Observing Microorganisms Through a Microscope

Transcription

1 Chapter 3 Observing Microorganisms Through a Microscope Units of Measurement Microorganisms are measured by metric units unfamiliar to many of us. The micrometer ( m), formerly known as the micron, is equal to (10 6 ) meter. The prefix micro indicates that the unit following should be divided by one million. A nanometer (nm), formerly known as a millimicron (m ) is equal to (10 9 ) meter. Nano tells us that the unit should be divided by one billion. An angstrom (Å) is equal to (10 10 ) meter. Microscopy: The Instruments Compound Light Microscopy The compound light microscope has two sets of lenses: the objective and the ocular. Specimens magnified by the objective lens magnified 100 times, for example are remagnified by the ocular, usually 10 times. Thus, the total magnification is 1000 times. Most microscopes provide magnifications of 100, 400, and A magnification of 2000 times is about the highest obtainable. The specimen is illuminated by visible light from the light source the illuminator that is passed through a condenser, which directs the light rays through the specimen (Figure 3.1a). Resolution, or resolving power, is the ability of a microscope to distinguish between two points. The shorter the wavelength of the illumination, the better the resolution. The white light used in a compound light microscope limits resolving power to about 0.2 m. The lenses closest to the specimen are the objective lenses. For the highest magnification, it is necessary to use oil immersion objectives. Immersion oil has the same refractive index as glass; that is, the relative velocity of light passing through it is the same. Without immersion oil filling the space between the slide bearing the specimen and the objective, the image will be fuzzy, with poor resolution (Figure 3.1b). Darkfield Microscopy Some microorganisms, such as the thin spirochete Treponema pallidum, which causes syphilis, are best seen with darkfield microscopy. In the darkfield microscope, an opaque disk blocks light from entering the objective directly. The light hits only the sides of the specimen, and scattered light enters the objective and reaches the eyes. The specimen appears white against a black background. Phase-Contrast Microscopy Living microorganisms do not show up well in the ordinary compound light microscope. The phasecontrast microscope takes advantage of subtle differences in the refractive index of different parts of 25

2 26 Chapter 3 Ocular lens (eyepiece) Remagnifies the image formed by the objective lens Body tube Transmits the image from the objective lens to the ocular lens Arm Objective lenses Primary lenses that magnify the specimen Stage Holds the microscope slide in position Condenser Focuses light through specimen Line of vision Body tube Objective lenses Specimen Condenser lenses Ocular lens Path of light Prism Diaphragm Controls the amount of light entering the condenser Coarse focusing knob Illuminator Light source Base Illuminator Base with source of illumination Fine focusing knob (a) Principal parts and functions (b) The path of light (bottom to top) FIGURE 3.1 The compound light microscope. the living cell and its surrounding medium. As light is slowed down in portions of differing density, it travels slightly different pathways. When recombined for viewing, the phase differences are seen as areas of differing brightness. The microorganism (and many of its internal structures) is seen in its natural state, alive and unstained. Differential Interference Contrast (DIC) Microscopy Differential interference contrast microscopy is similar to phase-contrast microscopy. It uses differences in refractive indexes but uses two beams of light instead of one. Prisms split each light beam, adding contrasting colors. Compared to standard phase-contrast microscopes, DIC images are more brightly colored, are nearly three-dimensional in appearance, and have higher resolution. Fluorescence Microscopy Certain fluorochrome dyes, which glow with visible light yellow, for example when illuminated by ultraviolet light, can be used to view and identify microorganisms. This is fluorescence; certain substances, when illuminated by a short wavelength, emit light of a longer wavelength. Fluorescence microscopy techniques use a special microscope with ultraviolet light illumination; this light illuminates the specimen but is not permitted to reach the eye. The stained microorganism is highly visible against a dark background in such a microscope. However, the principal use of these dyes and microscopes is in the fluorescent-antibody technique, or immunofluorescence. In this technique, the organisms are allowed to react on a slide with antibodies (highly specific proteins produced by the body s defense

3 Observing Microorganisms Through a Microscope 27 Antibodies Fluorochrome Antibodies combined with fluorochrome Unknown bacterium Cell-surface antigen molecules Bacterial cell with bound antibodies combined with fluorochrome FIGURE 3.2 The principle of immunofluorescence. A type of fluorochrome is combined with antibodies against a specific type of bacterium. When the preparation is added to bacterial cells on a microscope slide, the antibodies attach to the bacterial cells and the cells fluoresce when illuminated with ultraviolet light. system). A fluorescent dye is attached to the antibody. The combination of the antibody, the attached dye, and the microorganism for which the antibody is specific (called an antigen; it stimulates the body to produce these antibodies) allows the microorganism s presence to be detected (Figure 3.2). Because the antibody is specific for a particular microorganism, this is a very useful diagnostic technique. It is often used for diagnosis of syphilis and rabies. Confocal Microscopy One plane of part of a specimen in confocal microscopy is illuminated with a laser, which passes the returned light through an aperture aligned with the illuminated region. Successive planes and regions can be scanned and a clear two-dimensional image obtained. When used with computers, these images can be used to construct three-dimensional images.

4 28 Chapter 3 Electron Microscopy The wavelengths of electrons, which travel in waves much as light does, are only about 1/100,000 as long as those of visible light and therefore have much better resolving power. They can be focused by magnetic lenses in an electron microscope. Transmission Electron Microscopy. In transmission electron microscopy, a beam of electrons is passed through ultrathin sections of the specimen and focused on a fluorescent screen, where it is visible to the eye and can be photographed. Objects are generally magnified 10,000 times to 100,000 times, and structures, called artifacts, may appear as a result of the method of preparation. Salts of heavy metals may be fixed to the specimen (positive staining) to increase the density and make a darker image. If the metals are used on the surrounding field, it is termed negative staining, which is useful for viewing exceptionally small specimens. The technique of shadow casting produces a three-dimensional effect by spraying a heavy metal at an angle, accumulating on one side and leaving a clear area on the other. This also provides an idea of the size and shape. Scanning Electron Microscope. In scanning electron microscopy, the electron beam is directed at the intact specimen from the top, rather than passing through a section, and electrons leaving the surface of the specimen (secondary electrons) are viewed on a televisionlike screen. Spectacular pictures of seemingly three-dimensional, intact organisms are possible. Objects are generally magnified 1000 times to 10,000 times with a resolving power of about 20 nm. Scanning Tunneling and Atomic Force Microscopy Scanning tunneling microscopy uses a thin metal probe that scans a specimen and produces an image of the bumps and depressions of the atoms on the surface of the specimen. In atomic force microscopy a metal-and-diamond probe is moved along the surface of the specimen. The recorded movements yield a three-dimensional image. Preparation of Specimens for Light Microscopy Preparing Smears and Staining Most microorganisms are viewed in stained preparations; that is, they are colored with a dye to make them visible or to emphasize certain structures. A thin film of a microbial suspension, called a smear, is spread on the surface of a slide. Flaming the air-dried smear coagulates the microbial proteins and fixes the microorganisms to the slide so they do not wash off. The smear can then be stained. Basic dyes have a colored ion that is positive, helping them adhere to bacteria, which are slightly negative. Examples of basic dyes are crystal violet, methylene blue, and safranin. Acidic dyes, having a negative color ion, are more attracted to the background than to the negatively charged bacteria; thus, a field of colorless bacteria is presented against a stained background. This is an example of negative staining. An example of an acidic dye is eosin. Simple Stains To visualize shapes and arrangements of cells, a simple stain is usually sufficient. A chemical called a mordant may be added to make the microorganism stain more intensely or increase its size to enhance visibility. Differential Stains The most useful differential stain is the Gram stain, developed by Hans Christian Gram. It divides bacteria into two large groups: gram-positive and gram-negative. In preparing a Gram stain (1) apply a

5 Observing Microorganisms Through a Microscope 29 purple dye, crystal violet, to a heat-fixed smear. This stains all the cells and is called the primary stain. After a water rinse, (2) an iodine mordant is added. When a smear stained in this manner is (3) washed with ethanol or an ethanol-acetone solution, some species of bacteria are decolorized and others are not. If the smear retains the purple dye, the organism is gram-positive. If the alcohol removes the dye, the colorless microorganisms are no longer visible. (4) Safranin, a red dye, is then applied and the decolorized, or gram-negative, bacteria appear pink. Safranin is used here as a counterstain. The Gram stain reflects a basic difference in the cell wall structure of bacteria. It is a first step in identification, and the susceptibility of microorganisms to antibiotics is often related to the Gram reaction. Acid-Fast Stain. Members of the genera Mycobacterium (which includes the causes of tuberculosis and leprosy) and Nocardia possess a cell wall with waxy components. The red dye carbolfuchsin is more soluble in these waxes than in acid-alcohol and is retained by the cell. Therefore, the acid-fast stain, in which carbolfuchsin is applied and gently steamed for several minutes, will stain them red. This dye is held so firmly that the cells are not decolorized by acid-alcohol, which does remove the dye from bacteria that are not acid-fast. A methylene blue counterstain will produce a slide in which acid-fast organisms are red and others are blue. The acid-fast stain is an invaluable aid in the diagnosis of tuberculosis and leprosy. Special Stains A colloidal suspension of dark particles such as India ink or nigrosin can be used as a capsule stain. The capsule will appear around each bacterial cell as a halo from which the India ink carbon particles are excluded. Endospores do not stain by ordinary methods, but the Schaeffer Fulton endospore stain, which uses malachite green as a primary stain and safranin as a counterstain, shows endospores as green within red or pink cells. Flagella are too small to be resolved by light microscopes. In a flagella stain, a mordant can be used to increase the diameter of the flagella until they are visible in a light microscope.

6 30 Chapter 3 Self-Tests In the matching section, there is only one answer to each question; however, the lettered options (a, b, c, etc.) may be used more than once or not at all. I. Matching 1. The electrons pass through a thin section of the specimen. 2. Visible light passes through the specimen; uses separate objective and ocular lenses. 3. Details become visible because of differences in the refractive index of different parts of the cell. 4. Visible light is scattered after striking the specimen, and the specimen is visible against a darkened background. 5. A special microscope using ultraviolet illumination. a. Compound light microscope b. Scanning electron microscope c. Phase-contrast microscope d. Transmission electron microscope e. Fluorescence microscope f. Darkfield microscope 6. The electrons strike the surface of the specimen, and secondary electrons leaving the surface are viewed on a televisionlike screen. II. Matching 1. Pertaining to the relative velocities of light through a substance. 2. Involves the use of antibodies and ultraviolet light. 3. One millionth of a meter. 4. One ten-billionth of a meter. 5. The ability to separate two points in a microscope field. a. Micrometer b. Nanometer c. Ångstrom d. Resolving power e. Refractive index f. Immersion oil g. Immunofluorescence

7 Observing Microorganisms Through a Microscope 31 III. Matching 1. Adhere(s) best to bacteria, which have a negative charge, because the color molecule has a positive charge. 2. Used in diagnosis of tuberculosis. 3. Involve(s) the use of a negative stain made from India ink particles. 4. Schaeffer Fulton stain. 5. Use(s) carbolfuchsin dye. a. Basic dyes b. Acidic dyes c. Gram stain d. Acid-fast stain e. Capsule stain f. Endospore stain 6. Use(s) malachite green. 7. Reflect(s) a basic difference between microbial cell walls; ethanol will not remove stain from bacteria. IV. Matching 1. A microscope that uses laser illumination. 2. Extremely thin microbes, for example, the spirochete Treponema pallidum, are best seen with this type of light microscope. 3. This type of electron microscope yields images with seemingly three-dimensional views of the specimen. a. Confocal b. Phase contrast c. Darkfield d. Transmission e. Scanning 4. Light rays that pass through different portions of the specimen reach the eye with their wave-peaks reinforced or cancelled, making structures of the specimen relatively light or dark. V. Matching 1. Formerly known as a micron. 2. Formerly known as a millimicron. 3. This is of a meter. 4. A billionth of a meter. a. Micrometer b. Nanometer c. Ångstrom d. Millimeter

8 32 Chapter 3 Fill in the Blanks 1. About the highest magnification possible in a compound light microscope is. 2. Immersion oil has about the same refractive index as. 3. Fluorochrome dyes glow with visible light when illuminated by light. 4. Electron wavelengths are only about 1/100,000 as long as visible light and therefore have much resolving power. (better, poorer) 5. Bacteria tend to have a slightly electrical charge. (positive, negative) 6. The thin film of a microbial suspension spread on the surface of a slide is called a. 7. Flaming the slide before applying the stain is called. 8. Transmission electron microscopy permits magnifications as high as about 10,000 times to. 9. In the flagella stain, a is used to increase the diameter of the flagella. 10. Two bacterial genera that are acid-fast are and. 11. A disease for which the acid-fast stain is useful in diagnosis is. 12. In order to see shapes and arrangements of cells, a stain is usually sufficient. 13. dyes have a negative color ion. (acidic, basic)

9 Observing Microorganisms Through a Microscope 33 Label the Art a. Line of vision Path of light Prism Body tube b. Specimen c. Illuminator Base with source of illumination Critical Thinking 1. The equation that describes the resolving power of a microscope is: Resolving power = Wavelength of illumination/2 Numerical Aperture (The numerical aperture of an oil immersion objective is usually 1.30.) If the wavelength of light is 0.52 µm, what is the resolving power of this objective? 2. What type of microscopy would be most appropriate for viewing the following specimens or for the following situations? a. To identify pathogenic bacteria in clinical specimens. b. To view objects smaller than 0.2 m, such as viruses. c. To view heat-fixed, stained bacterial cells. d. To view microorganisms that can t be stained by standard methods, such as Treponema pallidum. e. To view the internal structure of living microorganisms.

10 34 Chapter 3 3. For each of the following specimens or situations, indicate which stain(s) or technique would be most appropriate. a. To detect bacterial capsules and evaluate an organism s virulence. b. To provide the necessary contrast for viewing specimens with a compound light microscope. c. To diagnose infections of Mycobacterium or Nocardia. d. To help determine what antibiotic will be most effective against a certain disease organism. 4. Why do gram-positive cells retain the crystal violet through the alcohol wash of Gram staining, whereas gram-negative cells do not? Answers Matching I. 1. d 2. a 3. c 4. f 5. e 6. b II. 1. e 2. g 3. a 4. c 5. d III. 1. a 2. d 3. e 4. f 5. d 6. f 7. c IV. 1. a 2. c 3. e 4. b V. 1. a 2. b 3. c 4. b Fill in the Blanks glass 3. ultraviolet 4. better 5. negative 6. smear 7. fixing , mordant 10. Mycobacterium; Nocardia 11. tuberculosis or leprosy 12. simple 13. acidic Label the Art a. ocular lens b. objective lenses c. condenser lenses Critical Thinking 1. It would be about 0.2 µm. (The figure for the wavelength of light is for green light, changed from 520 nm.) 2. a. Fluorescence microscopy b. Electron microscopy c. Brightfield microscopy d. Darkfield microscopy e. Phase-contrast microscopy

11 Observing Microorganisms Through a Microscope a. A negative stain using India ink or nigrosin. The India ink (or nigrosin) stains the background but doesn t penetrate the capsule. The capsule shows up as a halo surrounding the cell against a dark background. b. For this purpose a simple stain such as safranin or methylene blue will work fine. c. Acid-fast staining would be appropriate to diagnose infections of Mycobacterium and Nocardia. The red dye, carbolfuchsin, binds strongly to a waxy substance in the cell wall of these organisms but not to other nonacid-fast bacteria. d. The Gram staining reaction is helpful information when choosing an antibiotic, which often shows specificity for either gram-positive or gram-negative bacteria. 4. When iodine is added to a smear after previous staining with crystal violet, they combine to form a complex (CV-I complex) that is larger than the crystal violet molecule that initially entered the cells. The CV-I complex is too large to be washed out of the intact peptidoglycan layer of gram-positive cells. When decolorizing gram-negative cells, the alcohol washes away the outer lipoprotein layer and the crystal violet from the thin layer of peptidoglycan.

12