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☰ Search Explore Log in Create new account Upload × CHAPTER 4 Microscopy, Staining, and Classification Microscopy (pp. 96–105) Microscopy refers to the use of light or electrons to magnify objects. General Principles of Microscopy The same general principles apply to both light and electron microscopy. Wavelength of Radiation Various forms of radiation differ in wavelength, which is the distance between two corresponding parts of a wave. The human eye distinguishes different wavelengths of light as different colors. Moving electrons also act as waves, with wavelengths dependent on the voltage of an electron beam. Electron wavelengths are much smaller than those of visible light, and thus their use results in enhanced microscopy. Magnification Magnification is the apparent increase in size of an object and is indicated by a number followed by an “”, which is read “times.” Magnification results when a beam of radiation refracts (bends) as it passes through a lens. Resolution Resolution (also called resolving power) is the ability to distinguish between objects that are close together. The better the resolution, the better the ability to distinguish two objects that are close to one another. A principle of microscopy is that resolution distance is dependent on (1) the wavelength of the electromagnetic radiation and (2) the numerical aperture of the lens, which is its ability to gather light. Contrast Contrast refers to differences in intensity between two objects, or between an object and its background. Since most microorganisms are colorless, they are stained to increase contrast. The use of light that is in phase may also be used to enhance contrast. Light Microscopy Several classes of microscopes use various types of light to examine specimens. 22 Copyright © 2014 Pearson Education, Inc. Bright-Field Microscopes The most common microscopes are bright-field microscopes, in which the background (or field) is illuminated. There are two basic types: Simple microscopes contain a single magnifying lens and are similar to a magnifying glass. Compound microscopes use a series of lenses for magnification. Light rays pass through a specimen and into an objective lens immediately above the object being magnified. The objective lens is really a series of lenses, and several objective lenses are mounted on a revolving nosepiece. An oil immersion lens increases the magnification and the resolution. Immersion oil is used to fill the space between the specimen and a lens to reduce light refraction and increase the numerical aperture and thus the resolution. The lenses closest to the eyes are ocular lenses, whereas condenser lenses lie beneath the stage of the microscope and direct light through the slide. The total magnification of a compound microscope is determined by multiplying the magnification of the objective lens by that of the ocular lens. A photograph of a microscopic image is a micrograph. Dark-Field Microscopes Pale objects are best observed with dark-field microscopes, which utilize a dark-field stop in the condenser that prevents light from directly entering the objective lens. Instead, light passes into the slide at an oblique angle. Only light rays scattered by the specimen enter the objective lens and are seen, so the specimen appears light against a dark background. Phase Microscopes Phase microscopes use a phase plate to retard light rays passing through the specimen so that they are ½ wavelength out of phase with neighboring light waves, thereby producing contrast. Phase-contrast microscopes produce sharply defined images in which fine structures can be seen in living cells. Differential interference contrast microscopes create phase interference patterns and use prisms to split light beams into their component colors, giving images a dramatic three-dimensional or shadowed appearance. Fluorescent Microscopes Fluorescent microscopes use an ultraviolet (UV) light source to cause objects to fluoresce. Because UV light has a shorter wavelength than visible light, resolution is increased. Contrast is improved because fluorescing structures are visible against a black background. Confocal Microscopes Confocal microscopes use fluorescent dyes in conjunction with ultraviolet lasers to illuminate the fluorescent chemicals in only one thin plane of a specimen at a time. Several images are taken and digitized, and then computers construct three-dimensional images of the entire specimen. Electron Microscopy Because the shortest wavelength of visible light is about 400 nm, structures closer together than about 200 nm cannot be distinguished using light microscopy. By contrast, electrons traveling as waves have wavelengths between 0.01 nm and 0.001 nm; thus, their resolving power is much greater, and they typically magnify objects 10,000 to 100,000. There are two general types. Copyright © 2014 Pearson Education, Inc. CHAPTER 4 Microscopy, Staining, and Classification 23 Transmission Electron Microscopes A transmission electron microscope (TEM) generates a beam of electrons that passes through a thinly sliced, dehydrated specimen, through magnetic fields that manipulate and focus the beam, and then onto a fluorescent screen that changes the electrons’ energy into visible light. Scanning Electron Microscopes In a scanning electron microscope (SEM), the surface of the specimen is visualized. The SEM then focuses the beam of electrons back and forth across the surface of the coated specimen, scanning it rather than penetrating it. Electrons scattered off the surface of the specimen pass through a detector and a photomultiplier, producing a signal displayed on a monitor. Probe Microscopy Probe microscopes use miniscule electronic probes to magnify specimens more than 100,000,000. There are two types. Scanning tunneling microscopes pass a pointed metallic probe across and above the surface of a specimen and measure the amount of electron flow. They can reveal details on a specimen surface at the atomic level. Atomic force microscopes traverse the tip of the probe lightly on the surface of the specimen. Deflection of a laser beam aimed at the probe’s tip measures vertical movements translated by computer to reveal the specimen’s atomic topography. Staining (pp. 105–112) Both light and electron microscopy use staining—the coloring of specimens with dyes—to increase contrast. Preparing Specimens for Staining Preparing specimens for staining involves making a thin film of organisms—or smear—of the specimen on a slide, and then either passing the slide through a flame (heat fixation) or applying a chemical (chemical fixation) to attach the specimen firmly to the slide. Principles of Staining The colored portion of a dye, known as the chromophore, typically binds to chemicals via covalent, ionic, or hydrogen bonds. Anionic chromophores called acidic dyes or cationic chromophores known as basic dyes are used to stain different portions of an organism to aid viewing and identification. Simple Stains Simple stains are composed of a single basic dye such as crystal violet and involve no more than soaking the smear in the dye and rinsing. Differential Stains 24 MICROBIOLOGY WITH DISEASES BY TAXONOMY, 4e Copyright © 2014 Pearson Education, Inc. Differential stains use more than one dye so that different cells, chemicals, or structures can be distinguished. The Gram stain differentiates between purple-staining Grampositive cells and pink-staining Gram-negative cells. The procedure has four steps: 1. Flood the smear with the primary stain, crystal violet, and rinse. 2. Flood the smear with the mordant, iodine, and rinse. 3. Flood the smear with the decolorizing agent—a solution of ethanol and acetone—and rinse. 4. Flood the smear with the counterstain, safranin, and rinse. The acid-fast stain is used to differentiate cells with waxy cell walls such as cells of Mycobacterium and Nocardia. Endospores cannot be stained by normal techniques because their walls are practically impermeable to all chemicals. Two histological stains are commonly used on tissue samples. The Gomori methenamine silver (GMS) stain is used to detect fungi, while the hematoxylin and eosin (HE) stain reveals the presence of cancer cells. Special Stains Acidic dyes are repulsed by the negative charges on the surface of cells and therefore do not stain them. Such dyes that stain the background and leave the cells colorless are called negative (or capsule) stains. Flagellar stains bind to flagella, increase their diameter, and change their color, all of which increases contrast and makes them visible. Staining for Electron Microscopy Stains used for TEM are chemicals containing atoms of heavy metals, such as lead, which absorb electrons. Classification and Identification of Microorganisms (pp. 112–119) Scientists sort organisms on the basis of mutual similarities into nonoverlapping groups called taxa. Taxonomy is the science of classification and nomenclature (rules of naming). Linnaeus and Taxonomic Categories Carolus Linnaeus invented a system of taxonomy, grouping similar interbreeding organisms into species, species into genera, genera into families, families into orders, orders into classes, classes into phyla, and phyla into kingdoms. He gave each species a descriptive name consisting of a genus name and specific epithet. This practice of naming organisms with two names is called binomial nomenclature. Domains Carl Woese proposed the existence of three taxonomic domains based on three cell types revealed by rRNA sequencing: Eukarya, Bacteria, and Archaea. Cells of the three domains differ in many other characteristics, including their cell membrane lipids, transfer RNA (tRNA) molecules, and sensitivity to antibiotics. Copyright © 2014 Pearson Education, Inc. CHAPTER 4 Microscopy, Staining, and Classification 25 Taxonomic and Identifying Characteristics Taxonomists use one or more of five procedures to identify and classify microorganisms: 1. Many physical characteristics are used to identify microorganisms. For example, scientists can usually identify protozoa, fungi, algae, and parasitic worms based solely on their morphology. The appearance of bacterial colonies also gives clues to help identify microorganisms. 2. Microbiologists also use biochemical tests, noting a particular microbe’s ability to utilize or produce certain chemicals. 3. Serological tests using antiserum (serum containing antibodies) can determine whether a microorganism produces an antigen-antibody reaction in the laboratory. In an agglutination test, antiserum is mixed with a sample that may be antigenic: clumping of antigen with antibodies (agglutination) indicates the presence of the target cells. 4. Bacteriophages (or simply phages) are viruses that infect and usually destroy bacterial cells. Whenever a specific phage is able to infect and kill bacteria, the resulting lack of bacterial growth produces within the bacterial lawn a clear area called a plaque. 5. Scientists also analyze a specimen’s nucleic acid content and sequence. Taxonomic Keys Microbiologists use dichotomous keys, which involve stepwise choices between paired characteristics, to assist in identifying microorganisms. 26 MICROBIOLOGY WITH DISEASES BY TAXONOMY, 4e Copyright © 2014 Pearson Education, Inc. Download 1. Science 2. Biology 3. Microbiology ch_04.doc Chapter 13 Preparing A Bacterial Smear Early recognition and successful treatment of pulmonary Chapter 3 Observing Microorganisms Through A Microscope Lenses and Magnification Methods of Microbiology Gram stain reagents - Bakersfield College Unit 1.2 - Antibiotic Treatment LAB 2: Staining and Streaking Unit II: Bacterial Morphology and Cellular Structures studylib © 2017 DMCA Report