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Chapter 2 : Sampling and Staining for Light Microscopy

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Abstract:

Specific morphological details are required to characterize a microorganism; these are usually determined by means of light microscopy, but some require more sophisticated techniques such as laser scanning microscopy or electron microscopy. This chapter concentrates on the general means of characterizing bacteria by light microscopy because it includes the cytological approach to making the best of preparations for study and photomicrography. The field of aerobiology encompasses both indoor and outdoor components and focuses on a wide range of microorganisms, including pathogenic and nonpathogenic varieties. Although the numbers in a unit of volume may be quite small, the simplest approach to air sampling is the use of open petri dishes containing a suitable nutrient agar near a suspected contamination source and is applicable when the concentration of organisms is relatively high. Translational movement of bacteria by flagellar propulsion (swimming) may be observed in wet mounts of specimens by use of in most cases, the low-power or high-dry objectives. To ascertain the presence of flagella in doubtful cases, as well as to determine flagellar distribution (polar, peritrichous, or lateral), staining procedures and electron microscopy may be required. Gram staining is the most important differential technique applied to bacteria. Mature, dormant endospores of bacteria, when viewed unstained, are sharp edged, even sized, and strongly refractile, shining brightly in a plane slightly above true focus. A great number of techniques based on light microscopy are special to particular areas of bacteriology. The chapter gives examples of some special methods which are not routinely used.

Citation: Beveridge T, Lawrence J, Murray R. 2007. Sampling and Staining for Light Microscopy, p 19-33. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch2

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References

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1. Beveridge, T. J. 2001. Use of Gram stain in microbiology. Biotech. Histochem. 76: 111 118.
2. Brock, T. D. 1983. Membrane Filtration: a User’s Guide and Manual. Science Tech Inc., Madison, WI. A complete guide and instruction in the use and interpretation of membrane filter techniques.
3. Brock, T. D., 1987. The study of microorganisms in situ: progress and problems, p. 1 17. In M. Fletcher,, T. R. G. Gray,, and J. G. Jones (ed.), Ecology of Microbial Communities. Cambridge University Press, Cambridge, United Kingdom. An assessment of the applications of counting techniques in microbial ecology.
4. Clark, G. 1973. Staining Procedures Used by the Biological Stain Commission, 3rd ed. The Williams & Wilkins Co., Baltimore, MD. A resource for methods used in association with H. A. Conn’s Biological Stains (10).
5. Hall, G. H.,, J. G. Jones,, R. W. Pickup,, and B. M. Simon. 1990. Methods to study the bacterial ecology of freshwater environments. Methods Microbiol. 23: 181 210. A modern compilation of methods for bacteriological assessment of freshwaters.
6. Hendrickson, D. A.,, and M. M. Krenz,. 1991. Reagents and stains, p. 1289 1314. In A. Balows,, W. J. Hausler, Jr.,, K. L. Herrmann,, H. D. Isenberg,, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 5th ed. American Society for Microbiology, Washington, DC. A part of this chapter gives details of staining methods useful in clinical microbiology.
7. Herbert, R. A. 1990. Methods for enumerating microorganisms and determining biomass in natural environments . Methods Microbiol. 22: 1 39. A useful review and assessment of approaches to estimating biomass in environments.
8. Karl, D. M., 1986. Determination of in situ biomass, viability, metabolism, and growth, p. 85 176. In J. S. Poindexter, and E. R. Leadbetter (ed.), Bacteria in Nature, vol. 2. Plenum Press, New York, NY. Descriptions and assessments of the methods applied to determination of biomass and viable counts in the field.
9. Lawrence, J. R.,, D. R. Korber,, and G. M. Wolfaardt,. 2001. Digital image analyses of microorganisms. In G. Bitton (ed.), Encyclopedia of Environmental Microbiology, John Wiley and Sons, New York, NY. A useful review with clear examples of the application of imageprocessing and analysis techniques.
10. Lillie, R. D. 1977. H. A. Conn’s Biological Stains, 9th ed. The Williams & Wilkins Co., Baltimore, MD. An extensive compendium of stains and dyes with discussion of their chemical structures and applications. Also see reference 4.
11. McKinney, R. M.,, and W. B. Cherry,. 1985. Immunofluorescence microscopy, p. 891 897. In E. H. Lennette,, A. Balows,, W. J. Hausler, Jr.,, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 4th ed. A merican Society for Microbiology, Washington, D.C. A useful source of advice on the practice of immunofluorescence microscopy.
12. Murray, R. G. E.,, R. N. Doetsch,, and C. F. Robinow,. 1994. Determinative and cytological light microscopy, p. 21 41. In P. Gerhardt,, R. G. E. Murray,, W. A. Wood,, and N. R. Kreig (ed.), Methods for General and Molecular Bacteriology, ASM Press, Washington, D.C. A more expanded source for observation of live culures grown on agar, for chemical fixations, and for nucleoid visualization. We highly recommend that you read this chapter!.
13. Newell, S. Y.,, R. D. Fallon,, and P. S. Tabor,. 1986. Direct microscopy of natural assemblages, p. 1 48. In J. S. Poindexter, and E. R. Leadbetter (ed.), Bacteria in Nature, vol. 2. Plenum Press, New York, N.Y. A survey with useful references to studies in the field.
14. Norris, J. R.,, and D. W. Ribbons (ed.). 1971. Methods in Microbiology, vol. 5A. Academic Press, Inc., New York, N.Y. This is an old but useful volume; the material presented in the first part is particularly useful as a source of information supplementary to that presented here. The relevant chapters are as follows. I. Microscopy and micrometry, by L. B. Quesnel, p. 1-103. II. Staining bacteria, by J. R. Norris and H. Swain, p. 105-134. III. Techniques involving optical brightening agents, by A. M. Paton and S. M. Jones, p. 135-144. IV. Motility, by T. Iino and M. Enomoto, p. 145-163.
15. Pickup, R. W. 1991. Development of molecular methods for the detection of specific bacteria in the environment. J. Gen. Microbiol. 137: 1009 1019. A discussion of the strategies for sampling and detecting bacteria in the environment with special reference to genetically modified bacteria.
16. Robinow, C. F., 1960. Morphology of bacterial spores, their development and germination, p. 207 248. In I. C. Gunsalus, and R. Y. Stanier (ed.), The Bacteria, vol. 1. Academic Press, Inc., New York, N.Y.. A classic account of bacterial spores and their study using light microscopy. The same volume contains other illustrated articles on bacterial structure.
17. Robinow, C. F. 1975. The preparation of yeasts for light microscopy. Methods Cell Biol. 11: 1 22. Practical advice on using gelatin-agar slide cultures.
18. Rosebrook, J. A., 1991. Labeled-antibody techniques: fluorescent, radioisotopic, and immunochemical, p. 79 86. In A. Balows,, W. J. Hausler, Jr.,, K. L. Herrmann,, H. D. Isenberg,, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 5th ed. American Society for Microbiology, Washington, D.C. The applications of immunomicroscopy in clinical microbiology.
19. Barer, R.,, R. F. A. Ross,, and S. Thczk. 1953. Refractometry of living cells. Nature (London) 171: 720 724.
20. Bartholomew, J. W. 1962. Variables influencing results, and the precise definition of steps in Gram staining as a means of standardizing the results obtained. Stain Technol. 37: 139 155.
21. Bartholomew, J. W.,, and T. Mittwer. 1952. The Gram stain. Bacteriol. Rev. 16: 1 29.
22. Beveridge, T. J. 1997. The response of S-layered bacteria to the Gram stain. FEMS Microbiol. Rev. 20: 2 10.
23. Beveridge, T. J. 1990. Mechanism of gram variability in select bacteria. J. Bacteriol. 172: 1609 1620.
24. Beveridge, T. J.,, and S. Schultze-Lam. 1997. The response of selected members of the Archaea to the Gram stain. Microbiology 142: 2887 2895.
25. Beveridge, T. J.,, and J. A. Davies. 1983. Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain. J. Bacteriol. 156: 846 858.
26. Buttner, M. P.,, K. Willeke,, and S. A. Grinshpun,. 1996. Sampling and analysis of airborne microorganisms, p. 629 640. In C. J. Hurst,, G. R. Knudsen,, M. McInerney,, L. D. Stetzenbach,, and M. V. Walter (ed.), Manual of Environmental Microbiology. ASM Press, Washington, D.C..
27. Davies, J. A.,, G. K. Anderson,, T. J. Beveridge,, and H. C. Clark. 1983. Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain. J. Bacteriol. 156: 837-– 845. References 22 through 25 and 27 provide an up-to-date explanation of how the Gram stain works and why some prokaryotes stain in an unexpected way. Also see references 42 and 46.
28. Dorner, W. 1926. Un procédé simple pour la coloration des spores. Lait 6: 8 12.
29. Duguid, J. P. 1951. Th e demonstration of bacterial capsules and slime. J. Pathol. Bacteriol. 63: 673.
30. Grossart, H.-P.,, G. F. Steward,, J. Martinez,, and F. Azam. 2000. A simple, rapid method for demonstrating bacterial flagella. Appl. Environ. Microbiol. 66: 3632 3636.
31. Heimbrook, M. E.,, W. L. L. Wang,, and G. Campbell. 1989. Staining bacterial flagella easily. J. Clin. Microbiol. 27: 2612 2615.
32. Henrichsen, J. 1972. Bacterial surface translocation: a survey and a classification. Bacteriol. Rev. 36: 478 503.
33. Hobble, J. E.,, R. J. Daley,, and S. Jasper. 1977. Use of Nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33: 1225 1228.
34. Hotchkiss, R. D. 1948. A microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem. 16: 131 141.
35. Joux, F.,, and P. LeBaron. 1997. Ecological implications of an improved direct viable count method for aquatic bacteria. Appl. Environ. Microbiol. 63: 3643 3647.
36. Leifson, E. 1951. Staining, shape, and arrangement of bacterial flagella. J. Bacteriol. 62: 377 389.
37. Lisle, J. T.,, P. S. Stewart,, and G. A. McFeters,. 1999. Fluorescent probes applied to physiological characterization of bacterial biofilms. p. 166 178. In R. J. Doyle (ed.), Biofilms: Methods in Enzymology. Academic Press, New York, N.Y..
38. Lundgren, B. 1981. Fluorescein diacetate as a stain of metabolically active bacteria in soil. Oikos 36: 17 22.
39. Murray, R. G. E.,, and J. F. Whitfield. 1956. The effects of the ionic environment on the chromatin structures of bacteria. Can. J. Microbiol. 2: 245 260.
40. Newell, S. Y.,, and R. R. Christian. 1981. Frequency of dividing cells as an estimator of bacterial productivity. Appl. Environ. Microbiol. 42: 23 31.
41. Noller, E. C.,, and N. N. Durham. 1968. Sealed aerobic slide culture for photomicrography. Appl. Microbiol. 16: 439 440.
42. Popescu, A.,, and R. J. Doyle. 1996. The Gram stain after more than a century. Biotech. Histochem. 71: 1415 1451.
43. Robinow, C.,, and E. Kellenberger. 1994. The bacterial nucleoid revisited. Microbiol. Rev. 58: 211 232.
44. Robinow, C. E.,, and R. G. E. Murray. 1953. The differentiation of cell wall, cytoplasmic membrane and cytoplasm of Gram-positive bacteria by selective staining. Exp. Cell Res. 4: 390 407.
45. Rogers, F. G.,, and A. W. Pasculle,. 1991. Legionella, p. 442 453. In A. Balows,, W. J. Hausler, Jr.,, K. L. Herrmann,, H. D. Isenberg,, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 5th ed. American Society for Microbiology, Washington, D.C..
46. Salton, M. R. J. 1963. The relationship between the nature of the cell wall and the Gram stain. J. Gen. Microbiol. 30: 223 235.
47. Schaeffer, A. B.,, and M. Fulton. 1933. A simplified method of staining endospores. Science 77: 194.
48. Seto, S.,, G. Layh-Schmitt,, T. Kenri,, and M. Miyata. 2001. Visualization of the attachment organelle and cytoadherence proteins of Mycoplasma pneumoniae by immunofluorescence microscopy. J. Bacteriol. 183: 1621 1630.
49. Tabor, P. S.,, and R. A. Neihof. 1982. Improved method for determination of respiring individual microorganisms in natural waters. Appl. Environ. Microbiol. 43: 1249 1255.
50. Traxler, R. W.,, and J. L. Arceneaux. 1962. Method for staining cells from small inocula. J. Bacteriol. 84: 380.
51. Truant, J. P.,, W. A. Brett,, and W. Thomas. 1962. Fluorescence microscopy of tubercle bacilli stained with auramine and rhodamine. Henry Ford Hosp. Med. Bull. 10: 287 296.
52. Vela, G. R.,, and O. Wyss. 1964. Improved stain for visualization of Azotobacter encystment. J. Bacteriol. 87: 476 477.
53. Wilson, D. R.,, and T. J. Beveridge. 1993. Bacterial flagellar filaments and their component flagellins. Can. J. Microbiol. 39: 451 472.

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