8e2 Examples of Dark-Field and (X17X00 6bharand the b rium respo le for b with its side. Py (100)
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)
er” oter 2 The Study of Micr l Structure:Microscopy and Specimen Preparation geeod ⑧ 一Phase ring Mestdnacadaysotgpnasto miss the phase ring. Figure2 Phase-Contrast Microscopy.The optics of a dark-phase-contrast microscope phase and will cancel each other when they come together to form an and detecting bacterial components such as endo res and in that poly-B-hydrxybyrate.oly contrast mi Phasc
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
22 The Light Microncope 25 acterium ackground. slide.After passing through the specimen,the two beams are com her to form an imag.A live.un pores,granules. vacuoles,and nucleiare clearly visible. The Fluorescence Microscope The microscopes thus far considered produce an image from light that passes through a specimen.An object also can be seen be- ergy a mage contains The emicms light.A mercury vapor ar The Differential Interference Contrast Microscope that transmits ony the desired length orochromes,that fluo e th ugh the spe some mi The mic men,while the reference beam passes through a clear area of the
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 The Differential Interference Contrast Microscope 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 are combined and interfere with each other to form an image. A live, unstained specimen appears brightly colored and three-dimensional (figure 2.11). Structures such as cell walls, endospores, granules, vacuoles, and eucaryotic nuclei are clearly visible. The Fluorescence Microscope The microscopes thus far considered produce an image from light that passes through a specimen. An object also can be seen because it actually emits light, and this is the basis of fluorescence microscopy. When some molecules absorb radiant energy, they become excited and later release much of their trapped energy as light. Any light emitted by an excited molecule will have a longer wavelength (or be of 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 (figure 2.12) exposes a specimen to ultraviolet, violet, or blue light and forms an image of the object with the resulting fluorescent light. A mercury vapor arc lamp or other source produces an intense beam, and heat transfer is limited by a special infrared filter. The light passes through an exciter filter that transmits only the desired wavelength. A darkfield condenser provides a black background against which the fluorescent objects glow. Usually the specimens have been stained with dye molecules, called fluorochromes, that fluoresce brightly upon exposure to light of a specific wavelength, but some microorganisms are autofluorescing. The microscope forms an image of the fluorochrome-labeled microorganisms 2.2 The Light Microscope 25 Phase plate Bacterium Ray deviated by specimen is 1/4 wavelength out of phase. Deviated ray is 1/2 wavelength out of phase. Deviated and undeviated rays cancel each other out. Figure 2.10 The Production of Contrast in Phase Microscopy. The behavior of deviated and undeviated or undiffracted light rays in the darkphase-contrast microscope. Because the light rays tend to cancel each other out, the image of the specimen will be dark against a brighter background. Figure 2.11 Differential Interference Contrast Microscopy. A micrograph of the protozoan Amoeba proteus. The three-dimensional image contains considerable detail and is artificially colored (160)
er” The of Microbial 26 The Study of Microbial Structure:Micmoscopy and Specimen Preparation ng Figure 2.12 Fluore The pri tion of a flu ence microscope. from the light emitted when they fluoresce(figure 2.13). A bar maining traviolet ligh ochromes such as acridine orange and DAPI (diamidino- The fluore scence microscope has become an essential tool of other particulate material.It is even possible to distinguish se of tu he micro organisms can be vic edanddiroctwycountedinarel oy(pp781.831-32
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 from the light emitted when they fluoresce (figure 2.13). A barrier filter positioned after the objective lenses removes any remaining ultraviolet light, which could damage the viewer’s eyes, or blue and violet light, which would reduce the image’s contrast. The fluorescence microscope has become an essential tool in medical microbiology and microbial ecology. Bacterial pathogens (e.g., Mycobacterium tuberculosis, the cause of tuberculosis) can be identified after staining them with fluorochromes or specifically labeling them with fluorescent antibodies using immunofluorescence procedures. In ecological studies the fluorescence microscope is used to observe microorganisms stained with fluorochrome-labeled probes or fluorochromes such as acridine orange and DAPI (diamidino-2- phenylindole, a DNA-specific stain). The stained organisms will fluoresce orange or green and can be detected even in the midst of other particulate material. It is even possible to distinguish live bacteria from dead bacteria by the color they fluoresce after treatment with a special mixture of stains (figure 2.13d). Thus the microorganisms can be viewed and directly counted in a relatively undisturbed ecological niche. Immunofluorescence and diagnostic microbiology (pp. 781, 831–32). 26 Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation 6. Barrier filter Removes any remaining exciter wavelengths (up to about 500 nm) without absorbing longer wavelengths of fluorescing objects 5. Specimen stained with fluorochrome Emits fluorescence when activated by exciting wavelength of light 4. Dark-field condenser Provides dark background for fluorescence Mirror 3. Exciter filter Allows only short wavelength light (about 400 nm ) through 2. Heat filter 1. Mercury vapor arc lamp Eyepiece Objective lens Figure 2.12 Fluorescence Microscopy. The principles of operation of a fluorescence microscope
213 (x bodies tosho dead bacteria are red. 1.List the parts of a light microscope and their functions. 2.Dfine resolution.numricurworking distance.and I,acetic 3.How docs 4.Briefly describe hov wdark-field, Dyes and Simple Staining kind of ime provided by each.each type The many types of dyes used to stain have o regr0Olo charged dye binds to nega- 2.3 Preparation and Staining of Specimens onhkae由edmotogamaldsead ged group specific morphological features,and 1.Basic dyes-methylene blue,basic fuchsin,crystal violet preserve them for future study Fixation The stained cells seen in a microscope should resemble livin is th by which th egatively charged,basic dyes are most ofn used in erved ition.It inactivates enzymes that migh negatively charged groups h as ns cel so tha ther negaivecharge bind cel ng fi structures re two fundamentally different ty The pH mav sfcti since the na s heat I-fix b change i油pH.Thus an but not within cells.(2 Chemical must be used to protect fine cellular substructure and the morpho effective at higher pHs
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 1. List the parts of a light microscope and their functions. 2. Define resolution, numerical aperture, working distance, and fluorochrome. 3. How does resolution depend upon the wavelength of light, refractive index, and the numerical aperture? What are the functions of immersion oil and the substage condenser? 4. Briefly describe how dark-field, phase-contrast, differential interference contrast, and fluorescence microscopes work and the kind of image provided by each. Give a specific use for each type. 2.3 Preparation and Staining of Specimens Although living microorganisms can be directly examined with the light microscope, they often must be fixed and stained to increase visibility, accentuate specific morphological features, and preserve them for future study. Fixation The 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 cells and microorganisms 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. (1) Bacteriologists heat-fix bacterial smears by gently flame heating an air-dried film of bacteria. This adequately preserves overall morphology but not structures within cells. (2) Chemical fixation must be used to protect fine cellular substructure and the morphology of larger, more delicate microorganisms. Chemical fixatives penetrate cells and react with cellular components, usually proteins and lipids, to render them inactive, insoluble, and immobile. Common fixative mixtures contain such components as 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. (1) They have chromophore groups, groups with conjugated double bonds that give the dye its color. (2) They can bind with cells by ionic, covalent, or hydrophobic bonding. For example, a positively charged dye binds to negatively charged structures on the cell. Ionizable dyes may be divided into two general classes based on the nature of their charged group. 1. Basic dyes—methylene blue, basic fuchsin, crystal violet, safranin, malachite green—have positively charged groups (usually some form of pentavalent nitrogen) and are generally sold as chloride salts. Basic dyes bind to negatively charged molecules like nucleic acids and many proteins. Because the surfaces of bacterial cells also are negatively charged, basic dyes are most often used in bacteriology. 2. Acid dyes—eosin, rose bengal, and acid fuchsin—possess negatively charged groups such as carboxyls (—COOH) and phenolic hydroxyls (—OH). Acid dyes, because of their negative charge, bind to positively charged cell structures. The pH may alter staining effectiveness since the nature and degree of the charge on cell components change with pH. Thus anionic dyes stain best under acidic conditions when proteins and many other molecules carry a positive charge; basic dyes are most effective at higher pHs. 2.3 Preparation and Staining of Specimens 27 Figure 2.13 Examples of Fluorescence Microscopy. (a) Escherichia coli stained with fluorescent antibodies (600). The green material is debris. (b) Paramecium tetraurelia conjugating; acridine-orange fluorescence (125). (c) The flagellate protozoan Crithidia luciliae stained with fluorescent antibodies to show the kinetoplast (1,000). (d) A mixture of Micrococcus luteus and Bacillus cereus (the rods). The live bacteria fluoresce green; dead bacteria are red. (a) (b) (c) (d)