Laboratory Procedures in Clinical Microbiology Second Edition Laboratory Procedures in Clinical Microbiology Second Edition Edited by John A. Washington With Contributions by Members of the Section of Clinical Microbiology Department of Laboratory Medicine Mayo Clinic, Rochester, Minnesota With 117 Figures (24 in Color) Springer-Verlag New York Heidelberg Berlin Tokyo John A. Washington II Section of Clinical Microbiology Mayo Clinic 200 Southwest First Street Rochester, Minnesota 55905 Library of Congress Cataloging in Publication Data Main entry under title: Laboratory procedures in clinical microbiology. Includes bibliographies and index. 1. Diagnostic microbiology-Laboratory manuals. I. Washington, John A., 1936- . II. Mayo Clinic. Section of Clinical Microbiology. [DNLM: 1. Diagnosis, Laboratory. 2. Microbiology-laboratory manuals. QW 25 L12348] QR67.L33 1985 616'.01'028 84-23659 © 1981, 1985 by Mayo Foundation. Softcover reprint of the hardcover 2nd edition 1985 All rights reserved. No part of this book may be translated or reproduced in any form without written permission from Springer-Verlag, 175 Fifth Avenue, New York, New York 10010, USA. The use of general descriptive names, trade names, trademarks, etc. in this publication, even if the former are not especially identified, is not to be taken as a sign that such names, as understood by the Trade Marks and Merchandise Marks Act, may accordingly be used freely by anyone. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Typeset by Kingsport Press, Kingsport, Tennessee. 9 8 7 6 5 4 3 2 1 ISBN-13: 978-1-4612-9550-1 e-ISBN-13: 978-1-4612-5070-8 001: 10.1007/978-1-4612-5070-8 To the Staff, Fellows, Supervisors, Technologists and Technicians, and Secretaries and Clerical Personnel of the Section of Clinical Microbiology who have contributed so much and without whom none of this would be possible. Preface Although there are a number of comprehensive books in clinical micro biology, there remains a need for a manual that can be used in the clinical laboratory to guide the daily performance of its work. Most of the existing publications provide detailed and precise information, for example, by which a microorganism can be characterized and identified beyond any doubt; however, the number of tests involved in this process exceeds the capabilities and resources of most clinical laboratories and are irrele vant for patient care. It is, therefore, necessary in any clinical laboratory to extract from reference manuals, textbooks, and journals those tests and procedures that are to be used to complete the daily workload as efficiently and accurately as possible. It is also essential in the clinical laboratory to determine, on the basis of the kind of specimen being exam ined, which microorganisms are clinically relevant and require isolation and identification and which should either be excluded selectively or simply regarded as indigenous flora and, therefore, not specifically identi fied. Cost and time limit a laboratory's resources, and priorities must be established for handling the workload. The procedures described in the second edition of this manual are those selected by our staff for use in the clinical laboratory on the basis of clinic~l relevance, accuracy, reproducibility, and efficiency. Alternative procedures, when considered equivalent on the basis of personal or pub lished experience, have been included where appropriate. Since the out put or reports from the laboratory are only as good or valid as the quality of its input, since microbiologists are often consulted about the types of specimens recommended for particular examinations, and since most lab oratory procedures are based on the kind of specimen submitted to the vii viii Preface laboratory, emphasis has been placed on proper techniques of specimen selection, collection, transport, and processing. Clearly, not all the procedures we have described will be universally applicable. As has been recognized in all inspection and accreditation programs, laboratories vary considerably in the levels or extents of their work. Since, however, the rational basis for selecting procedures should be based on evaluations performed in laboratories with a sufficiently large workload to generate statistically valid data, it is hoped that the proce dures described in this manual will provide a basis for other laboratories' selection of procedures. Little emphasis has been placed in this manual on the use of kits or automated equipment since the reasons for replacing conventional tech niques with such devices vary so widely among laboratories. Nonetheless, it must be emphasized that the validation of any device is based on com parisons of its results with those of conventional techniques. It, therefore, remains essential for users of devices to understand the principles and applications of conventional techniques. Since the last edition of this manual, substantial changes have occurred in the taxonomy and nomenclature of the Enterobacteriaceae. Novel approaches have provided increased sensitivity and speed in detecting bacteremia and fungemia, as well as for detecting infections due to Yer sinia enterocolitica, Legionella, Mycobacterium, Chlamydia trachoma tis, herpes viruses, and cytomegaloviruses. Simple, rapid: and specific immu noassays are rapidly replacing biological and chromatographic assays for determining aminoglycoside and vancomycin levels in serum and the body fluids. Immunological procedures for rapid detection of all sorts of microbial antigens are in various stages of development and evaluation. The extent to which the sensitivity and specificity of such procedures approaches those of cultures will determine their role in the clinicallabo ratory of the future. Specific products and, in certain instances, their manufacturers are cited when their performance has been proved satisfactory or when their use has been shown to be essential for a test's accuracy. Acceptable substi tutes may often be available; however, generic equivalence in product performance should never be assumed because of differences in produc tion among manufacturers. A special note of gratitude is due to Lola Jaeger and Roberta Kondert for their care in setting and their patience and perseverance in resetting our ideas into manuscript form. John A. Washington II, M.D. Contents Preface vii Contributors xiii Chapter 1 Collection and Handling of Specimens 1 I. General Considerations 1 II. General Instructions for Specimen Collecting 5 III. Specific Guidelines for Specimen Collection 24 References 67 Chapter 2 Direct Examination of SpeCimens 71 I. General Considerations 71 II. Specific Indications 73 III. Stains and Wet Mount Preparations 85 References 92 Chapter 3 Initial Processing for Cultures of SpeCimens 95 I. Blood Cultures 95 II. Cultures of Normally Sterile Body Fluids, Tissue, Wounds, and Abscesses 97 III. Cultures of Specimens from the Upper Respiratory Tract 103 IV. Cultures of Specimens from the Lower Respiratory Tract 106 V. Cultures of Urine llO VI. Cultures of Feces 113 VII. Cultures of Genital Tract Infections 115 VIII. Cultures of Integumentary Infections 119 ix X Contents IX. Cultures of Material for Epidemiological Purposes 119 References 121 Chapter 4 Aerobic and Facultatively Anaerobic Bacteria 125 Section 4.1 General Classification of Bacteria 127 Section 4.2 Identification of Aerobic and Facultatively Anaerobic Bacteria 131 I. Gram-Positive Cocci 131 II. Gram-Negative Cocci 157 III. Gram-Positive Bacilli 164 IV. Gram-Negative Bacilli 179 References 241 Section 4.3 Fluorescent Antibody Procedures and Counterimmunoelectrophoresis 251 I. Overview 251 II. Fluorescent Antibody Techniques 253 III. Counterimmunoelectrophoresis 270 References 278 Section 4.4 Antimicrobial Susceptibility Tests of Aerobic and Facultatively Anaerobic Bacteria 281 I. Overview 281 II. Preparation of Antimicrobial Solutions 288 III. Testing Procedures 291 References 310 Chapter 5 Anaerobic Bacteria 315 I. General Considerations 315 II. Isolation of Anaerobic Bacteria 321 III. Identification of Anaerobic Bacteria 330 IV. Antimicrobial Susceptibility Testing of Anaerobic Bacteria 373 References 377 Chapter 6 Mycobacteria and Nocardia 379 I. General Considerations 379 II. Identification 381 III. Antimicrobial Susceptibility Testing 408 IV. Common Aerobic Actinomycetes 411 References 417 Chapter 7 Fungi 419 I. General Considerations 419 II. Identification of Yeasts 426 III. Definitive Tests 440 Contents xi IV. Identification of Filamentous Fungi 446 V. Serodiagnosis of Fungal Infections 487 VI. Antimicrobial Susceptibility Tests of Fungi 487 References 497 Chapter 8 Chlamydiae 501 I. General Considerations 501 II. Cultures for Chlamydia 503 References 514 Chapter 9 Mycoplasmas 519 I. General Considerations 519 II. Identification 521 References 532 Chapter 10 Viruses 537 I. General Considerations 537 II. Processing of Specimens and Cultures 539 III. Cell Cultures 539 IV. Identification of RNA-Containing Viruses 543 V. Identification of DNA-Containing Viruses 585 VI. Reporting of Viral Isolates 611 VII. Viral Serology 611 References 616 Chapter 11 Parasites 625 I. General Considerations 625 II. Procedures Suggested for Use in Examination of Clinical Specimens for Parasitic Infection 634 III. Processing of Stool Specimens 637 IV. Examination for Blood and Tissue Parasites 659 V. Examination of Specimens Other Than Feces and Blood 669 VI. Serology for Parasitic Diseases 674 VII. Miscellaneous Procedures 686 References 685 Chapter 12 Special Tests of Antimicrobial Activity 689 Section 12.1 Antimicrobial Assays 691 I. Overview 691 II. Bioassays 695 III. Liquid Chromatographic Assays 709 References 726 xii Contents Section 12.2 Bactericidal Tests 731 I. Minimal Bactericidal Concentration (MBC) 732 II. Serum Antibacterial Titer (SIT) and Serum Bactericidal ru~~n 7~ III. Combination Studies 739 References 743 Appendix A Laboratory Safety 747 I. Introduction 747 II. Routes of Laboratory-Acquired Infections 748 III. Dangerous Chemicals 749 IV. Procedures following Exposure or Accident 750 V. Guidelines for Prevention or Containment of Contamination, Biohazards, and Infectious Agents 752 VI. Environmental Control 763 VII. Occupational Health 765 References 766 Appendix B Media and Reagents 769 I. Media for Aerobic and Facultatively Anaerobic Bacteria 770 II. Media and Reagents for Anaerobic Bacteria 800 III. Media and Reagents for Mycobacteria and Nocardia 808 IV. Media for Fungi 811 V. Media, Reagents, and Buffers for Chlamydiae, Mycoplasmas, and Viruses 816 References 829 Appendix C Quality Control 831 I. Overview 831 II. Media and Reagents 833 III. Equipment 861 References 864 Index 865