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Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 2. The Study of Microbial Structure: Microscopy and Specimen Preparation © The McGraw−Hill Companies, 2002 (figure 2.8a,b); because the background is dark, this type of microscopy is called dark-field microscopy. Considerable internal structure is often visible in larger eucaryotic microorganisms (figure 2.8b). The dark-field microscope is used to identify bacteria like the thin and distinctively shaped Treponema pallidum (figure 2.8a), the causative agent of syphilis. The Phase-Contrast Microscope Unpigmented living cells are not clearly visible in the brightfield microscope because there is little difference in contrast between the cells and water. Thus microorganisms often must be fixed and stained before observation to increase contrast and create variations in color between cell structures. A phase-contrast microscope converts slight differences in refractive index and cell density into easily detected variations in light intensity and is an excellent way to observe living cells (figure 2.8c–e). The condenser of a phase-contrast microscope has an annular stop, an opaque disk with a thin transparent ring, which produces a hollow cone of light (figure 2.9). As this cone passes through a cell, some light rays are bent due to variations in density and refractive index within the specimen and are retarded by about 1 4 wavelength. The deviated light is focused to form an image of the object. Undeviated light rays strike a phase ring in the phase plate, a special optical disk located in the objective, while the deviated rays miss the ring and pass through the rest of the plate. If the phase ring is constructed in such a way that the undeviated light passing through it is advanced by 1 4 wavelength, the deviated and undeviated waves will be about 1 2 wavelength out of 22 Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation Dark-field stop Abbé condenser Specimen Objective Figure 2.7 Dark-Field Microscopy. The simplest way to convert a microscope to dark-field microscopy is to place (a) a dark-field stop underneath (b) the condenser lens system. The condenser then produces a hollow cone of light so that the only light entering the objective comes from the specimen. (a) (b)
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Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 2. The Study of Microbial Structure: Microscopy and Specimen Preparation © The McGraw−Hill Companies, 2002 2.2 The Light Microscope 23 (a) (b) (c) (d) (e) Figure 2.8 Examples of Dark-Field and Phase-Contrast Microscopy. (a) Treponema pallidum, the spirochete that causes syphilis; dark-field microscopy (500). (b) Volvox and Spirogyra; dark-field microscopy (175). Note daughter colonies within the mature Volvox colony (center) and the spiral chloroplasts of Spirogyra (left and right). (c) Spirillum volutans, a very large bacterium with flagellar bundles; phase-contrast microscopy (210). (d) Clostridium botulinum, the bacterium responsible for botulism, with subterminal oval endospores; phase-contrast microscopy (600). (e) Paramecium stained to show a large central macronucleus with a small spherical micronucleus at its side; phase-contrast microscopy (100)
eh” eeaeaeoee Phase plate the phasea ass the Figure2 Phase-Contrast Microscopy.The optics ofa dark-phase-contrast microscope. phase and will can eleach other when they come together to form an and detecting bacterial components such as endospores and in m and well-detined These are clearly visible (figure2.8d)because they havere Phas otrast microscon y is especially useful for study- ntras microscopes also are widely used in studying eucary ing microbial motility,determining the shape of living cells. otic cells
Prescott−Harley−Klein: Microbiology, Fifth Edition I. Introduction to Microbiology 2. The Study of Microbial Structure: Microscopy and Specimen Preparation © The McGraw−Hill Companies, 2002 phase and will cancel each other when they come together to form an image (figure 2.10). The background, formed by undeviated light, is bright, while the unstained object appears dark and well-defined. This type of microscopy is called dark-phase-contrast microscopy. Color filters often are used to improve the image (figure 2.8c,d). Phase-contrast microscopy is especially useful for studying microbial motility, determining the shape of living cells, and detecting bacterial components such as endospores and inclusion bodies that contain poly- -hydroxybutyrate, polymetaphosphate, sulfur, or other substances (see chapter 3). These are clearly visible (figure 2.8d) because they have refractive indexes markedly different from that of water. Phasecontrast microscopes also are widely used in studying eucaryotic cells. 24 Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation Dark image with bright background results Image plane Amplitude contrast is produced by light rays that are in reverse phase. Phase ring Phase plate Most diffracted rays of light pass through phase plate unchanged because they miss the phase ring. Diffracted rays are retarded 1/4 wavelength after passing through objects. Annular stop Condenser Direct light rays are advanced 1/4 wavelength as they pass through the phase ring. Figure 2.9 Phase-Contrast Microscopy. The optics of a dark-phase-contrast microscope