MCB3020 Textbook Chapter 2: Microscopy PDF

2 Microscopy ©Kenneth Lambert/AP Images Anthrax Bioterrorism Attack Table 2.1 Common Units of Measurement W hile on a trip to North Carolina in late September 2001, a sixty-three-year-old photojournalist and outdoor enthusiast from Florida began feeling ill. His muscles ached, he felt nauseous,...

2 Microscopy ©Kenneth Lambert/AP Images Anthrax Bioterrorism Attack Table 2.1 Common Units of Measurement W hile on a trip to North Carolina in late September 2001, a sixty-three-year-old photojournalist and outdoor enthusiast from Florida began feeling ill. His muscles ached, he felt nauseous, and he had a fever. His symptoms were annoying and varied in severity during his trip. After returning home, his symptoms worsened considerably. On October 2, he awoke with vomiting. Even more alarming was his mental confusion. When his wife took him to a local emergency room, doctors found that he was unable to answer simple questions not only about his illness but also about where he was and what time it was. His doctors performed a spinal tap and collected cerebral spinal fluid (CSF). The fluid was sent to the hospital laboratory where it was stained using the Gram-staining technique. Much to the surprise of the clinical lab scientists, the CSF contained long, Gram-positive rods that formed chains, a morphology unlike that of typical meningitis-causing bacteria. Based on the Gram-stain results, doctors made an initial diagnosis of inhalation anthrax. What made this diagnosis so surprising was that inhalation anthrax is extremely rare in the United States. Indeed, the last diagnosed case was in 1976. Doctors immediately began treatment with antibiotics, but the photojournalist’s condition worsened, and he died October 5. Thus began the first anthrax bioterrorism attack in the United States. Over the next several weeks, 16 other people developed inhalation anthrax; four of these individuals died. Much was learned about the disease from this attack. Much was also learned about the nation’s readiness to deal with bioterrorism. This event is also a reminder of the continuing importance of microscopy in microbiology and in diagnosing disease. In this chapter, we introduce some of the most commonly used types of microscopy. We begin with light microscopes and then describe other common types of microscopes, as well as how specimens are prepared for examination by microscopes. Readiness Check: Based on what you have learned previously, you should be able to: ✓ Explain why microscopy is a major tool used by microbiologists (sections 1.1 and 1.3) ✓ Define magnification ✓ Express the size of organisms using the metric system (table 2.1) 2.1 Lenses Create Images by Bending Light After reading this section, you should be able to: a. Relate the refractive indices of glass and air to the path light takes when it passes through a prism or convex lens b. Correlate lens strength and focal length Light microscopes were the first microscopes invented and they continue to be the most commonly used type. To understand light microscopy, we must consider the way lenses bend and focus light to form images. When a ray of light passes from one medium to another, refraction occurs; that is, the ray is bent at the interface. The refractive index is a measure of how much a substance slows the velocity of light; the direction and magnitude of bending are determined by the refractive indices of the two media forming the interface. For example, when light passes from air into glass, which has a greater refractive index, it is slowed and bent toward the normal, a line perpendicular to the surface (figure 2.1). As light leaves glass and returns to air, a medium with a lower refractive index, it accelerates and is bent away from the normal. Thus a prism bends light because glass has a different refractive index from air and the light strikes its surface at an angle. Lenses act like a collection of prisms operating as a unit. When the light source is distant so parallel rays of light 20 wil11886_ch02_020-039.indd 20 22/10/18 7:18 pm 2.2 There Are Several Types of Light Microscopes 21 2.2 There Are Several Types of Light Microscopes After reading this section, you should be able to: Figure 2.1 The Bending of Light by a Prism. Lines perpendicular to the surface of the prism are called normal; they are indicated by dashed lines. As light enters the glass, it is bent toward the first normal. When light leaves the glass and returns to air, it is bent away from the second normal. strike the lens, a convex lens focuses the rays at a specific point, the focal point (F in figure 2.2). The distance between the center of the lens and the focal point is called the focal length ( f in figure 2.2). Our eyes cannot focus on objects nearer than about 25 cm (i.e., about 10 inches). This limitation may be overcome by using a convex lens as a simple magnifier (or microscope) and holding it close to an object. A magnifying glass provides a clear image at much closer range, and the object appears larger. Lens strength is related to focal length; a lens with a short focal length magnifies an object more than a lens having a longer focal length. Comprehension Check 1. How do refraction, refractive index, focal point, and focal length differ? 2. Draw the path of a light ray through a thin and thick lens. 3. If the lenses in the above question were in corrective eyeglasses, which would be used for reading and which would improve distance vision? F f Figure 2.2 Lens Function. A lens functions somewhat like a collection of prisms. Light rays from a distant source are focused at the focal point F. The focal point lies a distance f, the focal length, from the lens center. MICRO INQUIRY How would the focal length change if the lens shown here were thicker? wil11886_ch02_020-039.indd 21 a. Evaluate the parts of a light microscope in terms of their contributions to image production and use of the microscope b. Predict the relative degree of resolution based on light wavelength and numerical aperture of the lens used to examine a specimen c. Create a table that compares and contrasts the various types of light microscopes in terms of their uses, how images are created, and the quality of images produced Microbiologists currently employ a variety of light microscopes in their work, each designed for specific applications. Modern microscopes are compound microscopes; that is, they have two sets of lenses. The objective lens is the lens closest to the specimen. It forms a magnified image that is further enlarged by one or more additional lenses. Bright-Field Microscope: Dark Object, Bright Background The bright-field microscope is routinely used to examine both stained and unstained specimens. It forms a dark image against a lighter background, thus it has a “bright field.” It consists of a metal stand composed of a base and an arm to which the remaining parts are attached (figure 2.3). A light source, either a mirror or an electric illuminator, is located in the base. Two focusing knobs, the fine and coarse adjustment knobs, are located on the arm and move either the stage or the nosepiece vertically to focus the image. The stage is positioned about halfway up the arm. Microscope slides are clipped to the stage, which can be moved during viewing by rotating control knobs. The substage condenser lens (or simply, condenser) is within or beneath the stage and focuses a cone of light on the slide. Its position may be fixed in simpler microscopes but can be adjusted vertically in more advanced models. The curved upper part of the arm holds the body assembly, to which a nosepiece and one or more ocular lenses (also called eyepieces) are attached. Binocular microscopes have eyepieces for both eyes. The body assembly contains a series of mirrors and prisms so the barrel holding the eyepiece may be tilted for ease in viewing. The nosepiece holds three to five objective lenses of differing magnifying power and can be rotated to change magnification. Ideally a microscope should be parfocal; that is, the image should remain in focus when objective lenses are changed. The image seen when viewing a specimen with a compound microscope is created by the objective and ocular lenses working together. Light from the illuminated specimen is focused by the objective lens, creating an enlarged image within the 22/10/18 7:18 pm 22 CHAPTER 2 | Microscopy Ocular (eyepiece) Body assembly Nosepiece Arm Objective lens Light intensity control Mechanical stage Aperture diaphragm control Substage condenser Coarse focus adjustment knob Fine focus adjustment knob Base with light source Stage adjustment knobs Figure 2.3 A Bright-Field Microscope. ©McGraw-Hill Education/James Redfearn, photographer microscope. The ocular lens further magnifies this primary image. The total magnification is calculated by multiplying the objective and eyepiece magnifications together. For example, if a 45× objective lens is used with a 10× eyepiece, the overall magnification of the specimen is 450×. Better Microscope Resolution Means a Clearer Image The most important part of the microscope is the objective lens, which must produce a clear image, not just a magnified one. Resolution is the ability of a lens to separate or distinguish between small objects that are close together. At best, the resolution of a bright-field microscope is 0.2 µm, which is about the size of a very small bacterium. Why is this the case? Resolution is in part dependent on the numerical aperture (n sin θ) of a lens. Numerical aperture is defined by two components: n is the refractive index of the medium in which the lens works (e.g., air = 1) and θ is 1/2 the angle of the cone of light entering an objective (figure 2.4). A cone with a narrow angle does not adequately separate the rays of light emanating from closely packed objects, and the images are not resolved. A cone of light with a very wide angle is able to separate the rays, and the closely packed objects appear widely separated and resolved. Recall that the refractive indices of all materials through which light wil11886_ch02_020-039.indd 22 Objective Working distance Slide with specimen θ θ Figure 2.4 Numerical Aperture in Microscopy. The numerical aperture of a lens is related to a value called the angular aperture (symbolized by θ), which is 1/2 the angle of the cone of light that enters a lens from a specimen. The equation for numerical aperture is n sin θ. In the right-hand illustration, the lens has larger angular and numerical apertures; its resolution is greater and its working distance smaller. waves pass determine the direction of the light rays emanating from the specimen. Some objective lenses work in air; since sin θ cannot be greater than 1 (the maximum θ is 90° and sin 90° is 1.00), no lens working in air can have a numerical aperture greater than 1.00. The only practical way to raise the numerical aperture above 1.00, and therefore achieve higher resolution, is 22/10/18 7:18 pm 2.2 There Are Several Types of Light Microscopes 23 Table 2.2 Properties of Objective Lenses to increase the refractive index with immersion oil, a colorless liquid with the same refractive index as glass (table 2.2). If air is replaced with immersion oil, many light rays that would otherwise not enter the objective due to reflection and refraction at the surfaces of the objective lens and slide will now do so (figure 2.5). This results in an increase in numerical aperture and resolution. Resolution is described mathematically by an equation developed in the 1870s by Ernst Abbé (1840–1905), a German physicist responsible for much of the optical theory underlying microscope design. The Abbé equation states that the minimal distance (d) between two objects that reveals them as separate entities depends on the wavelength of light (λ) used to illuminate the specimen and on the numerical aperture of the lens (n sin θ), which is the ability of the lens to gather light. d= 0.5 λ n sin θ The smaller d is, the better the resolution, and finer detail can be discerned in a specimen; d becomes smaller as the wavelength of light used decreases and as the numerical aperture increases. Thus the greatest resolution is obtained using a lens with the largest possible numerical aperture and light of the shortest wavelength, at the blue end of the visible spectrum (in the range of 450 to 500 nm). Examination of figure 2.4 shows that one objective is much closer to the specimen than the other. Specifically, the objective on the right has a higher numerical aperture and a shorter working distance. The working distance of an objective is the distance between the surface of the lens and the surface of the cover glass (if one is used) or the specimen when it is in sharp focus. As illustrated here, objectives with large numerical apertures and great resolving power have short working distances (table 2.2). Numerical aperture is also an important feature of a microscope’s condenser. The condenser is a large, light-gathering lens used to project a wide cone of light through the slide and into the objective lens. Although most condensers have a numerical aperture between 1.2 and 1.4, the numerical aperture will not exceed about 0.9 unless the top of the condenser is oiled to the bottom of the slide. During routine microscope operation, the condenser usually is not oiled, and this limits the overall resolution, even with an oil immersion objective. The most accurate calculation of a microscope’s resolving power considers both the numerical aperture of the objective lens and that of the condenser, as is evident from the following equation, where NA is the numerical aperture. dmicroscope = Slide Air Oil Cover glass Figure 2.5 The Oil Immersion Objective. An oil immersion objective operating in air and with immersion oil. wil11886_ch02_020-039.indd 23 λ (NAobjective + NAcondenser) However, in most cases the limit of resolution of a light microscope is calculated using the Abbé equation, which considers the objective lens only. We now see why the maximum theoretical resolving power of a microscope when viewing a specimen using an oil immersion objective (numerical aperture of 1.25) and bluegreen light is approximately 0.2 μm. d= (0.5)(530 nm) = 212 nm or 0.2 μm 1.25 Given the limit of resolution of a light microscope, the largest useful magnification—the level of magnification needed to 22/10/18 7:18 pm 24 CHAPTER 2 | Microscopy increase the size of the smallest resolvable object to be visible with the light microscope—can be determined. Our eye can just detect a speck 0.2 mm in diameter. When the acuity of the eye and the resolution of the microscope are considered together, it is calculated that the useful limit of magnification is about 1,000 times the numerical aperture of the objective lens. Most standard microscopes have 10× eyepieces and have an upper limit of about 1,000× with oil immersion. A 15× eyepiece may be used with good objective lenses to achieve a useful magnification of 1,500×. Any further magnification does not enable a person to see more detail. Indeed, a light microscope can be built to yield a final magnification of 10,000×, but it would simply be magnifying a blur. Only the electron microscope provides sufficient resolution to make higher magnifications useful. Objective Specimen Abbé condenser Dark-field stop Visualizing Living, Unstained Microbes Bright-field microscopes are probably the most common microscope found in teaching, research, and clinical laboratories. However, many microbes are unpigmented so are not clearly visible because there is little difference in contrast between the cells, subcellular structures, and water. As we discuss in section 2.3, one solution to this problem is to stain cells before observation. Unfortunately staining procedures usually kill cells. But what if an investigator wishes to view living cells? Three types of light microscopes create detailed, clear images of living specimens: dark-field microscopes, phase-contrast microscopes, and differential interference contrast microscopes. Figure 2.6 Dark-Field Microscopy. In dark-field microscopy, a dark-field stop (inset) is placed underneath the condenser lens system. The condenser then produces a hollow cone of light so that the only light entering the objective is reflected or refracted by the specimen. The dark-field microscope produces detailed images of living, unstained cells and organisms by simply changing the way in which they are illuminated. A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective. Only light that has been reflected or refracted by the specimen forms an image (figure 2.6). The field surrounding a specimen appears black, while the object itself is brightly illuminated (figure 2.7). The dark-field microscope can reveal considerable internal structure in larger eukaryotic microorganisms (figure 2.7b). It also can be used to identify certain bacteria such as the thin and distinctively shaped Treponema pallidum, the causative agent of syphilis (figure 2.7a). (a) T. pallidum (b) Volvox Dark-Field Microscope: Bright Object, Dark Background Figure 2.7 Examples of Dark-Field Microscopy. (a) Treponema pallidum, the spirochete that causes syphilis (×400). (b) The protist Volvox. Note daughter colonies within the mature Volvox colony. (a) Source: CDC/Schwartz; (b) ©McGraw-Hill Education/Stephen Durr, photographer wil11886_ch02_020-039.indd 24 22/10/18 7:18 pm 2.2 There Are Several Types of Light Microscopes 25 outside the cell. Thus both deviated light waves that interact with bacterial cell structures and undeviated light waves that pass around and through the cell are produced. Because the deviated light waves are slowed relative to the undeviated light waves, they are said to be out of phase. That is, the crests and troughs of the deviated and undeviated waves do not align. Typically the deviated light waves are slowed by about ¼ wavelength compared to the undeviated light (figure 2.8). Phase-contrast microscopes take advanDeviated ray is Ray deviated by Bacterium in a tage of this phenomenon to create differences Deviated and 1/2 wavelength specimen is 1/4 wet mount undeviated rays in light intensity that provide contrast to allow out of phase. wavelength out cancel each other the viewer to see a clearer, more detailed imof phase. out. age of the specimen (figure 2.9). They do so Figure 2.8 The Production of Contrast in Phase-Contrast Microscopy. The behavior of deviated by separating the two types of light so that the and undeviated (i.e., undiffracted) light rays in the positive-phase-contrast microscope. Because undeviated light (primarily from the surthe light rays tend to cancel each other out, the image of the specimen will be dark against a roundings) can be manipulated and then rebrighter background. combined with the deviated light (from the bacterium) to form an image. Two components allow this to occur: a condenser annulus and a phase plate Phase-Contrast Microscope (figure 2.10). The condenser annulus is an opaque disk with a thin To understand phase-contrast microscopy, consider a bacterium transparent ring. A ring of light is directed by the condenser anin a drop of water on a microscope slide (i.e., a wet mount) as ilnulus to the condenser, which focuses the light on the specimen as lustrated in figure 2.8. The refractive indices of bacterial cell shown in figure 2.10. Deviated and undeviated light then pass structures are greater than that of water. Therefore, light waves through the objective toward the phase plate. The phase plate has passing through a cell structure will be diffracted and slowed a thin ring through which the undeviated light (i.e., from the surmore than light waves passing through the water inside and roundings) is focused (figure 2.8). In a common type of phasecontrast microscopy (positive phase contrast), the ring is coated with a substance that advances the phase of the undeviated light by ¼ wavelength. The deviated light is focused on the rest of the phase plate, which lets the deviated light pass through unchanged. Wave trough Wave crest Phase plate Micronucleus (a) An amoeba Macronucleus (b) Paramecium sp. Figure 2.9 Examples of Phase-Contrast Microscopy. (a) An amoeba, a eukaryotic microbe that moves by means of pseudopodia, which extend out from the main part of the cell body. (b) Paramecium sp. stained to show a large central macronucleus with a small spherical micronucleus at its side (×400). (a) ©McGraw-Hill Education/Stephen Durr, photographer; (b) ©McGraw-Hill Education/James Redfearn, photographer wil11886_ch02_020-039.indd 25 22/10/18 7:18 pm 26 CHAPTER 2 | Microscopy Image plane Undeviated light Phase plate Objective Deviated light Figure 2.11 Differential Interference Contrast Microscopy. This image of the protozoan Amoeba proteus appears three-dimensional and contains considerable detail. Specimen ©McGraw-Hill Education/Stephen Durr, photographer Condenser widely used to study eukaryotic cells. Bacterial endospores are a survival strategy (section 3.9); Inclusions (section 3.6) Differential Interference Contrast Microscope Condenser annulus Figure 2.10 Phase-Contrast Microscopy. The optics of a positivephase-contrast microscope. MICRO INQUIRY What is the purpose of the annular stop in a phase- contrast microscope? Is it found in any other kinds of light microscopes? After leaving the phase plate, the deviated and undeviated light are now out of phase by ½ wavelength (figure 2.8). When the two rays of light recombine to form an image, they cancel each other out, a phenomenon called destructive interference. Destructive interference is also seen if the crest of a wave of water meets the trough of another wave—the two cancel each other out and the surface of the water remains calm at the point where they meet. In our example, the resulting image consists of a darker bacterium against a lighter background. Phase-contrast microscopy is especially useful for studying microbial motility, determining the shape of living cells, and detecting bacterial structures such as endospores and inclusions. These are clearly visible because they have refractive indices markedly different from that of water. Phase-contrast microscopes also are wil11886_ch02_020-039.indd 26 The differential interference contrast (DIC) microscope is similar to the phase-contrast microscope in that it creates an image by detecting differences in refractive indices and thickness. Two beams of plane-polarized light at right angles to each other are generated by prisms. In one design, the object beam passes through the specimen, while the reference beam passes through a clear area of the slide. After passing through the specimen, the two beams combine and interfere with each other to form an image. A live, unstained specimen appears brightly colored and seems to pop out from the background, giving the viewer the sense that a three-dimensional image is being viewed (figure 2.11). Structures such as cell walls, endospores, granules, vacuoles, and nuclei are clearly visible. Fluorescence Microscopes Use Emitted Light to Create Images The light microscopes thus far considered produce an image from light that passes through a specimen. An object also can be seen because it emits light. This is the basis of fluorescence microscopy. When some molecules absorb radiant energy, they become excited and release much of their trapped energy as light. Any light emitted by an excited molecule has a longer wavelength (i.e., has lower energy) than the radiation originally absorbed. Fluorescent light is emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable state. The fluorescence microscope excites a specimen with a specific wavelength of light that triggers the emission of fluorescent light by the object, which forms the image. The most 22/10/18 7:18 pm 2.2 There Are Several Types of Light Microscopes 27 Long wavelengths Exciter filter (removes long wavelengths) Barrier filter (blocks ultraviolet radiation but allows visible light through) Table 2.3 Commonly Used Fluorochromes Dichromatic mirror (reflects short wavelengths; transmits longer wavelengths) Mercury arc lamp Short wavelengths Long wavelengths Fluorochrome-stained specimen (absorbs short-wavelength radiation and emits longer-wavelength light) Figure 2.12 Epifluorescence Microscopy. The principles of operation of an epifluorescence microscope. fluorochrome-labeled probes or fluorochromes that bind specific cell constituents (table 2.3). In addition, microbial ecologists use epifluorescence microscopy to visualize photosynthetic microbes, as their pigments naturally fluoresce when excited by light of specific wavelengths. It is even possible to distinguish live bacteria from dead bacteria by the color they fluoresce after treatment with a specific mixture of stains (figure 2.13a). Genetic methods are used to assess microbial diversity (section 29.2); Identification of microorganisms from specimens (section 37.2) Another important use of fluorescence microscopy is the localization of specific proteins within cells. One approach is to use genetic engineering techniques that fuse the gene for the protein of interest to a gene isolated from jellyfish belonging commonly used fluorescence microscopy is epifluorescence microscopy, also called incident light or reflected light fluorescence microscopy. Epifluorescence (Greek epi, upon) microscopes illuminate specimens from above. The objective lens also acts as a condenser (figure 2.12). A mercury vapor arc lamp or other source produces an intense beam of light that passes through an exciter filter. The exciter filter transmits only the desired wavelength of light. The excitation light is directed down the microscope by the dichromatic mirror. This mirror reflects light of shorter wavelengths (i.e., the excitation light) but allows light of longer wavelengths to pass through. The excitation light continues down, passing through the objective lens to the specimen, which is usually stained with molecules called fluorochromes (table 2.3). The fluorochrome absorbs light energy from the excitation light and emits fluorescent light that travels up through the objective lens into the microscope. Because the emitted fluorescent light has a longer wavelength, it passes through the dichromatic mirror to a barrier filter, which blocks out any residual excitation light. Finally, the emitted light passes through the barrier filter to the eyepieces. (b) (a) The fluorescence microscope has be10 µm come an essential tool in microbiology. Figure 2.13 Fluorescent Dyes and Tags. (a) Dyes that cause live cells to fluoresce green and Bacterial pathogens can be identified after dead ones red. (b) Fluorescent antibodies tag specific molecules. In this case, the antibody binds to a staining with fluorochromes or specifimolecule that is unique to Yersinia pestis, the bacterium that causes plague. cally tagging them with fluorescently (a) ©Dr. Rita B. Moyes; (b) Source: CDC/Courtesy of Larry Stauffer, Oregon State Public Health Laboratory labeled antibodies using immunofluorescence procedures. In ecological studies, MICRO INQUIRY How might the fluorescently labeled antibody used in figure 2.13b be used to fluorescence microscopy is used to diagnose strep throat? observe microorganisms stained with wil11886_ch02_020-039.indd 27 22/10/18 7:18 pm 28 CHAPTER 2 | Microscopy fluorescent. GFP has been used extensively in studies on bacterial cell division and related phenomena (figure 2.14). In fact, the 2008 Nobel Prize in Chemistry was awarded to Osamu Shimomura of Japan and Americans Martin Chalfie and Roger Tsien for their development of this important tool. Fluorescence labeling (section 17.4) Figure 2.14 Green Fluorescent Protein. Visualization of Mbl, a cytoskeletal protein of Bacillus subtilis. The Mbl protein has been fused with green fluorescent protein and therefore fluoresces green. ©Jeff Errington/Centre for Bacterial Cell Biology/Newcastle University to the genus Aequorea. This jellyfish gene encodes a protein that naturally fluoresces green when exposed to light of a particular wavelength and is called green fluorescent protein (GFP). Thus when the protein is made by the cell, it is (a) All objects are defined by three axes: x, y, and z. The confocal scanning laser microscope (CSLM) creates images of planes formed by the x and y axes (x-y planes) and planes formed by the x and z axes (x-z planes). (b) The light microscope image of the two beads shown in (a). Note that neither bead is clear, because the image is generated from light emanating from multiple planes of focus. Confocal Microscopy Like the large and small beads illustrated in figure 2.15a, biologi cal specimens are three-dimensional. When threedimensional objects are viewed with traditional light microscopes, light from all areas of the object, not just the plane of focus, enters the microscope and is used to create an image. The resulting image is murky and fuzzy (figure 2.15b). This problem has been solved by the confocal scanning laser microscope (e) Views are also generated using different planes. The top left panel is the image of a single x-y plane (i.e., looking down from the top of the beads). The two lines represent the two x-z planes imaged in the other two panels. The vertical line indicates the x-z plane shown in the top right panel (i.e., a view from the right side of the beads) and the horizontal line indicates the x-z plane shown in the bottom panel (i.e., a view from the front face of the beads). (f) A three-dimensional reconstruction of a Pseudomonas aeruginosa biofilm. The biofilm was exposed to an antibacterial agent and then stained with dyes that distinguish living (green) from dead (red) cells. The cells on the surface of the biofilm have been killed, but those in the lower layers are still alive. This image clearly demonstrates the difficulty of killing all the cells in a biofilm. (c) A CSLM uses light from a single plane of focus to generate an image. A composite image of the two beads is generated using digitized information collected from multiple planes within the beads. (d) Digitized information is collected from multiple planes within the beads to generate a three-dimensional reconstruction. Figure 2.15 Light and Confocal Microscopy. (a–e) Two beads examined by light and confocal microscopy. (f) Three-dimensional reconstruction of a biofilm. (b–f) ©P. Dirckx/Center for Biofilm Engineering/Montana State University wil11886_ch02_020-039.indd 28 22/10/18 7:18 pm 2.3 Staining Specimens Helps to Visualize and Identify Microbes 29 (CSLM), or simply, confocal microscope. The confocal microscope uses a laser beam to illuminate a specimen that has been fluorescently stained. A major component of the confocal microscope is an opening (that is, an aperture) placed above the objective lens. The aperture eliminates stray light from parts of the specimen that lie above and below the plane of focus (figure 2.16). Thus the only light used to create the image is from the plane of focus, and a much sharper image is formed. To generate a confocal image, a computer interfaced with the confocal microscope receives digitized information from each plane in the specimen. This information can be used to create a composite image that is very clear and detailed (figure 2.15c) or a three-dimensional reconstruction of the specimen (figure 2.15d). Images of x-z plane cross sections of the specimen can also be generated, giving the observer views of the specimen from three perspectives (figure 2.15e). Confocal microscopy has numerous applications. One is the study of biofilms, which can form on many different types of surfaces, including indwelling medical devices such as hip joint replacements. As shown in figure 2.15f, it is difficult to kill all cells in a biofilm. This makes biofilms a particular concern because their formation on medical devices can result in infections that are difficult to treat. Biofilms are common in nature (section 7.6) Comprehension Check 1. Compare and contrast the function of the condenser, objective, and eyepiece lenses. 2. What happens to resolution if each of the following increase: wavelength of light, refractive index, and numerical aperture? How are resolution and magnification related? 3. What might a microscopist see through an oil immersion objective if no immersion oil is present? 4. Why don’t most light microscopes use 30× ocular lenses for greater magnification? 5. Compare and contrast how dark-field, phase-contrast, differential interference contrast, epifluorescence, and confocal microscopes work and the kind of image or images provided by each. Give a specific use for each type. 6. Which type of microscope(s) would be used to examine the following specimens? Photosynthetic bacteria, swimming bacteria, GFPlabeled microorganisms, unstained microbes, and pond scum; explain each of your answers. 2.3 Staining Specimens Helps to Visualize and Identify Microbes After reading this section, you should be able to: a. Recommend a fixation process to use when the microbe is a bacterium or archaeon and when the microbe is a protist b. Plan a series of appropriate staining procedures to describe an unknown bacterium as fully as possible c. Compare what happens to Gram-positive and Gramnegative bacterial cells during each step of the Gram-staining procedure Laser light Lens Mirror Aperture Scanner Detector Objective Cell Fixation Plane of focus Figure 2.16 Ray Diagram of a Confocal Microscope. The yellow lines represent laser light used for illumination. Red lines symbolize the light arising from the plane of focus, and the blue lines stand for light from parts of the specimen above and below the focal plane. MICRO INQUIRY How does the light source differ between a confocal light microscope and other light microscopes? wil11886_ch02_020-039.indd 29 As noted in section 2.2, specimens examined by bright-field microscopy are often fixed and stained before being examined. Such preparation serves to increase the visibility of the microorganisms, accentuate specific morphological features, and preserve them for future study. Importantly, some staining procedures help microbiologists identify the organism being examined. Stained cells seen in a microscope should resemble living cells as closely as possible. Fixation is the process by which the internal and external structures of specimens are preserved and fixed in position. It inactivates enzymes that might disrupt cell morphology and toughens cell structures so that they do not change during staining and observation. A microorganism usually is killed and attached firmly to the microscope slide during fixation. There are two fundamentally different types of fixation: heat fixation and chemical fixation. Heat fixation is routinely used to observe bacteria and archaea. Typically, a film of cells (a smear) is 22/10/18 7:18 pm 30 CHAPTER 2 | Microscopy gently heated. Heat fixation preserves overall morphology and inactivates enzymes. However, it also destroys proteins in subcellular structures, which may distort their appearance. Chemical fixation is used to protect fine cellular substructure as well as morphology. It is used when examining microorganisms by many electron microscopy techniques. Chemical fixatives penetrate cells and react with cellular components, usually proteins and lipids, to render them inactive, insoluble, and immobile. Common fixative mixtures contain ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde. Dyes and Simple Staining The many types of dyes used to stain microorganisms have two features in common. First, they have chromophore groups— chemical moieties with conjugated double bonds that give the dye its color. Second, they bind cells by ionic, covalent, or hydrophobic bonds. Dyes that bind cells by ionic interactions are probably the most commonly used dyes. These ionizable dyes may be divided into two general classes based on the nature of their charged group: basic dyes and acidic dyes (table 2.4). The staining effectiveness of ionizable dyes may be altered by pH, since the nature and number of the charged moieties on cell components change with pH. Thus acidic dyes stain best under acidic conditions when proteins and many other molecules carry a positive charge; basic dyes are most effective at higher pH values. Dyes that bind through covalent bonds or that have certain solubility characteristics are also useful. For instance, DNA can be stained by the Feulgen procedure in which the staining compound (Schiff’s reagent) is covalently attached to the deoxyribose sugars of DNA. Sudan III (Sudan Black) selectively stains lipids because it is lipid soluble but does not dissolve in aqueous portions of the cell. Microorganisms can be stained by simple staining, in which a single dye is used (figure 2.17). Simple staining’s value lies in its ease of use. The fixed smear is covered with stain for a short time, excess stain is washed off with water, and the slide is blotted dry. Basic dyes such as crystal violet, methylene blue, and carbolfuchsin are frequently used in simple staining to determine the size, shape, and arrangement of bacterial and archaeal cells. Table 2.4 wil11886_ch02_020-039.indd 30 Crystal violet stain of slender, rod shaped bacteria. Figure 2.17 Simple Staining Illustrates Cell Size and Morphology. ©Dr. Rita B. Moyes While most dyes directly stain the cell or object of interest, some dyes (e.g., India ink and nigrosin) are used in negative staining. In negative staining, the background is stained, not the cell; instead, the unstained cells appear as bright objects against a dark background. Differential Staining The Gram stain, developed in 1884 by the Danish physician Christian Gram, is the most widely employed staining method in bacteriology. The Gram stain is an example of differential staining—procedures that distinguish organisms based on their staining properties. Use of the Gram stain classifies most bacteria into one of two groups—Gram negative or Gram positive, based on the composition of their cell walls. The Gram-staining procedure is illustrated in figure 2.18. In the first step, the smear is stained with a primary stain (crystal violet). This is followed by treatment with an iodine solution. Iodine functions as a mordant, a substance that helps bind the dye tightly to the cell wall. The smear is then decolorized by washing with alcohol or acetone. This step generates the differential aspect of the Gram stain; the thick cells walls of Gram-positive bacteria retain the crystal violet, whereas Gram-negative bacteria lose the crystal violet and become colorless. Finally, the smear is counterstained, usually Ionizable Dyes 22/10/18 7:18 pm 2.3 Staining Specimens Helps to Visualize and Identify Microbes 31 Steps in Staining State of Bacteria Step 1: Crystal Cells stain purple. violet (primary stain) for 1 minute. Water rinse. Step 2: Iodine (mordant) for 1 minute. Water rinse. Cells remain purple. Step 3: Alcohol (decolorizer) for 10–30 seconds. Water rinse. Gram-positive cells remain purple. Gram-negative cells become colorless. Step 4: Safranin (counterstain) for 30–60 seconds. Water rinse. Blot dry. Gram-positive cells remain purple. Gram-negative cells appear red. (a) Gram stain Purple cells are Gram positive. Red cells are Gram negative. (b) Acid-fast stain Red cells are acid-fast. Blue cells are non-acid-fast. (c) Capsule stain of Klebsiella pneumoniae (d) Flagellar stain of Bacillus brevis Figure 2.18 Gram Stain. Steps in the Gram-staining procedure. MICRO INQUIRY Why is the decolorization step considered the most critical in the Gram-staining procedure? Figure 2.19 Differential Stains. (a, c) ©McGraw-Hill Education/Lisa Burgess, photographer; (b) ©McGraw-Hill Education/James Redfearn, photographer; (d) Source: CDC/Dr. William A. Clark with safranin, which colors Gram-negative bacteria pink to red and leaves Gram-positive bacteria dark purple (figure 2.19a). There are two main types of bacterial cell walls (section 3.4) Gram staining Acid-fast staining is another important differential staining procedure. It can be used to identify Mycobacterium tuberculosis and M. leprae (figure 2.19b), the pathogens responsible for tuberculosis and leprosy, respectively. These bacteria, as well as other mycobacteria, have cell walls containing lipids constructed from mycolic acids, a group of branched-chain hydroxy fatty acids, which prevent dyes from readily binding to the cells (see figure 23.7). A commonly used staining procedure, the cold Ziehl-Neelsen method, uses high concentrations of phenol and carbol fuchsin, as well as a wetting agent, to drive the stain carbol fuchsin into mycobacterial cells. Once this dye has penetrated, the cells are not easily decolorized by acidified alcohol (acid-alcohol) and thus are said to be acidfast. Non-acid-fast bacteria are easily decolorized by acidalcohol and thus are stained another color by a second dye called a counterstain. One of the simplest staining procedures is capsule staining (figure 2.19c), a technique that reveals the presence of capsules, wil11886_ch02_020-039.indd 31 a network usually made of polysaccharides that surrounds many bacteria and some fungi. Cells are mixed with India ink or nigrosin dye and spread out in a thin film on a slide. After airdrying, cells appear as bodies surrounded by a halo of capsule in the midst of a blue-black background because ink and dye particles cannot penetrate either the cell or its capsule. Thus capsule staining is an example of negative staining. There is little distortion of cell shape, and the cell can be counterstained for even greater visibility. Capsules and slime layers (section 3.5) Flagella staining provides taxonomically valuable information about the presence and distribution pattern of flagella on bacterial and archaeal cells (figure 2.19d; see also figure 3.37). Their flagella are fine, threadlike organelles of locomotion that are so slender (about 10 to 30 nm in diameter) they can only be seen directly using an electron microscope (although bundles of flagella can be visualized by dark-field microscopy). To observe bacterial flagella with a light microscope, their thickness is increased by coating them with mordants such as tannic acid and potassium alum and then staining with a dye such as basic fuchsin. Bacterial flagella (section 3.7) 6/13/19 4:39 PM 32 CHAPTER 2 | Microscopy Comprehension Check 1. Describe the two general types of fixation. Which would you use when Gram staining a bacterium? Which would you use before observing the organelles of a protist? 2. Why would basic dyes be more effective under alkaline conditions? 3. What procedural error(s) might be responsible if you Gram stained a mixture of bacteria known to be Gram-positive and Gram-negative and see only red cells? 4. Are capsular and flagellar staining differential staining procedures? Explain your answer. 2.4 Electron Microscopes Use Beams of Electrons to Create Highly Magnified Images After reading this section, you should be able to: a. Create a concept map, illustration, or table that compares transmission electron microscopes (TEMs) to light microscopes b. Decide when it would be best to examine a microbe by TEM, scanning electron microscopy (SEM), and electron cryotomography For centuries the light microscope has been the most important instrument for studying microorganisms. However, even the best light microscopes have a resolution limit of about 0.2 μm, which greatly compromises their usefulness for detailed studies of many microorganisms (figure 2.20). Viruses, for example, are too small to be seen with light microscopes (with the exception of some recently discovered giant viruses). Bacteria and archaea can be observed, but because they are usually only 1 to 2 μm in diameter, only their general shape and major morphological features are visible. The detailed internal structure of microorganisms therefore cannot be effectively studied by light microscopy. Recall that the resolution of a light microscope increases as the wavelength of the light it uses for illumination decreases. In electron microscopes, electrons replace light as the illuminating beam. The electron beam can be focused, much as light is in a light microscope, but its wavelength is about 100,000 times shorter than that of visible light. Therefore electron microscopes have a practical resolution roughly 1,000 times better than the light microscope; with many electron microscopes, points closer than 0.5 nm can be distinguished, and the useful magnification is well over 100,000× (figure 2.20). The value of the electron microscope is evident on comparison of the photographs in figure 2.21. Microbial morphology can now be studied in great detail. We briefly review the most common types of electron microscopy. Transmission Electron Microscope A transmission electron microscope (TEM) uses a heated tungsten filament in the electron gun to generate a beam of Range of light microscope Range of electron microscope Colonial alga (Pediastrum) 100 µm Amoeba Nucleus 10 µm 1 µm White blood cell Red blood cell Rickettsia bacteria Rod-shaped bacteria (Escherichia coli ) Mycoplasma bacteria 100 nm Coccus-shaped bacterium (Staphylococcus) Poxvirus Human Immunodeficiency Virus (HIV) Poliovirus Scanning tunneling microscope 10 nm (100 Å) 1 nm (10 Å) 0.1 nm (1 Å) wil11886_ch02_020-039.indd 32 Proteins Amino acid (small molecule) Hydrogen atom Figure 2.20 The Limits of Microscopic Resolution. Dimensions are indicated with a logarithmic scale (each major division represents a 10-fold change in size). To the right side of the scale are the approximate sizes of cells, viruses, molecules, and atoms. 22/10/18 7:19 pm 2.4 Electron Microscopes Use Beams of Electrons to Create Highly Magnified Images 33 Photosynthetic membrane vesicle Nucleoid (a) cut such a thin slice, specimens must be embedded in a supportive plastic matrix. To prepare specimens, they are first fixed with chemicals such as glutaraldehyde and osmium tetroxide to stabilize cell structure. The specimen is then dehydrated with organic solvents (e.g., acetone or ethanol). Next the specimen is soaked in unpolymerized, liquid epoxy plastic until it is completely permeated, and then the plastic is hardened to form a solid block. Thin sections are skillfully cut from the block with a glass or diamond knife using a device called an ultramicrotome. As with bright-field light microscopy, cells are usually stained so they can be seen clearly with a TEM. The probability of electron scattering is determined by the density (atomic number) of atoms in the specimen. Biological molecules are composed primarily of atoms with low atomic numbers (H, C, N, and O), and electron scattering is fairly constant throughout an unstained cell or virus. Therefore specimens are further (b) Figure 2.21 Light and Electron Microscopy. A comparison of light and electron microscopic resolution. (a) The proteobacterium Spirillum volutans in phasecontrast light microscope (×1,000). (b) A thin section of another spiral-shaped proteobacterium Rhodospirillum rubrum in transmission electron microscope (×100,000). (a) ©McGraw-Hill Education/James Redfearn, photographer; (b) ©Biology Media/Science Source electrons that is focused on the specimen by the condenser (figure 2.22). Since electrons cannot pass through a glass lens, doughnut-shaped electromagnets called magnetic lenses focus the beam. The column containing the lenses and specimen must be under vacuum to obtain a clear image because electrons are deflected by collisions with air molecules. The specimen scatters some electrons, but those that pass through are used to form an enlarged image of the specimen on a fluorescent screen that interfaces with a computer monitor (figure 2.23). Denser regions in the specimen scatter more electrons and therefore appear darker because fewer electrons strike that area of the screen; these regions are said to be “electron dense.” In contrast, electron-transparent regions are brighter. Table 2.5 compares some of the important features of light and transmission electron microscopes. The TEM has distinctive features that place harsh restrictions on the nature of samples that can be viewed and the means by which those samples must be prepared. Specimens must be viewed in a vacuum and only extremely thin slices (20 to 100 nm) of a specimen can be viewed because electron beams are easily absorbed and scattered by solid matter. To wil11886_ch02_020-039.indd 33 Transmission Electron Microscope Light Microscope Electron gun Lamp Condenser lens Glass Electromagnet Electron beams Light rays Specimen Electromagnet Objective lens Glass Image Ocular lens Glass Eye Electromagnet Viewing screen Figure 2.22 A Comparison of Light and Transmission Electron Microscopes. 22/10/18 7:20 pm 34 CHAPTER 2 | Microscopy Figure 2.23 A Transmission Electron Microscope. The electron gun is at the top of the central column, and the magnetic lenses are within the column. The image on the fluorescent screen is also viewed on a computer monitor. ©McGraw-Hill Education prepared by soaking thin sections with solutions of heavy metal salts such as lead citrate and uranyl acetate. The lead and uranium ions bind to structures in the specimen and make them more electron opaque, thus increasing contrast in the material. Heavy osmium atoms from the osmium tetroxide fixative also stain specimens and increase their contrast. The stained thin sections are then mounted on tiny copper grids and viewed. Table 2.5 Two other important techniques for preparing specimens for TEM include negative staining and shadowing. In negative staining, the specimen is spread out in a thin film with either phosphotungstic acid or uranyl acetate. Just as in negative staining for light microscopy, the specimen appears bright against a dark background, in this case because the heavy metals do not penetrate biological material. Negative staining enables visualization of viruses and cellular microbes, but unlike thin sections, internal structures cannot be discerned (figure 2.24a). In shadowing, a specimen is coated with a thin film of platinum or other heavy metal by evaporation at an angle of about 45 degrees from horizontal so that the metal strikes the microorganism on only one side. In one commonly used imaging method, the area coated with metal appears dark in photographs, whereas the uncoated side and the shadow region created by the object are light (figure 2.24b). This technique is particularly useful in studying virus particle morphology, bacterial and archaeal flagella, and DNA. The process of chemical fixation and dehydration can introduce artifacts that can alter cellular morphology. This can be minimized or avoided by using a freeze-etching procedure. When cells are rapidly frozen in liquid nitrogen, they become very brittle and can be broken along lines of greatest weakness, usually down the middle of internal membranes (figure 2.25). The exposed surfaces are then shadowed and coated with layers of platinum and carbon to form a replica of the surface. After the specimen has been removed chemically, this replica is studied in the TEM, providing a detailed view of intracellular structure. Scanning Electron Microscope Transmission electron microscopes form an image from radiation that has passed through a specimen. The scanning electron microscope (SEM) produces an image from electrons released Characteristics of Light and Transmission Electron Microscopes 1 The resolution limit of a human eye is about 0.2 mm. wil11886_ch02_020-039.indd 34 22/10/18 7:20 pm 2.4 Electron Microscopes Use Beams of Electrons to Create Highly Magnified Images 35 Head Cytoplasm Tail sheath Long tail fibers Cell envelope Flagellum 100 nm Figure 2.25 Example of Freeze-Etching. A freeze-etched preparation (a) T4 virion—negative stain of the bacterium Nitrospira sp. ©Dr. Kari Lounatmaa/SPL/Science Source (b) P. fluorescens—after shadowing Figure 2.24 Stainied Microorganisms Visualized by TEM. (a) T4 is a virus that infects Escherichia coli. (b) Pseudomonas fluorescens with its polar flagella. (a) ©AMI Images/Science Source; (b) ©Dr. Tony Brain/SPL/Science Source Electron gun MICRO INQUIRY Why are all electron micrographs black and white (although they are sometimes artificially colorized after printing)? from an object’s surface. The surfaces of microorganisms are visualized in great detail; most SEMs have a resolution of about 10 nm. Specimen preparation for SEM is relatively easy, and in some cases, air-dried material can be examined directly. However, microorganisms usually must first be fixed, dehydrated, and dried to preserve surface structure and prevent collapse of cells when they are exposed to the SEM’s vacuum. Before viewing, dried samples are mounted and coated with a thin layer of metal to prevent the buildup of an electrical charge on the surface and to give a better image. To create an image, the SEM scans a narrow, tapered electron beam back and forth over the specimen (figure 2.26). When the beam strikes a particular area, surface atoms discharge a tiny shower of electrons called secondary electrons, and these are trapped by a detector. Secondary electrons strike a material in the detector that emits light when struck by electrons (the material is called a scintillator). The flashes of light are converted to an electrical current and amplified by a photomultiplier. The signal is digitized and sent to a computer, where it can be viewed. wil11886_ch02_020-039.indd 35 Scanning coil Condenser lenses Scanning circuit Primary electrons Detector Photomultiplier Secondary electrons Specimen Specimen holder Vacuum system Figure 2.26 The Scanning Electron Microscope. 22/10/18 7:20 pm 36 CHAPTER 2 | Microscopy (a) (b) Figure 2.27 Scanning Electron Micrograph of Mycobacterium tuberculosis. Colorized image (×15,549). Source: CDC/Janice Haney Carr The number of secondary electrons reaching the detector depends on the nature of the specimen’s surface. When the electron beam strikes a raised area, a large number of secondary electrons enter the detector; in contrast, fewer electrons escape a depression in the surface and reach the detector. Thus raised areas appear lighter on the screen and depressions are darker. A realistic three-dimensional image of the microorganism’s surface results (figure 2.27). A single instrument can house both transmission and scanning electron microscopes (S/TEM). SL OM PG PM P FIL ST RIB Figure 2.28 TEM and Electron Cryotomography. A comparison of (a) a thin section of a Caulobacter crescentus cell prepared using conventional TEM procedures and (b) a central slice from a three-dimensional reconstruction of an intact C. crescentus cell. FIL, a bundle of filaments; OM, outer membrane; P, a presumed inclusion; PG, peptidoglycan; PM, plasma membrane; RIB, ribosome; SL, S-layer; ST, stalk. (a) ©AMI Images/SPL/Science Source; (b) ©Grant J. Jensen Electron Cryotomography Comprehension Check Beginning approximately 40 years ago, a series of advances in electron microscopy paved the way for the development of electron cryotomography, a technique that since the 1990s has been providing exciting insights into the structure and function of cells and viruses. Cryo- refers to sample preparation and visualization. Samples are prepared by rapidly plunging the specimen into an extremely cold liquid (e.g., ethane) and the sample is kept frozen while being examined. Rapid freezing of the sample forms vitreous ice rather than ice crystals. Vitreous ice is a glasslike solid that preserves the native state of structures and immobilizes the specimen so that it can be viewed in the high vacuum of the electron microscope. Tomography refers to the method used to create images. The object is viewed from many directions, referred to as a tilt series. The individual images are recorded and processed by computer programs, and finally merged to form a three-dimensional reconstruction of the object. Threedimensional views, slices, and other types of representations of the object can be derived from the reconstruction (figure 2.28). The ultrastructure of bacterial and archaeal cells has been the focus of numerous studies using electron cryotomography. Some of these studies have revealed new cytoskeletal elements, such as those associated with magnetosomes—the inclusions used by some bacteria to orient themselves in magnetic fields (see figure 22.14). 1. Why does an electron microscope have much greater resolution than a light microscope? 2. Where is the electron gun located relative to the sample? Why must a TEM use high vacuum and very thin sections? 3. Under what circumstances would it be desirable to prepare specimens for a TEM by thin section? Negative staining? Shadowing? Freeze-etching? 4. How does a scanning electron microscope differ from a TEM? If you were shown a micrograph, how would you know if it were a TEM, SEM, or electron cryotomography? wil11886_ch02_020-039.indd 36 2.5 Scanning Probe Microscopy Can Visualize Molecules and Atoms After reading this section, you should be able to: a. Distinguish scanning tunneling from atomic force microscopes in terms of how they create images and their uses b. Compare and contrast light microscopy, electron microscopy, and scanning probe microscopy in terms of their uses, resolution, and the quality of the images created Among the most powerful microscopes are scanning probe microscopes (SPMs). These microscopes measure surface features of an object by moving a sharp probe over the object’s surface. One type of 22/10/18 7:20 pm 2.5 Scanning Probe Microscopy Can Visualize Molecules and Atoms 37 SPM is the scanning tunneling microscope. It can achieve magnifications of 100 million times, and it allows scientists to view atoms on the surface of a solid. The scanning tunneling microscope has a needlelike probe with a point so sharp that often there is only one atom at its tip. The probe is lowered toward the specimen surface until its electron cloud just touches that of the surface atoms. When a small voltage is applied between the tip and specimen, electrons flow through a narrow channel in the electron clouds. This tunneling current, as it is called, is extraordinarily sensitive to distance and will decrease about a thousandfold if the probe is moved away from the surface by a distance equivalent to the diameter of an atom. The arrangement of atoms on the specimen surface is determined by scanning the probe tip back and forth over the surface while keeping the probe at a constant height above the specimen. As the tip follows the surface contours by moving up and down, its motion is recorded and analyzed by a computer to create an accurate three-dimensional image of the surface atoms. The surface map can be displayed on a computer screen or plotted on paper. The resolution is so great that individual atoms are observed easily. Even more exciting is that the microscope can examine objects when they are immersed in water. Therefore it can be used to study biological molecules such as DNA (figure 2.29). The microscope’s inventors, Gerd Binnig and Heinrich Rohrer, shared the 1986 Nobel Prize in Physics for their work, together with Ernst Ruska, the designer of the first transmission electron microscope. A second type of SPM is the atomic force microscope, which moves a sharp probe over the specimen surface while keeping the distance between the probe tip and the surface constant. It does this by exerting a very s