Ultrastructure Techniques for Microorganisms

The modem microbiologist is often a real specialist who has difficulty under­ standing and applying many of the techniques beyond those in his or her own immediate field. On the other hand, most benefits to modem microbiology are obtained when a broad spectrum of scientific approaches can be focused on a problem. In early studies, electron microscopy was pivotal in understanding bacterial and viral morphology, and we still feel that we will understand a disease better if we have seen an electron micrograph of the causative agent. Today, because there is an increased awareness of the need to understand the rela­ tionships between microbial structure and function, the electron microscope is still one of the most important tools microbiologists can use for detailed analysis of microorganisms. Often, however, the aforementioned modem microbiologist still thinks of ultrastructure as involving negative staining or ultrathin sectioning in order to get a look at the shape of a "bug. " Many of the newer ultrastructure techniques, such as gold-labeled antibody localization, freeze-fracture, X-ray microanalysis, enzyme localization, and even scanning electron microscopy, are poorly under­ stood by, and therefore forbidding to, the average microbiologist. Even many cell biologists admit to having difficulty staying in touch with current develop­ ments in the fast-moving field of electron microscopy techniques.

Table of Contents:
1 Preparation of Microfungi for Scanning Electron Microscopy.- 1. Introduction.- 2. Microfungi in Pure Culture.- 3. Fungus-Natural Substrate Associations.- 4. Correlation of Morphological Data.- 5. Limitations of Preparatory Procedures.- 6. References.- 2 Low-Temperature Scanning Electron Microscopy.- 1. Introduction.- 2. Historical Review.- 3. The Apparatus and Methods Used.- 4. A Comparative Evaluation of the Technique.- 5. The Value and Importance of the Technique.- 6. Conclusions.- 7. References.- 3 Effects of Specimen Preparation on the Apparent Ultrastructure of Microorganisms.- 1. Specimen Preparation for Examination of Thin Sections.- 2. Fixation Additives to Improve Detection of Microbial Structures 903..- 3. Mesosomes and Fixation.- 4. Images of Gonococcal Pili.- 5. References.- 4 Secrets of Successful Embedding, Sectioning, and Imaging.- 1. Introduction.- 2. Historical Aspects.- 3. Fixation.- 4. Shrinkage.- 5. Flat Embedment for Light Microscope Selection of Cells.- 6. Other Embedding Tricks.- 7. Microtomy.- 8. Stability of Epoxy Resins.- 9. Tissue and Section Contaminants.- 10. Resin Modifiers for Improving Sectioning.- 11. Image.- 12. Evaluation of Diamond Knives.- 13. Safety.- 14. Appendix: Suggested Reading.- 15. References.- 5 Computer-Aided Reconstruction of Serial Sections.- 1. Introduction.- 2. Literature Review.- 3. Serial Sectioning.- 4. Obtaining the Ultrastructural Data for Computer-Aided Reconstruction.- 5. Entry of Ultrastructural Data into the Computer System.- 6. Production and Manipulation of Computer-Generated Reconstructions.- 7. Conclusions.- 8. References.- 6 Electron Microscopy of Nucleic Acids.- 1. Introduction and History.- 2. Sample Mounting Techniques.- 3. Sample Preparation Requirements.- 4. DNA Analyses.- 5. RNA Analyses.- 6. DNA-RNA Interactions.- 7. Digitizing and Computer Analysis.- 8. Summary and Conclusions.- 9. References.- 7 Freeze-Substitution of Fung.- 1. Introduction.- 2. Fixation Methods Used for Fungi.- 3. Freeze-Substitution.- 4. Recommended Protocol for Preserving Fungi Using Quenching Methodologies.- 5. Typical Results and Criteria for Good Preservation.- 6. Advantages of Using Freeze-Substitution.- 7. Disadvantages of Using Freeze-Substitution.- 8. Cell Systems to Be Studied Using Freeze-Substitution.- 9. References.- 8 Freeze-Fracture (-Etch) Electron Microscopy.- 1. Introduction.- 2. A Brief History.- 3. Methods and Recommendations.- 4. Typical Results.- 5. References.- 9 Preparation of Freeze-Dried Specimens for Electron Microscopy.- 1. Introduction.- 2. Historical Origins.- 3. Methods and Instrumentation.- 4. Examples of Results.- 5. Concluding Remarks.- 6. References.- 10 Low-Temperature Embedding.- 1. Introduction.- 2. Technical Procedures.- 3. Results.- 4. Perspectives.- 5. References.- 11 High-Voltage Electron Microscopy.- 1. Introduction.- 2. The High-Voltage Electron Microscope.- 3. Theoretical Advantages and Disadvantages of High Accelerating Voltages.- 4. Examination of Thick Sections.- 5. Examination of Whole Cells.- 6. Conclusions.- 7. References.- 12 Computer Analysis of Ordered Microbiological Objects.- 1. Introduction.- 2. Diffraction Patterns and Fourier Transforms.- 3. Image Processing.- 4. Three-Dimensional Reconstruction.- 5. Conclusions.- 6. References.- 13 Digitizing and Quantitation.- 1. Introduction.- 2. Historical Background.- 3. Definitions and Symbols.- 4. Digitizing Tablets, Image Analyzers, and Point-Counting.- 5. Methods of Measurement.- 6. References.- 7. Suggested Reading.- 14 Localization of Carbohydrate-Containing Molecules.- 1. Introduction.- 2.Cationic Reagents.- 3. PAS-Based Reactions.- 4. Lectins.- 5. Glycosidic Enzymes.- 6. Carbohydrate-Specific Antibodies.- 7. References.- 15 Cytochemical Techniques for the Subcellular Localization of Enzymes in Microorganisms.- 1. Introduction.- 2. Ion Capture of Enzyme Product.- 3. Ferricyanide Reduction.- 4. Diaminobenzidine Oxidation.- 5. Conclusions.- 6. References.- 16 Localization of Nucleic Acids in Sections.- 1. Introduction.- 2. “Selective” Stains.- 3. “Teulgen”-Type Stains for DNA.- 4. Distinguishing between DNA and RNA.- 5. Gold-Labeled Antibody Staining of Specific Types of RNA.- 6. Gold-Labeled DNase and RNase.- 7. Extraction Methods.- 8. In Situ Hybridization.- 9. Gautier’s ????? Technique.- 10. Summary and Perspectives.- 11. References.- 17 Colloidal Gold Labels for Immunocytochemical Analysis of Microbes.- 1. Introduction.- 2. Production of Gold Colloids.- 3. Coupling Proteins to the Colloidal Gold Sols.- 4. Types of Labeling Made Possible Using Colloidal Gold Complexes.- 5. Application—Analysis of Microbial Surfaces and Surface Structure.- 6. Application—Analysis of Internal Structures and Components.- 7. Applications in Other Types of Microscopy.- 8. Concluding Comments.- 9. References.- 18 X-Ray Microanalysis.- 1. Introduction.- 2. Principles.- 3. Instruments and Their Capabilities.- 4. Specimen Preparation.- 5. Recommendations and Outlook.- 6. References.