Chapter 2 : Sampling and Staining for Light Microscopy

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $30.00

Preview this chapter:
Zoom in

Sampling and Staining for Light Microscopy, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap02-1.gif /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap02-2.gif


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

Key Concept Ranking

Scanning Electron Microscopy
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


1. Beveridge, T. J. 2001. Use of Gram stain in microbiology. Biotech. Histochem. 76:111118.
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. 117. 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. 23:181210. A modern compilation of methods for bacteriological assessment of freshwaters.
6. Hendrickson, D. A.,, and M. M. Krenz,. 1991. Reagents and stains, p. 12891314. 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. 22:139. 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. 85176. 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. 891897. In E. H. Lennette,, A. Balows,, W. J. Hausler, Jr.,, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 4th ed. American 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. 2141. 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. 148. 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. 137:10091019. 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. 207248. 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. 11:122. Practical advice on using gelatin-agar slide cultures.
18. Rosebrook, J. A., 1991. Labeled-antibody techniques: fluorescent, radioisotopic, and immunochemical, p. 7986. 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) 171:720724.
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. 37:139155.
21. Bartholomew, J. W.,, and T. Mittwer. 1952. The Gram stain. Bacteriol. Rev. 16:129.
22. Beveridge, T. J. 1997. The response of S-layered bacteria to the Gram stain. FEMS Microbiol. Rev. 20:210.
23. Beveridge, T. J. 1990. Mechanism of gram variability in select bacteria. J. Bacteriol. 172:16091620.
24. Beveridge, T. J.,, and S. Schultze-Lam. 1997. The response of selected members of the Archaea to the Gram stain. Microbiology 142:28872895.
25. Beveridge, T. J.,, and J. A. Davies. 1983. Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain. J. Bacteriol. 156:846858.
26. Buttner, M. P.,, K. Willeke,, and S. A. Grinshpun,. 1996. Sampling and analysis of airborne microorganisms, p. 629640. 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 6:812.
29. Duguid, J. P. 1951. The 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. 66:36323636.
31. Heimbrook, M. E.,, W. L. L. Wang,, and G. Campbell. 1989. Staining bacterial flagella easily. J. Clin. Microbiol. 27:26122615.
32. Henrichsen, J. 1972. Bacterial surface translocation: a survey and a classification. Bacteriol. Rev. 36:478503.
33. Hobble, J. E.,, R. J. Daley,, and S. Jasper. 1977. Use of Nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33:12251228.
34. Hotchkiss, R. D. 1948. A microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem. 16:131141.
35. Joux, F.,, and P. LeBaron. 1997. Ecological implications of an improved direct viable count method for aquatic bacteria. Appl. Environ. Microbiol. 63:36433647.
36. Leifson, E. 1951. Staining, shape, and arrangement of bacterial flagella. J. Bacteriol. 62:377389.
37. Lisle, J. T.,, P. S. Stewart,, and G. A. McFeters,. 1999. Fluorescent probes applied to physiological characterization of bacterial biofilms. p. 166178. 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 36:1722.
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. 2:245260.
40. Newell, S. Y.,, and R. R. Christian. 1981. Frequency of dividing cells as an estimator of bacterial productivity. Appl. Environ. Microbiol. 42:2331.
41. Noller, E. C.,, and N. N. Durham. 1968. Sealed aerobic slide culture for photomicrography. Appl. Microbiol. 16:439440.
42. Popescu, A.,, and R. J. Doyle. 1996. The Gram stain after more than a century. Biotech. Histochem. 71:14151451.
43. Robinow, C.,, and E. Kellenberger. 1994. The bacterial nucleoid revisited. Microbiol. Rev. 58:211232.
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. 4:390407.
45. Rogers, F. G.,, and A. W. Pasculle,. 1991. Legionella, p. 442453. 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. 30:223235.
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. 183:16211630.
49. Tabor, P. S.,, and R. A. Neihof. 1982. Improved method for determination of respiring individual microorganisms in natural waters. Appl. Environ. Microbiol. 43:12491255.
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. 10:287296.
52. Vela, G. R.,, and O. Wyss. 1964. Improved stain for visualization of Azotobacter encystment. J. Bacteriol. 87:476477.
53. Wilson, D. R.,, and T. J. Beveridge. 1993. Bacterial flagellar filaments and their component flagellins. Can. J. Microbiol. 39:451472.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error