Electron Microscopy

Terry J. Beveridge; Dianne Moyles; Bob Harris

doi:10.1128/9781555817497.ch4

Chapter 4 : Electron Microscopy

Authors: Terry J. Beveridge, Dianne Moyles, Bob Harris

Content Type: Reference

Publication Year: 2007

Category: Microbial Genetics and Molecular Biology

Book DOI: 10.1128/9781555817497

Chapter DOI: 10.1128/9781555817497.ch4

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Abstract:

This chapter explains some of the mysteries of electron microscopy (EM), and makes the various techniques more user friendly to researchers who have lost the skills and recognized the importance of its use. The first level of ultrastructural information is provided by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Many new and complex derivatives of EM are available, all with impressive and complicated names, e.g., scanning transmission electron microscopy, energy-dispersive X-ray spectroscopy (EDS), electron energy loss spectroscopy (EELS), electron spectroscopic imaging (ESI), cryoTEM, and cryoSEM; often these techniques can be intermixed with one another. The chapter describes a limited number of TEM methods that are considered useful to the worker who has need of basic structural information about microorganisms. Emphasis is placed on the use of negatively stained preparations and thin-sectioned materials for examination by TEM. The chapter outlines a simple plunging freeze-substitution method. Shadowing was among the first techniques used to visualize bacteria by TEM. The chapter presents several methods of using shadowing (e.g., shadow casting, freeze-etching, and rotary shadowing). It also focuses on image acquisition. The main advantage of digital imaging is the amount of time saved. As the price of electron microscope plate film doubles and triples, the charge-coupled device (CCD) option becomes more attractive. The identities and positions of bacterial macromolecules, either within the cytoplasm or on the surfaces of cells, are often matters of importance to research and require appropriate TEM methods. Finally, the chapter provides a catalog of useful items for EM.

Citation: Beveridge T, Moyles D, Harris B. 2007. Electron Microscopy, p 54-81. 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.ch4

Key Concept Ranking

Viruses: 0.55381846
Chemicals: 0.5391532
Transmission Electron Microscope: 0.5312558
Electron Energy Loss Spectroscopy: 0.45402414
Scanning Electron Microscope: 0.4190874
Scanning Probe Microscopy: 0.4092488

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Electron Microscopy

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FIGURE 1

Thin section of an unidentified gram-negative bacterium fixed via the glutaraldehyde-osmium tetroxide protocol (section 4.2.2.1) showing the gram-negative envelope, condensed nucleoid, and clustered ribosomes produced by this method. An S-layer and capsule or pili (fimbriae) are also present. With the thin sectioning technique, it is difficult to differentiate between a capsule and pili, and a negative stain would help in identification. This is a TEM image.

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FIGURE 1

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FIGURE 2

TEM image of a thin section of a freezesubstituted E. coli K-12 cell showing a well-preserved cytoplasm and cell envelope. Compare the cytoplasm and cell envelope of this cell with those shown in Fig. 1 . (Reprinted from reference 8 with permission of the publisher.)

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FIGURE 2

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FIGURE 3

SEM image of a gold-sputtered Chlorobium sp. covered with special surface appendages called spinae (arrow). Bar = 500 nm. (Reprinted from reference 8 with permission of the publisher.)

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FIGURE 3

SEM image of a gold-sputtered Chlorobium sp. covered with special surface appendages called spinae (arrow). Bar = 500 nm. (Reprinted from reference 8 with permission of the publisher.)

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FIGURE 4

Transmission electron microscope being operated by one of the authors (R. Harris) in the EDS mode to determine the elemental composition of a natural bacterial community.

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FIGURE 4

Transmission electron microscope being operated by one of the authors (R. Harris) in the EDS mode to determine the elemental composition of a natural bacterial community.

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FIGURE 5

TEM image of a negatively stained preparation of isolated membrane vesicles, pili, and pyocin particles from Pseudomonas aeruginosa. Negative stains are particularly good for visualizing small particles for identification.

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FIGURE 5

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FIGURE 6

ESEM image of a wet and unaltered sample of a biofilm growing in a sulfur spring located near Ancaster, Ontario, Canada. Double arrows show the chains of cells (cyanobacteria) growing in the biofilm which are partially obscured by exopolymeric substances. The single arrow points to a break in the exopolymeric substances. This image was kindly supplied by S. Douglas of the NASA-Jet Propulsion Laboratory, Pasadena, CA.

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FIGURE 6

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FIGURE 7

Unstained thin section of a Pseudomonas aeruginosa cell which has accumulated a lanthanum mineral phase on its surface. The lower inset uses SAED to show that it is a crystalline mineral phase due to the periodicity of the reflections in the diffractogram. The inset to the right is an EDS spectrum showing a high La concentration. The P and O could be due to the mineral, the cell, or the plastic. The high Cu is from the copper TEM grid. (From S. Langley and T. J. Beveridge, Can. J. Microbiol. 45:616–622, 1999.)

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FIGURE 7

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FIGURE 8

TEM image of a Mannheimia haemolytica cell expressing an acidic capsule that has been thin sectioned after labeling with PCF (electron-dense particles surrounding the bacterium) to both stabilize and label the capsule.

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FIGURE 8

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FIGURE 9

TEM image of a negatively stained flagellum isolated from Vibrio cholerae. Notice how the stain has penetrated between the flagellin subunits of the filament and the protein subunits of the hook (here artificially straightened). The flagellar rings of the basal body are also well differentiated. (From F. G. Ferris, T. J. Beveridge, M. L. Marseau-Day, and A. D. Larson, Can. J. Microbiol. 30:322–333, 1984.)

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FIGURE 9

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FIGURE 10

TEM image of reciprocal faces of two different dividing Bacillus subtilis cells showing the convex (right side) and concave (left side) fracture planes of the plasma membrane after freeze fracturing and -etching. The black arrows point to the growing septa, and the white arrows point to the edges of the cell walls. The white arrows with the circles denote the shadow direction of the platinum.

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FIGURE 10

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FIGURE 11

Flow diagram for the structural analysis of a microorganism. From the top down, the techniques go from simple to complex, and each has a section reference. The times given for each procedure suppose that all equipment is in place and ready for use. These techniques are for TEM, but SEM can also be appropriate for assessing general shape, growth habitat, and extracellular associations.

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FIGURE 11

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FIGURE 12

TEM image of a thin-sectioned E. coli cell that has been section stained. The cell has been stained by the indirect protein A-colloidal gold method to detect the presence and location of penicillin-binding protein 3 from Pseudomonas aeruginosa, which has been cloned into it. The arrow points to a colloidal gold particle, and many more particles can be seen around the cell periphery.

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FIGURE 12

References

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1. Aldrich, H. C.,, and W. J. Todd (ed.). 1986. Ultrastructure Techniques for Microorganisms. Plenum Press, New York, NY. One of the few books dedicated to EM techniques used on microorganisms, including chapters on conventional techniques (such as chemical fixation and embedding and freeze-etching) and also on more recent techniques (such as cryopreparation, image analysis, and immunolabeling). This book deserves a place in every microbiology EM laboratory.

2. Duke, P. J.,, and A. G. Michette (ed.). 1990. Modern Microscopies: Techniques and Applications. Plenum Press, New York, NY. Most chapters in this book are dedicated to X-ray and nuclear magnetic resonance microscopies, but there are a few worthwhile chapters on cryoTEM and image reconstruction.

3. Dykstra, M. J. 1992. Biological Electron Microscopy: Theory, Techniques, and Troubleshooting. Plenum Press, New York, NY. An up-to-date general overview of TEM and how it is used in biology. There is little information dedicated to microbiology.

4. Glauert, A. M.(ed.). 1972-1991. Practical Methods in Electron Microscopy, vol. 1-15. American Elsevier Publishing Co., Inc., New York, NY. Glauert, a distinguished electron microscopist with an interest in bacterial structure, has edited this remarkable series on state-ofthe- art EM techniques. Most university libraries have the entire set, and it is well worthwhile to look through it. Some of the volumes are available as paperbacks. In particular, Glauert’s part 1 of vol. 3, titled Fixation, Dehydration, and Embedding of Biological Specimens, is a handy 200-page paperback book which describes a full range of fixatives, buffers, stains, and plastics. Very good to have for laboratory recipes.

5. Hajibagheri, M. A. N. (ed.). 1999. Electron Microscopy Methods and Protocols. Humana Press, Totowa, NJ. A soft-cover book edited by Hajibagheri with chapters by many of the experts in EM. Techniques from negative staining to cryoTEM to colloidal gold labeling are covered.

6. Hayat, M. A. 1986. Basic Techniques for Transmission Electron Microscopy. Academic Press, Inc., New York, NY. This 400-page paperback book is filled with helpful hints and general recipes for processing of biological samples for TEM. It deserves a place in every EM laboratory.

7. Hayat, M. A. (ed.). 2000. Principles and Techniques of Electron Microscopy. Biological Applications. Cambridge University Press, Cambridge, United Kingdom. You cannot do EM in biology without running into Hayat’s name. During the 1970s through the 1990s, he published a very successful multivolume set which deals with a full range of important techniques. Like Glauert’s series, this set is available in most university libraries and should be examined. Here is a single book dedicated to biology that is full of useful information; it is 543 pages long.

8. Koval, S. F.,, and T. J. Beveridge,. 2000. Electron microscopy, p. 276– 287. In J. Lederberg (ed.), Encyclopedia of Microbiology. Academic Press, San Diego, CA. A good general overview of the various types of microscopy and how microscopy pertains to microbiology. Nicely illustrated with electron micrographs.

9. Moses, N.,, P. S. Handley,, H. J. Busscher,, and P. G. Rouxhet (ed.). 1991 . Structural and Physico-Chemical Methods for Microbial Cell Surface Analysis. VCH Publishing, New York, NY. Although techniques other than EM are described, this book is dedicated to the elucidation of bacterial surfaces and surveys the most up-to-date methodology.

10. Royal Microscopical Society. Royal Microscopical Society Microscopy Handbooks. Oxford University Press, New York, NY. This is a series of short, soft-covered books dedicated to microscopy. No. 3 (P. J. Goodhew, Specimen Preparation for Transmission Electron Microscopy, 1984), no. 8. (S. K. Chapman, Maintaining and Monitoring the Transmission Electron Microscope, 1988), and no. 20 (D. Chescoe and P. J. Goodhew, The Operation of the Transmission Electron Microscope and the Scanning Electron Microscope, 1990) are particularly good volumes to look at for basic operation of a transmission or scanning electron microscope and for troubleshooting. No. 43 (M. Hoppert and A. Holzenburg, Electron Microscopy in Microbiology , 1998) is particularly recommended because it details how to use SEM and TEM to visualize microorganisms.

11. Sommerville, J.,, and U. Scheer (ed.). 1987. Electron Microscopy in Molecular Biology: a Practical Approach. IRL Press, Oxford, United Kingdom. This book does not deal with microorganisms but offers several methods for visualizing nucleic acids and proteins once they have been extracted from cells.

12. Harris, R. J. 1997 . Royal Microscopical Society Handbook, no. 35. Negative Staining and Cryoelectron Microscopy: the Thin Film Techniques. BIOS Scientific Publishing Ltd., Oxford, United Kingdom. Another of the Royal Microscopical Society handbooks that is right up to date and written by one of the experts who has good microbiological experience.

13. Hobot, J. A. 1990. New aspects of bacterial ultrastructure as revealed by modern acrylics for electron microscopy. J. Struct. Biol. 104: 169– 177. A discourse, with an emphasis on bacteria, that explains the newer acrylic plastics that can be cured from −80 to +60°C.

14. Acetarin, J.-D.,, E. Carlemalm,, and W. Villiger. 1986. Developments of new Lowicryl resins for embedding biological specimens at even lower temperatures. J. Microsc. 143: 81– 88.

15. Armbruster, B. L.,, E. Carlemalm,, R. Chiovetti,, R. M. Caravito,, J. A. Hobot,, E. Kellenberger,, and W. Villiger. 1982. Specimen preparation for electron microscopy using low temperature embedding resins. J. Microsc. 126: 77– 85.

16. Carlemalm, E.,, M. Garavito,, and W. Villiger. 1982. Resin development for electron microscopy and an analysis of embedding at low temperature. J. Microsc. 126: 123– 143.

17. Carlemalm, E.,, W. Villiger,, J. A. Hobot,, J.-D. Acetarin,, and E. Kellenberger. 1985. Low temperature embedding with Lowicryl resins: two new formulations and some applications. J. Microsc. 140: 55– 63.

18. Coggins, L. W., 1987. Preparation of nucleic acids for electron microscopy, p. 1– 29. In J. Sommerville, and U. Scheer (ed.), Electron Microscopy in Molecular Biology: a Practical Approach. IRL Press, Oxford, United Kingdom.

19. Glenney, J. R., Jr., 1987. Rotary metal shadowing for visualizing rod-shaped proteins, p. 167– 178. In J. Sommerville, and U. Scheer (ed.), Electron Microscopy in Molecular Biology: a Practical Approach. IRL Press, Oxford, United Kingdom.

20. Zentgraf, H.,, C. T. Bock,, and M. Schrenk,. 1987. Chromatin spreading, p. 81– 100. In J. Sommerville, and U. Scheer (ed.), Electron Microscopy in Molecular Biology: a Practical Approach. IRL Press, Oxford, United Kingdom.

21. Chapman, R. L.,, and L. A. Staehelin,. 1986. Freezefracture (-etch) electron microscopy, p. 213– 240. In H. C. Aldrich, and W. J. Todd (ed.), Ultrastructure Techniques for Microorganisms. Plenum Press, New York, NY. Although old, this up-to-date chapter is devoted to microorganisms.

22. Hui, S. W. (ed.). 1989. Freeze-Fracture Studies of Membranes. CRC Press, Inc., Boca Raton, FL. Dedicated to the preparation of membranes for freeze fracture and the interpretation of their fracture planes.

23. Adrian, M.,, J. Dubochet,, J. Lepault,, and A. W. McDowall. 1984. Cryo-electron microscopy of viruses. Nature (London) 308: 32– 36. Thin, frozen films of virus particles exemplify exquisite detail against a background of vitreous ice.

24. Amako, K.,, Y. Meno,, and A. Takade. 1988. Fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli K-1. J. Bacteriol. 170: 4960– 4962.

25. Amako, K.,, K. Murata,, and A. Umeda. 1983. Structure of the envelope of Escherichia coli observed by the rapidfreezing and substitution fixation method. Microbiol. Immunol. 27: 95– 99.

26. Amako, K.,, K. Okada,, and S. Miake. 1984. Evidence for the presence of a capsule in Vibrio vulnificus. J. Gen. Microbiol. 130: 2741– 2743.

27. Amako, K.,, and A. Takade. 1985. The fine structure of Bacillus subtilis revealed by the rapid-freezing and substitution- fixation method. J. Electron Microsc. 34: 13– 17. References 24 to 27 are some of the earliest and best freezesubstitution studies on bacteria. Amako was one of the first to apply the technique to bacteria.

28. Beckett, A.,, and N. D. Read,. 1986. Low-temperature scanning electron microscopy, p. 45– 86. In H. C. Aldrich, and W. J. Todd (ed.), Ultrastructure Techniques for Microorganisms. Plenum Press, New York, NY. A helpful chapter on the advantages of low temperatures for SEM.

29. Carlemalm, E.,, W. Villiger,, J.-D. Acetarin,, and E. Kellenberger,. 1985. Low temperature embedding, p. 147– 164. In M. Willer,, R. P. Becker,, A. Boyde,, and J. J. Wolosewick (ed.), The Science of Biological Specimen Preparation for Microscopy and Microanalysis, 1985. Scanning Electron Microscopy, Inc., AMF O’Hare, IL. This Biozentrum group in Basel, Switzerland, developed the concept of low-temperature embedding and the use of Lowicryl resins.

30. Dubochet, J.,, A. W. McDowall,, B. Menge,, E. N. Schmid,, and K. G. Lickfeld. 1983. Electron microscopy of frozen-hydrated bacteria. J. Bacteriol. 155: 381– 390. Shows some of the first and best frozen thin sections of bacteria.

31. Graham, L. L.,, and T. J. Beveridge. 1990. Evaluation of freeze-substitution and conventional embedding protocols for routine electron-microscopic processing of eubacteria. J. Bacteriol. 172: 2141– 2149.

32. Graham, L. L.,, and T. J. Beveridge. 1990. Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freezesubstitution. J. Bacteriol. 172: 2150– 2159.

33. Graham, L. L.,, R. Harris,, W. Villiger,, and T. J. Beveridge. 1991. Freeze-substitution of gram-negative eubacteria: general cell morphology and envelope profiles. J. Bacteriol. 173: 1623– 1633. References 31 and 32 detail the biochemical preservation of bacteria and relate it to ultrastructural detail by comparing conventional methods with freeze-substitution. Reference 33 applies the freeze-substitution technique to a wide range of gramnegative bacteria. These articles will assist any microbiologist contemplating the use of freeze-substitution.

34. Hobot, J. A., 1991. Low temperature embedding techniques for studying microbial cell surfaces, p. 127– 150. In N. Mozes,, P. S. Handley,, H. J. Busscher,, and P. G. Rouxhet (ed.), Structural and Physico-Chemical Methods for Microbial Cell Surface Analysis. VCH Publishing, New York, NY. A good review of the current techniques that use low-temperature methacrylates to better preserve antigenicity and structure.

35. Hobot, J. A.,, E. Carlemahn,, W. Villiger,, and E. Kellenberger. 1984. Periplasmic gel: new concept resulting from the reinvestigation of bacterial cell envelope ultrastructure by new methods. J. Bacteriol. 160: 143– 152.

36. Hobot, J. A.,, W. Villiger,, J. Escaig,, M. Maeder,, A. Ryter,, and E. Kellenberger. 1985. Shape and fine structure of nucleoids observed on sections of ultrarapidly frozen and cryosubstituted bacteria. J. Bacteriol. 162: 960– 971. References 35 and 36 profoundly altered our perception of gram-negative periplasmic spaces (i.e., the concept of a “periplasmic gel”) and the cytoplasmic distribution of the bacterial chromosome.

36a.. Hunter, R. C., and T. J. Beveridge. 2005. High-resolution visualization of Pseudomonas aeruginosa PAO1 biofilms by freeze-substitution transmission electron microscopy. J. Bacteriol. 187: 7619– 7630. Here is an article that shows the power of preservation that the technique of freeze-substitution has on bacterial biofilms. Exopolymeric substances, O side chains of lipopolysaccharide, and cells are all exquisitely maintained.

37a.. Matias, V. R. F.,, A. Al-Amoudi,, J. Dubochet,, and T. J. Beveridge. 2003. Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa. J. Bacteriol. 185: 6112– 6118. An example of cryoTEM and cryosections of Escherichia coli and Pseudomonas aeruginosa emphasizing the structure of the gram-negative envelope and the mass distribution.

37b.. Matias, V. R. F.,, and T. J. Beveridge. 2005. Cryo-electron microscopy reveals native polymeric cell wall structure in Bacillus subtilis 168 and the existence of a periplasmic space. Mol. Microbiol. 56: 240– 251.

37c.. Matias, V. R. F.,, and T. J. Beveridge. 2006. Native cell wall organization shown by cryo-electron microscopy confirms the existence of a periplasmic space in Staphylococcus aureus. J. Bacteriol. 188: 1011– 1021. Both references 37b and c give examples of how gram-positive bacteria look after they have been vitrified by freezing and cryosectioned. Both Bacillus subtilis and Staphylococcus aureus are shown to have a periplasmic space.

37d.. Renelli, M.,, V. Matias,, R. Y. Lo,, and T. J. Beveridge. 2004. Characterization of DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential. Microbiology 150: 2161– 2169. An example of cryoTEM and frozen foils and one of the few articles that shows the mass distribution of intact outer and inner membrane vesicles while in their frozen vitrified state.

37e.. Robards, A. W.,, and U. B. Sleytr. 1985. Practical Methods in Electron Microscopy, vol. 10. Low Temperature Methods in Biological Electron Microscopy. Elsevier Science Publishing, Amsterdam, The Netherlands.

38. Roos, N.,, and A. J. Morgan. 1990. Royal Microscopical Society Handbook, no. 21. Cryopreparation of Thin Biological Specimens for Electron Microscopy. Methods and Applications. Oxford University Press, New York, NY.

39. Steinbrecht, R. A.,, and K. Zierold (ed.). 1987. Cryotechniques in Biological Electron Microscopy. Springer-Verlag KG, Berlin, Germany. References 37b, c, and e, 38, and 39 are books and chapters which describe the principles involved in rapid freezing, the properties of different ice forms, freeze-substitution, frozen thin sections, and thin frozen films.

40. Umeda, A.,, Y. Ueki,, and K. Amako. 1987. Structure of the Staphylococcus aureus cell wall determined by the freeze-substitution method. J. Bacteriol. 169: 2482– 2487. Other than Bacillus subtilis, few gram-positive vegetative cells have been subjected to freeze-substitution. Here we have Staphylococcus aureus.

41. Jacques, M.,, M. Gottschalk,, B. Foiry,, and R. Higgins. 1990. Ultrastructural study of surface components of Streptococcus suis. J. Bacteriol. 172: 2833– 2838. One of the more recent PCF studies on capsules.

42. Sára, M.,, and U. B. Sleytr. 1987. Charge distribution of the S layer of Bacillus stearothermophilus NRS 1536/3c and importance of charged groups for morphogenesis and function. J. Bacteriol. 169: 2804– 2809. Shows both negative stains and freeze-etching on PCF-labeled cell surfaces.

43. Sonnenfeld, E. M.,, T. J. Beveridge,, and R. J. Doyle. 1985. Discontinuity of charge on cell wall poles of Bacillus subtilis. Can. J. Microbiol. 31: 875– 877. Limiting concentrations of PCF are used to locate the most anionic sites on the cell wall.

44. Sonnenfeld, E. M.,, T. J. Beveridge,, A. Koch,, and R. J. Doyle. 1985. Asymmetric distribution of charge on the cell wall of Bacillus subtilis. J. Bacteriol. 163: 1167– 1171. Shows the use of PCF for labeling electronegative sites on Bacillus subtilis walls in thin section, before and after neutralization of charges. Eventually carboxyl and phosphoryl groups are shown to be the major wall anions.

45. Acker, G.,, D. Bitter-Suermann,, U. Meier-Dieter,, H. Peters,, and H. Mayer. 1986. Immunocytochemical localization of enterobacterial common antigen in Escherichia coli and Yersinia enterocolitica. J. Bacteriol. 168: 348– 356.

46. Acker, G.,, and C. Kammerer. 1990. Localization of enterobacterial common antigen immunoreactivity in the ribosomal cytoplasm of Escherichia coli cells cryosubstituted and embedded at low temperature. J. Bacteriol. 172: 1106– 1113. Immunogold studies using conventionally and cryoprepared samples with a distinct E. coli antigen.

47. Behnke, O.,, T. Ammitzboll,, J. Jessen,, M. Klokker,, K. Nflausen,, J. Tranum-Jensen,, and L. Olsson. 1986. Non specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry. Eur J. Cell Biol. 41: 326– 338.

48. Bendayan, M.,, and S. Garzon. 1988. Protein-G-gold complex: comparative evaluation with protein-A-gold for high-resolution immunocytochemistry. J. Histochem. Cytochem. 36: 597– 607. Shows that protein G is better for labeling of IgM antibodies, which make up the widest spectrum of monoclonal antibodies.

49. DeMey, J. 1984. Colloidal gold as a marker and tracer in light and electron microscopy. EMSA Bull. 14: 54– 66. A useful review including some of the most frequently used recipes.

50. Dürrenberger, M.,, M.-A. Bjornsti,, T. Uetz,, J. A. Hobot,, and E. Kellenberger. 1988. Intracellular localization of histone-like protein HU in Escherichia coli. J. Bacteriol. 170: 4757– 4768. This is one of the most carefully controlled studies of thinsection labeling dealing with bacteria. See also reference 54.

51. Dürrenberger, M. B. 1989. Removal of background label in immunocytochemistry with the apolar Lowicryls by using washed protein A-gold-precoupled antibodies in a one-step procedure. J. Electron Microsc. Tech. 11: 109– 116. References 47, 49, and 51 describe some of the problems to be aware of with colloidal gold labeling.

52. Hayat, M. A. (ed.). 1991. Colloidal Gold: Principles, Methods and Applications, vol. 3. Academic Press, Inc., New York, NY.

53. Hicks, D.,, and R. S. Molday,. 1984. Analysis of cell labeling for scanning and transmission electron microscopy, p. 203–219.. In J. R. Revel,, T. Barnard,, and G. H. Haggis (ed.), The Science of Biological Specimen Preparation for Microscopy and Microanalysis. Scanning Electron Microscopy, Inc., AMF O’Hare, IL. References 52 and 53 review the methodology used in immunolabeling. As an encompassing book on the topic, reference 50 is up to date.

54. Hobot, J. A.,, M.-A. Bjornsti,, and E. Kellenberger. 1987. Use of on-section immunolabeling and cryosubstitution for studies of bacterial DNA distribution. J. Bacteriol. 169: 2055– 2064. This goes together with reference 50.

55. Kellenberger, E.,, M. Dürrenberger,, W. Villiger,, E. Carlemalm,, and M. Wurtz. 1987. The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions. J. Histochem. Cytochem. 35: 959– 965. A good explanation of why Lowicryl resins are useful for immunolabeling.

56. Roth, J. 1986. Post embedding cytochemistry with goldlabeled reagents: a review. J. Microsc. 143: 125– 137. Roth and Bendayan are two of the pioneers in immunogold labeling. References 48 and 56 are, then, by the experts.

57. Slot, J. W.,, and H. J. Geuze. 1984. A new method of preparing gold probes for multiple-labeling cytochemistry. Eur. J. Cell Biol. 38: 87– 93.

58. Slot, J. W.,, and H. J. Geuze. 1991. Sizing of protein Acolloidal gold probes for immunoelectron microscopy. J. Cell Biol. 90: 533– 536. Recipes for making and sizing colloidal gold.

59. Smit, J.,, and W. J. Todd,. 1986. Colloidal gold labels for immunocytochemical analysis of microbes, p. 469– 517. In H. C. Aldrich, and W. J. Todd (ed.), Ultrastructure Techniques for Microorganisms. Plenum Press, New York, NY. One of the few review chapters which deals with the immunolabeling of microorganisms.

60. Aldrich, H. C., 1986. X-ray microanalysis, p. 517– 525. In H. C. Aldrich, and W. J. Todd (ed.), Ultrastructure Techniques for Microorganisms. Plenum Press, New York, NY. A chapter dedicated to EDS applied to microorganisms.

61. Beveridge, T. J.,, M. N. Hughes,, H. Lee,, K. T. Leung,, R. K. Poole,, I. Savvaidis,, S. Silver,, and J. T. Trevors. 1996. Metal-microbe interactions. Adv. Microb. Physiol. 38: 177– 243. EDS and many other techniques are discussed with an emphasis on metal ions and geomicrobiology.

62. Budd, P. M.,, and P. J. Goodhew. 1988. Royal Microscopical Society Handbook, no. 16. Light Element Analysis in the Transmission Electron Microscope: WEDS and EELS. Oxford University Press, New York, NY.

63. Chang, C.-F.,, H. Shuman,, and A. P. Somlyo. 1986. Electron probe analysis, X-ray mapping and electron energyloss spectroscopy of calcium, magnesium, and monovalentions in log-phase and in dividing Escherichia coli B cells. J. Bacteriol. 167: 935– 939. One of the few compositional studies of the natural electrolyte concentrations in E. coli. It applies EDS and EELS with a field emission electron source.

64. Johnstone, K.,, D. J. Ellar,, and T. C. Appleton. 1980. Location of metal ions in Bacillus megaterium spores by high-resolution electron probe X-ray microanalysis. FEMS Microbiol. Lett. 7: 97– 101. One of the first EDS studies applied to bacteria. In this case, endospores are analyzed.

65. Morgan, J. A. 1985. Royal Microscopical Society Handbook, no. 15. X-Ray Microanalysis in Electron Microscopy for Biologists. Oxford University Press, New York, NY. References 8, 60, 61, 65, and 67 have good overviews of the topic. The physics and theory are emphasized in references 65 and 67, but references 8, 60, and 61 may be better to start with.

66. Ottensmeyer, F. P., 1984. Electron energy loss analysis and imaging in biology, p. 340– 343. In G. W. Bailey (ed.), Proceedings of the 42nd Annual Meeting of the Electron Microscopy Society of America. San Francisco Press, Inc., San Francisco, CA. A short synopsis of ESI by its pioneering scientist.

67. Sigee, D. C.,, J. Morgan,, A. T. Sumner,, and A. Warley. 1993. X-Ray Microanalysis in Biology. Cambridge University Press, New York, NY.

68. Stewart, M.,, A. P. Somlyo,, A. V. Somlyo,, H. Shuman,, J. A. Lindsay,, and W. G. Murrell. 1980. Distribution of calcium and other elements in cryosectioned Bacillus cereus T spores, determined by high-resolution scanning electron probe X-ray microanalysis. J. Bacteriol. 143: 481– 491.

69. Stewart, M.,, A. P. Somlyo,, A. V. Somlyo,, H. Shuman,, J. A. Lindsay,, and W. G. Murrell. 1981. Scanning electron probe X-ray microanalysis of elemental distribution in freeze-dried cryosections of Bacillus coagulans spores. J. Bacteriol. 147: 670– 674. References 68 and 69 are two meticulous compositional studieson bacterial endospores. The researchers used frozen sections to point map the elements within sectioned spores by EDS.

70. Hayat, M. A. (ed.). 1974. Principles and Techniques of Scanning Electron Microscopy. Biological Applications. Van Nostrand Reinhold Co., New York, NY. A multivolume set, full of good information, that was started in 1974. See volumes 1, 3, and 6 for SEM of spores, microorganisms, and bacteriophages.

71. Joy, D. C., 1984. Resolution in the low-voltage SEM, p. 444– 449. In G. W. Bailey (ed.), Proceedings of the 42nd Annual Meeting of the Electron Microscopy Society of America. San Francisco Press, Inc., San Francisco, CA. Describes the advantages of using low-voltage SEM. Also see the article “Low Voltage SEM,” by J. Pawley, which immediately precedes this one.

72. Passmore, S. M.,, and B. Bole,. 1976. Scanning electron microscopy of microbial colonies, p. 19– 29. In R. Fuller, and D. W. Lovelock (ed.), Microbial Ultrastructure. The Use of the Electron Microscope. Academic Press, Inc., New York, NY. An old reference but still useful.

73. Yoshii, A.,, J. Tokumaga,, and J. Tawara. 1975. Atlas of Scanning Electron Microscopy in Microbiology. Igaku Shoin, Ltd., Tokyo, Japan.

74. Bayer, M. E. 1991. Zones of membrane adhesion in the cryofixed envelope of Escherichia coli. J. Struct. Biol. 107: 268– 280. Adhesion zones, or “Bayer’s patches,” are thought to be zones where secretory proteins and outer membrane constituents are trafficked. Here is an article by the researcher who first discovered them.

75. Bayer, M. E.,, and M. H. Bayer,. 1994. Periplasm, p. 447– 464. In J.-M. Ghuysen, and R. Hakenbeck (ed.), Bacterial Cell Wall. Elsevier Science B. V., Amsterdam, The Netherlands. Many researchers would suggest that this often neglected cell wall constituent, the periplasm, is amongst the most important regions of the cell. Also see references 79, 83, 85, 88, 89, and 91.

76. Bayer, M. E.,, and M. H. Bayer. 1994. Biophysical and structural aspects of the bacterial capsule. ASM News 60: 192– 198. Capsules are amongst the most difficult structures to preserve because most of their mass is water. This reference gives a nice general overview of them.

77. Beveridge, T. J. 1981. Ultrastructure, chemistry, and function of the bacterial wall. Int. Rev. Cytol. 72: 229– 317. A review that correlates bacterial surface chemistry with a structural perspective and discusses what it means in a functional context. Somewhat dated but still worthwhile.

78. Beveridge, T. J., 1989. The structure of bacteria, p. 1– 65. In E. R. Leadbetter, and J. S. Poindexter (ed.), Bacteria in Nature: a Treatise on the Interaction of Bacteria and Their Habitats, vol. 3. Plenum Press, New York, NY. One of the most comprehensive compilations of information on bacterial structure, it is useful reading before interpreting initial EM images.

79. Beveridge, T. J. 1999. Structures of gram-negative cell walls and their derived membrane vesicles. J. Bacteriol. 181: 4725– 4733.

80. Beveridge, T. J., 1999. The ultrastructure of Gram-positive cell walls, p. 3– 10. In V. Fischetti,, R. Novick,, J. Ferretti,, D. Potnoy,, and J. Rood (ed.), Gram-Positve Pathogens. ASM Press, Washington, DC.

81. Beveridge, T. J., 1999. Bacterial cells. In R. Atlas (ed.), Encyclopedia of Life Sciences. [Online.] Macmillan References Ltd., London, United Kingdom. http://www.els.net.

82. Beveridge, T. J., 1999. Archaeal cells. In R. Atlas (ed.), Encyclopedia of Life Sciences. [Online.] Macmillan References Ltd., London, United Kingdom. http://www.els.net.

83. Beveridge, T. J., 1999. Bacterial cell wall. In R. Atlas (ed.), Encyclopedia of Life Sciences. [Online.] Macmillan References Ltd., London, United Kingdom. http://www.els.net. If your university subscribes to the Encyclopedia of Life Sciences, references 81 to 83 should be readily accessible to you and are a good starting point for differentiating bacteria from archaea and for getting an understanding of cell wall structure. Many other good, up-to-date microbiology topics are also covered; in fact, ~3,000 of them will be covered once the online site is completed!

84. Beveridge, T. J.,, and J. W. Costerton (ed.). 1988. Shape and form. Can. J. Microbiol. 34: 363– 420. A compilation of short reviews by experts in the field of bacterial structure. The topics range from structural design strategies to growth and taxis. This compilation occurs in a special edition of the journal dedicated to R. G. E. Murray, who is one of the masters of bacterial structure.

85. Beveridge, T. J.,, and L. L. Graham. 1991. Surface layers of bacteria. Microbiol. Rev. 55: 684– 705. This is a review of the modern perception of the structure of bacterial surfaces. Cryotechniques, diffraction, image processing, immunogold labeling, and probes for charge are all covered.

86. Bohrmann, B.,, W. Villiger,, R. Johansen,, and E. Kellenberger. 1991. Coralline shape of the bacterial nucleoid after cryofixation. J. Bacteriol. 173: 3149– 3158. A wonderful blend of light microscopy and cryoTEM is used to define the shape of the bacterial nucleoid.

87. Costerton, J. W. 1979. The role of electron microscopy in the elucidation of bacterial structure and function. Annu. Rev. Microbiol. 33: 459– 479. A good overview which has stood the test of time.

88. Graham, L. L.,, T. J. Beveridge,, and N. Nanninga. 1991. Periplasmic space and the concept of the periplasm. Trends Biochem. Sci. 16: 328– 329.

89. Graham, L. L.,, R. Harris,, W. Villiger,, and T. J. Beveridge. 1991. Freeze-substitution of gram-negative eubacteria: general cell morphology and envelope profiles. J. Bacteriol. 173: 1623– 1633.

90. Hale, C. A.,, A. C. Rhee, and P. A. De Boer. 2000. Zip A-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains. J. Bacteriol. 182: 5153– 5166. There is increasing interest in the division machinery in gramnegative cells and the way in which various proteins form a primative cytoskeletal complex at the constriction site. Here is a recent reference that combines TEM and fluorescent light microscopy.

91. Hobot, J. A.,, E. Carlemalm,, W. Villiger,, and E. Kellenberger. 1984. Periplasmic gel: a new concept resulting from the reinvestigation of bacterial cell envelope ultrastructure by new methods. J. Bacteriol. 160: 143– 152. References 79, 85, 88, and 91 redefine the concept of the periplasm and the periplasmic space and show the impact freeze-substitution has had on bacterial structure.

92. Jones, C. J.,, and S.-I. Aizawa. 1991. The bacterial flagellum and flagellar motor: structure, assembly and function. Adv. Microb. Physiol. 32: 109– 172. There are more-recent reviews on flagella but this one combines TEM, biophysics, and molecular biology and is by a Japanese group that has performed many good structural studies on this fascinating motility organelle.

93. Krell, P. J.,, and T. J. Beveridge. 1987. The structure of bacteria and molecular biology of viruses. Int. Rev. Cytol. Suppl. 17: 15– 88. An overview of procaryotic and viral structures which was aimed at advanced university undergraduate studies.

94. Nanninga, N. 1998. Morphogenesis of Escherichia coli. Microbiol. Mol. Biol. Rev. 62: 110– 129. This is a review that encompasses most gram-negative structures and describes their functionality while at the same time contemplating what we know and what we still need to know. All students should read this one.

95. Remsen, C. C. 1982. Structural attributes of membranous organelles in bacteria. Int. Rev. Cytol. 76: 195– 223. Like reference 77, this one is rather old but has textbook structural information in it.

96. Robinow, C.,, and E. Kellenberger. 1994. The bacterial nucleoid revisited. Microbiol. Rev. 58: 211– 232. Our list of references would not be complete without one from these two experts Robinow and Kellenberger, who, together, have done more than any other researcher to describe and define that structure that clearly separates procaryotes from eucaryotes, the nucleoid. Light microscopy and TEM at their best are described in this review.

97. Sleytr, U. B.,, and T. J. Beveridge. 1999. Bacterial S-layers. Trends Microbiol. 7: 253– 260. A recent review by two researchers who have marveled at Slayers for over 30 years; it is dedicated to the expansive structural literature on procaryotic S-layers.

98. Stoltz, J. F. (ed.). 1991. Structure of Phototrophic Prokaryotes. CRC Press, Inc., Boca Raton, FL. A review of the structure of cyanobacteria, the purple and green bacteria, and their internal, photosynthetic membranes.

99. van Iterson, W. (ed.). 1984. Benchmark Papers in Microbiology, vol. 17. Inner Structures of Bacteria. Van Nostrand Reinhold Co., Inc., New York, NY.

100. van Iterson, W. (ed.). 1984. Benchmark Papers in Microbiology, vol. 18. Outer Structures of Bacteria. Van Nostrand Reinhold Co., Inc., New York, NY. References 99 and 100 are two wonderful volumes in which van Iterson has compiled the significant papers showing the progressive advance of the understanding of bacterial ultrastructure. Only the significant features of each paper are printed, and these are followed by editorial comments by an experienced microscopist.