Chapter 3 Study Guide PDF

3: Observing Microorganisms Through a Microscope LEARNING OBJECTIVES CHECK YOUR UNDERSTANDING 3-1 ​List the metric units of measurement If a microbe measures 10 µm in length, how long is it in that are used for microorganisms. nanometers? 3-2...

3: Observing Microorganisms Through a Microscope LEARNING OBJECTIVES CHECK YOUR UNDERSTANDING 3-1 ​List the metric units of measurement If a microbe measures 10 µm in length, how long is it in that are used for microorganisms. nanometers? 3-2 ​Diagram the path of light through a Through what lenses does light pass in a compound compound microscope. microscope? 3-3 ​Define total magnification and What does it mean when a microscope has a resolution resolution. of 0.2 nm? 3-4 ​Identify a use for darkfield, phase- How are brightfield, darkfield, phase-contrast, and contrast, differential interference contrast, fluorescence microscopy similar? fluorescence, confocal, two-photon, and scanning acoustic microscopy, and compare each with brightfield illumination. 3-5 ​Explain how electron microscopy Why do electron microscopes have greater resolution differs from light microscopy. than light microscopes? 3-6 ​Identify one use for the TEM, SEM, For what is TEM used? SEM? Scanned-probe and scanned-probe microscopes. microscopy? 3-7 ​Differentiate an acidic dye from a basic Why doesn’t a negative stain color a cell? dye. 3-8 ​Explain the purpose of simple staining. Why is fixing necessary for most staining procedures? 3-9 ​List the steps in preparing a Gram Why is the Gram stain so useful? stain, and describe the appearance of gram- positive and gram-negative cells after each step. 3-10 ​Compare and contrast the Gram stain Which stain would be used to identify microbes in the and the acid-fast stain. genera Mycobacterium and Nocardia? 3-11 ​Explain why each of the following is How do unstained endospores appear? Stained used: capsule stain, endospore stain, flagella endospores? stain. NEW IN THIS EDITION ​Coverage of two-photon microscopy. ​Addition of several new photos. CHAPTER SUMMARY Units of Measurement (p. 55) CHAPTER SUMMARY Units of Measurement (p. 55) ​1. ​The standard unit of length is the meter (m). ​2. ​Microorganisms are measured in micrometers, µm (10–6 m), and in nanometers, nm (10–9 m). Microscopy: The Instruments (p. 55) ​1. ​A simple microscope consists of one lens; a compound microscope has multiple lenses. Light Microscopy (pp. 56, 58–62) Compound Light Microscopy (pp. 56, 58–59) ​2. ​The most common microscope used in microbiology is the compound light microscope (LM). ​3. ​The total magnification of an object is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens. ​4. ​The compound light microscope uses visible light. ​5. ​The maximum resolution, or resolving power (the ability to distinguish two points) of a compound light microscope is 0.2 µm; maximum magnification is 2000x. ​6. ​Specimens are stained to increase the difference between the refractive indexes of the specimen and the medium. ​7. ​Immersion oil is used with the oil immersion lens to reduce light loss between the slide and the lens. ​8. ​Brightfield illumination is used for stained smears. ​9. ​ nstained cells are more productively observed using darkfield, phase-contrast, or DIC U microscopy. Darkfield Microscopy (p. 59) ​10. ​The darkfield microscope shows a light silhouette of an organism against a dark background. ​11. ​It is most useful for detecting the presence of extremely small organisms. Phase-Contrast Microscopy (pp. 59–60) ​12. ​A phase-contrast microscope brings direct and reflected or diffracted light rays together (in phase) to form an image of the specimen on the ocular lens. ​13. ​It allows the detailed observation of living organisms. Differential Interference Contrast (DIC) Microscopy (p. 60) ​14. ​The DIC microscope provides a colored, three-dimensional image of the object being observed. ​15. ​It allows detailed observations of living cells. Fluorescence Microscopy (pp. 61–62) ​16. ​In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet light source. ​17. ​The microorganisms appear as bright objects against a dark background. ​18. ​Fluorescence microscopy is used primarily in a diagnostic procedure called fluorescent- antibody (FA) technique, or immunofluorescence. Confocal Microscopy (p. 62) ​19. ​In confocal microscopy, a specimen is stained with a fluorescent dye and illuminated with short-wavelength light. ​20. ​Using a computer to process the images, two-dimensional and three-dimensional images of cells can be produced. Two-Photon Microscopy (p. 62) ​21. ​In TPM, a live specimen is stained with a fluorescent dye and illuminated with long- wavelength light.  ​21. ​In TPM, a live specimen is stained with a fluorescent dye and illuminated with long- wavelength light. Scanning Acoustic Microscopy (p. 63) ​22. ​Scanning acoustic microscopy (SAM) is based on the interpretation of sound waves through a specimen. ​23. ​It is used to study living cells attached to surfaces such as cancer cells, artery plaque, and biofilms. Electron Microscopy (pp. 63–65) ​24. ​Instead of light, a beam of electrons is used with an electron microscope. ​25. ​Instead of glass lenses, electromagnets control focus, illumination, and magnification. ​26. ​Thin sections of organisms can be seen in an electron micrograph produced using a transmission electron microscope (TEM). Magnification: 10,000–100,000x. Resolving power: 2.5 nm. ​27. ​Three-dimensional views of the surfaces of whole microorganisms can be obtained with a scanning electron microscope (SEM). Magnification: 1000–10,000x. Resolving power: 20 nm. Scanned-Probe Microscopy (p. 65) ​28. ​Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) produce three-dimensional images of the surface of a molecule. Preparation of Specimens for Light Microscopy (pp. 68–72) Preparing Smears for Staining (pp. 68–69) ​1. ​Staining means coloring a microorganism with a dye to make some structures more visible. ​2. ​Fixing uses heat or alcohol to kill and attach microorganisms to a slide. ​3. ​A smear is a thin film of material used for microscopic examination. ​4. ​Bacteria are negatively charged, and the colored positive ion of a basic dye will stain bacterial cells. ​5. ​The colored negative ion of an acidic dye will stain the background of a bacterial smear; a negative stain is produced. Simple Stains (p. 69) ​6. ​A simple stain is an aqueous or alcohol solution of a single basic dye. ​7. ​It is used to make cellular shapes and arrangements visible. ​8. ​A mordant may be used to improve bonding between the stain and the specimen. Differential Stains (pp. 69–71) ​9. ​Differential stains, such as the Gram stain and acid-fast stain, differentiate bacteria according to their reactions to the stains. ​10. ​The Gram stain procedure uses a purple stain (crystal violet), iodine as a mordant, an alcohol decolorizer, and a red counterstain. ​11. ​Gram-positive bacteria retain the purple stain after the decolorization step; gram-negative bacteria do not and thus appear pink from the counterstain. ​12. ​Acid-fast microbes, such as members of the genera Mycobacterium and Nocardia, retain carbolfuchsin after acid-alcohol decolorization and appear red; non–acid-fast microbes take up the methylene blue counterstain and appear blue. Special Stains (pp. 71–72) ​13. ​Negative staining is used to make microbial capsules visible. ​14. ​The endospore stain and flagella stain are special stains that color only certain parts of bacteria. THE LOOP THE LOOP Chapter 3 should provide a good reference for laboratory exercises on microscopy and staining. The test questions can be used as laboratory quizzes. ANSWERS Review 1. ​a. ​10–6 m b. ​nm c. ​103 nm 2. ​a. ​Compound light microscope b. ​Darkfield microscope c. ​ hase-contrast microscope P d. ​ luorescence microscope F e. ​Electron microscope f. ​Differential interference contrast microscope 3. ​ 4. ​Ocular lens magnification x oil immersion lens magnification = total magnification of specimen 1​ 0x x​ 1​ 00x ​ = 1​ 000x 5. ​a. 2​ ,000x c​. 0​.2 µm e​. ​ eeing three-dimensional detail S b. 1​ 00,000x d​. 0​.0025 µm 6. ​In a Gram stain, the mordant combines with the basic dye to form a complex that will not wash out of gram-positive cells. In a flagella stain, the mordant accumulates on the flagella so that they can be seen with a light microscope. 7. ​A counterstain stains the colorless non–acid-fast cells so that they are easily seen through a microscope. 8. ​In the Gram stain, the decolorizer removes the color from gram-negative cells. In the acid-fast stain, the decolorizer removes the color from non–acid-fast cells. 9. ​a. ​Purple d​. ​Purple g​. ​Colorless ​b. ​Purple e​. ​Purple h​. ​Red ​c. ​Purple ​f. ​Purple Critical Thinking 1. ​The counterstain safranin can be omitted. Gram-positive bacteria will appear purple, and gram- negative bacteria will be colorless. 2. ​You would be able to discern two objects separated by the four distances given because each is equal to or greater than the resolving power of the microscope. 3. ​The high lipid content of acid-fast cell walls makes them impermeable to most stains. If the primary stain penetrates, the Gram stain decolorizer will not decolorize the cell. Therefore, acid-fast bacteria would be gram-positive if they could be Gram stained.  primary stain penetrates, the Gram stain decolorizer will not decolorize the cell. Therefore, acid-fast bacteria would be gram-positive if they could be Gram stained. 4. ​Inclusions as well as endospores may not stain in a Gram stain. The endospore stain will identify the unstained structure as an endospore. Clinical Applications 1. ​Ehrlich observed that mycobacteria could not be decolorized with acid-alcohol, so he reasoned that an acidic disinfectant would not be able to penetrate the cell wall. 2. ​N. gonorrhoeae bacteria are gram-negative (red) diplococci, often found in the large human cells (phagocytes). 3. ​These are called clue cells. The large red cells are human mucosal cells; gram-positive bacteria on the surface of the human cells. 4. ​The presence of acid-fast rods suggests the elephant had a mycobacterial infection. Subsequent cultures verified that the elephant had tuberculosis. CASE STUDY: ELECTRON MICROSCOPY Background Samples prepared for transmission electron microscopy are embedded in an epoxy resin and sliced into ultrathin (100-nm) sections. The sections are usually stained with a heavy metal such as lead to enhance contrast. The sections are then examined with a transmission electron microscope. The photographs of these thin sections are put in order according to their position in the living cell and used to determine the shape of the original sample. Question Determine the appearance of the original, intact cell. The Solution A spirochete; see Figure 4.10. The solid area is the cross section of the cell body, and the “dots” are cross sections of axial filaments. The changing positions of the cell and axial filaments are due to the spiraling of the axial filaments around the cell.