Methods in Mycoplasmology V1

Methods in Mycoplasmology, Volume I: Mycoplasma Characterization is the first of a two-volume series that resulted from cumulative efforts to meet the need for standardized techniques in mycoplasmology. The book is organized into five sections. Section A provides an introduction to the mycoplasmas by discussing their characteristics and classification, mycoplasma-animal host interrelationships, and mycoplasma-plant-insect interrelationships. The chapters in Section B examine the morphological and ultrastructural features of mycoplasmas, using techniques such as phase-contrast microscopy, dark-field microscopy, and transmission electron microscopy. Section C discusses the cultivation and nutrition of mycoplasmas. Section D focuses on analytical methods for the characterization of mycoplasmas, including harvest and washing procedures, cell lysis and membrane isolation, and crossed immunoelectrophoresis. Section E describes tests designed to detect specific enzymatic activities or nutritional requirements of mycoplasmas. These include sugar fermentation and arginine hydrolysis tests as well as tests to establish the sterol requirement of mycoplasmas. Section F deals with serological tests. These tests either have specific application to detection of mycoplasma antigens or antibody in plant and animal tissues; are useful in identification of specific groups of mycoplasmas (spiroplasmas); or provide higher sensitivity in measurements of antibodies against mycoplasmas.

Contenu

Contributors
Preface
Contents of Volume II
Section A Introduction
A1 Characteristics of the Mycoplasmas as a Group
A2 Principles of Mycoplasma Classification and Taxonomy
A3 Mycoplasma-Animal Host Interrelationships
A4 Mycoplasma-Plant-lnsect Interrelationships
Section B Morphology and Ultrastructure
B1 Introductory Comments
B2 Phase-Contrast Microscopy
B3 Dark-Field Microscopy
B4 Gram Staining Technique
B5 Transmission Electron Microscopy: Basic Techniques
B6 Scanning Electron Microscopy of Mycoplasmas
B7 Specialized Electron Microscopic Techniques for Mycoplasmas in Animal Tissues
B8 Specialized Electron Microscopic Techniques for Mycoplasma-Like Organisms in Plant Tissues
B9 Specialized Electron Microscopic Techniques for Spiroplasmas in Plant and Insect Tissues
B10 Transfer of Spiroplasmas onto Collodion Membranes for Electron Microscopic Studies
B11 Identification of Mycoplasma Colonies
B12 Examination of Mycoplasmas for Motility
Section C Cultivation and Nutrition
C1 Introductory Remarks
C2 General Cultivation Techniques for Mycoplasmas and Spiroplasmas
C3 Collection and Transport of Specimens
C4 Growth Inhibitory Factors in Animal and Plant Tissues
C5 Precautions in Handling Infectious Material
C6 Sterility and Quality Control of Mycoplasma Culture Media
C7 Culture Media for Classical Mycoplasmas
C8 Culture Media for Ureaplasmas
C9 Culture Media for Spiroplasmas
C10 Culture Media for Anaeroplasmas
C11 Defined and Partly Defined Media
C12 Cloning and Filtration Techniques for Mycoplasmas
C13 Labeling of Cellular Components during Growth
C14 Methods for Direct and Indirect Measurement of Mycoplasma Growth
C15 Preservation of Mycoplasma Cultures and Culture Collections
C16 Bacterial and Fungal Inhibitors in Mycoplasma Culture Media
C17 Optimal Temperature and Atmospheric Conditions for Growth
Section D Analysis of Cellular Components
D1 Introductory Remarks
D2 Harvest and Washing of Mycoplasmas
D3 Cell Lysis and Isolation of Membranes
D4 Manipulation of Membrane Lipid Composition
D5 Electrophoretic Characterization of Mycoplasma Proteins
D6 Characterization of Membrane Proteins by Crossed Immunoelectrophoresis
D7 Characterization of Membrane Lipids
D8 Characterization of Membrane Lipoglycans
D9 Characterization of Carbohydrate Components of Mycoplasma Membranes
D10 DNA Extraction and Purification
D11 Determination of Guanine Plus Cytosine Content of DNA
D12 Genome Size Determination
D13 EcoRI Restriction Enzyme Analysis of Mycoplasma DNA
D14 Hybridation between Mycoplasma DNAs
D15 Localization of Enzymes in Mycoplasmas: Preparatory Steps
Section E Biochemical and Enzymatic Tests in Mycoplasma Identification
E1 Introductory Remarks
E2 Sugar Fermentation
E3 Arginine Hydrolysis
E4 Urea Hydrolysis
E5 Tests for Digitonin Sensitivity and Sterol Requirement
E6 Phosphatase Activity
E7 Proteolytic Activity
E8 Film and Spot Production
E9 Pigmented Carotenoids
E10 Tetrazolium Reduction
E11 Hemagglutination, Hemadsorption, and Hemolysis
E12 Detection of ß-D-Glucosidase: Hydrolysis of Esculin and Arbutin
E13 Isozyme Resolution in Mycoplasmas
Section F Serological Identification of Mycoplasmas
F1 Introductory Remarks
F2 Preparation of Antigens and Antisera
F3 Growth Inhibition Tests
F4 Metabolism Inhibition Tests
F5 Tetrazolium Reduction Inhibition
F6 The Mycoplasmacidal Test (MCT)
F7 Immunoflourescence
F8 Agar Precipitin and Immunoelectrophoretic Methods for Detection of Mycoplasmic Antigens
F9 Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Anti-Mycoplasmal Antibody
F10 Application of ELISA to Spiroplasma Detection and Classification
F11 The Combined Deformation-Metabolism Inhibition Test
F12 Single Radial Hemolysis Technique
F13 Growth Precipitation Test
Appendix: Tables 1-4
Index