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Category: Microbial Genetics and Molecular Biology
Electron Microscopy, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap04-1.gif /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap04-2.gifAbstract:
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.
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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.
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.
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.)
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.)
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.)
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.)
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.
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.
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.
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.
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.
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.
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.)
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.)
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.
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.
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.)
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.)
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.
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.
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.
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.
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.
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.