium has many surface proteins and carbohydrates that are antigenic because they are foreign to the rabbit. The rabbit responds to these foreign antigens by producing antibodies against them. These antibodies can be isolated from the rabbit’s serum and concentrated into a solution known as an antiserum (plural: antisera). Antisera bind to the antigens that triggered their production. In a procedure called an agglutination test, antiserum is mixed with a sample that potentially contains its target cells. If the antigenic cells are present, antibodies in the antiserum will clump (agglutinate) the antigen (FIGURE 4.23). Other antigens, and therefore other organisms, remain unaffected because antibodies are highly specific for their targets. Scientists and laboratory technicians use antisera to distinguish among species and even among serotypes (strains) of the same species. For example, Rebecca Lancefield (1895–1981) ▲ FIGURE 4.23 An agglutination test, one type of serological test. (a) In a positive agglutination test, visible clumps are formed by the binding of antibodies to their target antigens present on cells. (b) The processes involved in agglutination tests. In a negative result, antibody binding cannot occur because its specific target is not present; in a positive result, specific binding does occur. Note that agglutination occurs because each antibody molecule can bind simultaneously to two antigen molecules. increased our ability to distinguish different streptococcal bacteria and diagnose their diseases using a serological classification system that she devised. Her work is memorialized as what are now known as Lancefield groupings, a common way to identify streptococci. (Chapter 17 examines other serological tests, such as enzyme-linked immunosorbent assay, or ELISA, and immunoblotting.) Phage Typing Bacteriophages (or simply phages) are viruses that infect and usually destroy bacterial cells. Just as antibodies are specific for their target antigens, phages are specific for the hosts they can infect. Phage typing, like serological testing, works because of such specificity. One bacterial strain may be susceptible to a particular phage while a related strain is not. In phage typing, a technician spreads a solution containing the bacterium to be identified across a solid surface of growth medium and then adds small drops of solutions containing different types of bacteriophage. Wherever a specific phage is able to infect and kill bacteria, the resulting lack of bacterial growth produces within the bacterial lawn a clear area called a plaque (FIGURE 4.24). A microbiologist can identify an unknown bacterium by comparing the phages that form plaques with known phage-bacteria interactions. 118 CHAPTER 4 Microscopy, Staining, and Classification Bacterial lawn Plaques species based upon unique proteins that the microbes synthesize. The technique uses a matrix to stabilize ions of large organic molecules, such as proteins, which have been ionized with laser energy. The organic molecules remain stable and retain their structure so that scientists can sort them by mass spectrometry—a process that sorts chemicals based on their mass-to-charge ratios. Though the details of MALDI-TOF and mass spectrometry are beyond the scope of this text, scientists use MALDI-TOF to sort and identify proteins unique to specific microbes and thereby show that particular microbes are present in a sample. Scientists and physicians use MALDI-TOF to identify species, to predict resistance to particular antibiotics, and to rapidly diagnose diseases. Analysis of Nucleic Acids ▲ FIGURE 4.24 Phage typing. Drops containing bacteriophages were added to this plate after its entire surface was inoculated with a bacterium. After 12 hours of bacterial growth, clear zones, called plaques, developed where the phages killed bacteria. Given the great specificity of phages for infecting and killing its host, the strain of bacterium can be identified. MALDI-TOF Mass Spectrometry Medical laboratory scientists can use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, more simply known as MALDI-TOF (mal′de-toff) to identify microbial As we have discussed, the sequence of nucleotides in nucleic acid molecules (either DNA or RNA) provides a powerful tool for classifying and identifying microbes. In many cases, nucleic acid analysis has confirmed classical taxonomic hierarchies. In other cases, as in Worse and Fox’s discovery of archaea, curious new organisms and relationships not obvious from classical methodologies have come to light. Techniques of nucleotide sequencing and comparison, such as polymerase chain reaction (PCR) (Chapter 8), are best understood after we have discussed microbial genetics (Chapter 7). Determining the percentage of a cell’s DNA that is guanine and cytosine, a quantity referred to as the cell’s G + C content EMERGING DISEASE CASE STUDY Necrotizing Fasciitis Fever, chills, nausea, weakness, and general yuckiness. Carlos thought he was getting the flu. Further, he had pulled a cactus thorn from his arm the day before, and the tiny wound had swollen to a centimeter in diameter. It was red, extremely hot, and much more painful than such a puncture had a right to be. Everything was against him. He couldn’t afford to miss days at work, but he had no choice. He shivered in bed with fever for the next two days and suffered more pain than he had ever experienced, certainly more than the time he broke his leg. Even more than passing a kidney stone. The red, purple, and black inflammation on his arm had grown to the size of a baseball. It was hard to the touch and excruciatingly painful. He decided it was time to call his brother to take him to the doctor. That decision saved his life. Carlos’s blood pressure dropped severely, and he was unconscious by the time they arrived. The physician immediately admitted Carlos to the hospital, where the medical team raced to treat necrotizing fasciitis, commonly called “flesh-eating” disease. This reemerging disease is caused by group A Streptococcus, a serotype of Gram-positive bacteria also known as Streptococcus pyogenes. Group A strep invades through a break in the skin and travels along the fascia—the protective covering of muscles—producing toxins that destroy human tissues, affecting about 750 people each year in the United States. By cutting away all the infected tissue; using high-pressure, pure oxygen to inhibit bacterial growth; and applying antimicrobial drugs to kill the bacterium, the doctors stabilized Carlos. After months of skin grafts and rehabilitation, he returned to work, grateful to be alive. (For more about necrotizing fasciitis, see pp. 554–555.) 1. What color do cells of S. pyogenes appear after the Gram staining procedure? Classification and Identification of Microorganisms (or G + C percentage), has also become a part of prokaryotic taxonomy. Scientists express the content as follows: that only one of two choices applies to any particular organism (FIGURE 4.25). Depending on which of the two statements applies, the key either directs the user to another pair of statements or provides the name of the organism in question. Note that more than one key can be created to enable the identification of a given set of organisms, but all such keys involve mutually exclusive, “either/or” choices that send the user along a path that leads to the identity of Play Dichotomous Keys: Overview, Sample with the unknown Flowchart, Practice @ Mastering Microbiology organism. G + C * 100 A + T + G + C G + C content varies from 20% to 80% among prokaryotes. Often (but not always), organisms that share characteristics have similar G + C content. Organisms that were once thought to be closely related but have widely different G + C percentages are invariably not as closely related as had been thought. Taxonomic Keys MICRO CHECK As we have seen, taxonomists, medical clinicians, and researchers can use a wide variety of information—including morphology, chemical characteristics, nucleotide sequencing, and results from biochemical, serological, and phage typing tests—in their efforts to identify microorganisms, including pathogens. But how can all these characteristics and results be organized so that they can be used efficiently to identify an unknown organism? All this information can be arranged in dichotomous keys, which contain a series of paired statements worded so 16. What type of testing allows lab technicians to test pathogenic bacterial specimens for reaction with known antibodies? TELL ME WHY Why didn’t Linnaeus create taxonomic groups for viruses? 1a. Gram-positive cells………………………… Gram-positive bacteria 1b. Gram-negative cells……………………….. 2 2a. Rod-shaped cells…………………………… 3 2b. Non-rod-shaped cells……………………… Cocci and pleomorphic bacteria 3a. Can tolerate oxygen………………………… 4 3b. Cannot tolerate oxygen……………………. Obligate anaerobes 4a. Ferments lactose…………………………… 5 4b. Cannot ferment lactose…………………… Non-lactose-fermenters 5a. Can use citric acid as a sole carbon source……………………………… 6 5b. Cannot use citric acid alone………………. 8 Gram-positive cells? No Yes Rod-shaped cells? No Gram-positive bacteria Yes Cocci and pleomorphic bacteria Can tolerate oxygen? No Yes Obligate anaerobes Ferments lactose? 6a. Produces hydrogen sulfide gas……………. Salmonella 6b. Does not produce hydrogen sulfide gas…. 7 7a. Produces acetoin…………………………… Enterobacter 7b. Does not produce acetoin………………… Citrobacter 119 No Non-lactosefermenters 8a. Produces gas from glucose……………….. Escherichia 8b. Does not produce gas from glucose……... Shigella Yes Can use citric acid (citrate) as sole carbon source? No Yes Produces gas from glucose? (a) No Shigella Produces hydrogen sulfide gas? Yes No Escherichia No (b) ▲ FIGURE 4.25 Use of a dichotomous taxonomic key. The example presented here involves identifying the genera of potentially pathogenic intestinal bacteria. (a) A sample key. To use it, choose the one statement in a pair that applies to the organism to be identified, and then either refer to another key (as indicated by words) or go to the appropriate place within this key (as indicated by a number). (b) A flowchart that shows the various paths that might be followed in using the key presented in part (a). Highlighted is the path taken when the bacterium in question is Escherichia. Yes Produces acetoin? Citrobacter Salmonella Yes Enterobacter 120 CHAPTER 4 Microscopy, Staining, and Classification MICRO IN THE CLINIC FOLLOW-UP End of the Camping Trip? Upon the insistence of all her friends, Marisol agrees to end the trip early. When she gets back into town, she makes an appointment to see her doctor. Unlike Marisol’s friend, the doctor is concerned about the discolored area on the back of Marisol’s leg. The doctor tells Marisol that the area isn’t a bruise; it’s actually a rash. The presence of the rash, in combination with her other symptoms, makes him suspect that she may have Lyme disease. He draws blood for laboratory tests and to confirm the diagnosis. Marisol wants to know how her blood will be used to diagnose infection. The doctor explains that they will be using tests to detect the bacterium Borrelia burgdorferi. Marisol is surprised; she remembers learning about Lyme disease in her biology class but thought that a “bull’seye” rash was the telltale sign. The doctor explains that although the bull’s-eye rash is the best-known and perhaps the most distinctive sign, there are several rashes that are indicative of Lyme disease. The blood tests indicate that Marisol indeed has Lyme disease. Her doctor prescribes a course of the antimicrobial drug doxycycline and explains that Marisol should start feeling better in the next day or so. Within a couple of weeks she should be back to normal. He also reminds her that it’s important to use insect repellents containing the chemical DEET and thoroughly check for ticks when outdoors in areas where ticks may be present. 1. One of the tests used to positively diagnose Lyme disease is immunofluorescence microscopy. How can this method be used to identify a pathogen such as B. burgdorferi in a blood sample? 2. Identify and describe a serological test that could be used to detect B. burgdorferi in blood. Check your answers to Micro in the Clinic Follow-Up questions in the Mastering Microbiology Study Area. Make the connection among Chapters 2, 3, and 4. CHAPTER SUMMARY Play Microscopy and Staining: Overview @ Mastering Microbiology Watch MICRO MATTERS videos in the Mastering Microbiology Study Area. 6. The magnifications of the objective lens and the ocular lens are multiplied together to give total magnification. 7. A photograph of a microscopic image is a micrograph. Units of Measurement (p. 97) 1. The metric system is a decimal system in which each unit is onetenth the size of the next largest unit. 2. The basic unit of length in the metric system is the meter. Microscopy (pp. 97–106) 1. Microscopy refers to the passage of light or electrons of various wavelengths through lenses to magnify objects and provide resolution and contrast so that those objects can be viewed and studied. 2. Immersion oil is used in light microscopy to fill the space between the specimen and a lens to reduce light refraction and thus increase the numerical aperture and resolution. 3. Staining techniques and polarized light may be used to enhance contrast between an object and its background. 4. Simple microscopes contain a single magnifying lens, whereas compound microscopes use a series of lenses for magnification. 5. The lens closest to the object being magnified is the objective lens, several of which are mounted on a revolving nosepiece. The lenses closest to the eyes are ocular lenses. Condenser lenses lie beneath the stage and direct light through the slide. 8. Dark-field microscopes provide a dark background for small or colorless specimens. 9. Phase microscopes, such as phase-contrast microscopes and differential interference contrast microscopes (Nomarski microscopes), cause light rays that pass through a specimen to be out of phase with light rays that pass through the field, producing contrast. 10. Fluorescence microscopes use ultraviolet light and fluorescent dyes to fluoresce specimens and enhance contrast. 11. A confocal microscope uses fluorescent dyes in conjunction with computers to provide Play Light Microscopy three-dimensional images @ Mastering Microbiology of a specimen. 12. A transmission electron microscope (TEM) provides an image produced by the transmission of electrons through a thinly sliced, dehydrated specimen. 13. A scanning electron microscope (SEM) provides a threedimensional image by scattering electrons from the metal-coated surface of a specimen. 14. Minuscule electronic probes are used in scanning tunneling microscopes (STM) and in atomic force microscopes (AFM) to reveal details at the atomic level. Play Electron Microscopy @ Mastering Microbiology Questions for Review Staining (pp. 106–112) 1. Preparing to