Laboratory Procedures in Clinical Microbiology

Laboratory Procedures in Clinical Microbiology

Edited by

John A. Washington

With Contributions by Members of the Section of Clinical Microbiology Department of Laboratory Medicine Mayo Clinic, Rochester, Minnesota

With 195 Figures (43 in Color)

Springer-Verlag New York Heidelberg Berlin

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. Medical microbiology-Technique.

1. Washington, John A. QR46.L18 616'.01'028 81-459

AACR2

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.

All rights reserved.

No part of this book may be translated or reproduced in any form without written permission from Springer-Verlag.

© 1981 by Springer-Verlag New York Inc.

Softcover reprint ofthe hardcover 1st edition 1981

9 8 7 6 5 4 321

ISBN-13: 978-1-4684-0120-2 DOl: 10.1007/978-1-4684-0118-9

e-ISBN-13: 978-1-4684-0118-9

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 micro­biology, 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 irrelevant 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 !is efficiently and accurately as possible. It is also essential in the clinical laboratory to determine, on the basis of the kind of specimen being examined, which microorganisms are clinically relevant and require isolation and identifica­tion and which should either be excluded selectively or simply regarded as indigenous Hora and, therefore, not specifically identified. Cost and time limit a laboratory's resources, and priorities must be established for handling the workload.

The procedures described in this manual are those selected by our staff for use in the clinical laboratory on the basis of clinical relevance, accuracy, reproducibility, and efficiency.' Alternative procedures, when considered equivalent on the basis of personal or published experience, have been in­cluded where appropriate. Since the output 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 laboratory procedures are based on the kind of specimen submitted to the 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

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viii Preface

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 procedures 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 tech­niques with such devices vary so widely among laboratories. Nonetheless, it must be emphasized that the validation of any device is based on com­parisons 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.

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 substitutes may often be a.vailable; however, generic equivalence in product performance should never be assumed because of differences in production among manu­facturers.

A special note of gratitude is due to Mmes. Lola Jaeger, Linda Husser, 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

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 66

Chapter 2 Direct Microscopic Examination of Specimens 69 I. General Considerations 69 II. Specific Indications 70 III. Stains and Wet Mount Preparations 81 References 89

Chapter 3 Initial Processing for Cultures of Specimens 91 I. Blood Cultures 91 II. Cultures of Normally Sterile Body Fluids, Tissue,

Wounds, and Abscesses 95 III. Cultures of Specimens from the Upper Respiratory Tract 101 IV. Cultures of Specimens from the Lower Respiratory Tract 104 V. Cultures of Urine 109 VI. Cultures of Feces 112 VII. Cultures of Genital Tract Infections 113 VIII. Cultures of Integumentary Infections 115 IX. Cultures of Material for Epidemiological Purposes 115 References 124

Chapter 4 Aerobic and Facultatively Anaerobic Bacteria 127

Section 4.1 General Classification of Bacteria 129

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Section 4.2 Identification of Aerobic and Facultatively Anaerobic Bacteria 133

I. Gram-Positive Cocci 133 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 249

I. Overview 249 II. Fluorescent Antibody Techniques 250 III. Counterimmunoelectrophoresis 268 References 276

Section 4.4 Antimicrobial Susceptibility Tests of Aerobic and Facultatively Anaerobic Bacteria 279

I. Overview 279 II. Preparation of Antimicrobial Solutions 285 III. Testing Procedures 288 References 306

Chapter 5 Anaerobic Bacteria 309 I. General Considerations 309 II. Isolation of Anaerobic Bacteria 315 III. Identification of Anaerobic Bacteria 323 IV. Antimicrobial Susceptibility Testing of Anaerobic Bacteria 358 References 363

Chapter 6 Mycobacteria and Nocardia 365 I. General Considerations 365 II. Identification 367 III. Antimicrobial Susceptibility Testing 393 IV. Common Aerobic Actinomycetes 395 v. Identification 397 References 405

Chapter 7 Fungi 407 I. General Considerations 407 II. Identification of Yeasts 411 III. Preliminary Tests 424 IV. Definitive Tests 429 V. Identification of Filamentous Fungi 436 VI. Serodiagnosis of Fungal Infections 472 VII. Antimicrobial Susceptibility Tests of Fungi 473 References 488

Contents xi

Chapter 8 Chlamydiae 493 I. General Considerations 493 II. Cultures for Chlamydia 495 References 506

Chapter 9 Mycoplasmas 509 I. General Considerations 509 II. Identification 511 References 522

Chapter 10 Viruses 525 I. General Considerations 525 II. Processing of Specimens and Cultures 527 III. Cell Cultures 527 IV. Identification of RNA-Containing Viruses 531 V. Identification of DNA-Containing Viruses 576 VI. Reporting of Viral Isolates 598 References 603

Chapter 11 Parasites 609 I. General Considerations 609 II. Procedures Suggested for Use in Examination of Clinical

Specimens for Parasitic Infection 617 III. Processing of Stool Specimens 623 IV. Examination for Blood and Tissue Parasites 646 V. Examination of Specimens Other Than Feces and

Blood (Table 11-11) 657 VI. Serology for Parasitic Diseases 664 VII. Miscellaneous Procedures 665 References 676

Chapter 12 Special Tests of Antimicrobial Activity 679

Section 12.1 Antimicrobial Assays 681 I. Overview 681 II. Bioassays 684 III. Liquid Chromatographic Assays 702 References 713

Section 12.2 Bactericidal Tests 715 I. Minimal Bactericidal Concentration (MBC) 716 II. Serum Antibacterial Titer 719 III. Combination Studies 723 References 726

Chapter 13 Laboratory Safety 729 I. Introduction 729

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II. Routes of Laboratory-Acquired Infections 730 III. Dangerous Chemicals 731 IV. Procedures following Exposure or Accident 731 V. Guidelines for Prevention or Containment of Contamination,

Biohazards, and Infectious Agents 733 VI. Environmental Control 742 VII. Occupational Health 744 References 745

Appendix A Media and Reagents 747 I. Media for Aerobic and Facultatively Anaerobic Bacteria 748 II. Media and Reagents for Anaerobic Bacteria 776 III. Media and Reagents for Mycobacteria and Nocardia 783 IV. Media for Fungi 787 V. Media, Reagents, and Buffers for Chlamydiae,

Mycoplasmas, and Viruses 793 References 806

Appendix B Quality Control 807 I. Overview 807 II. Media and Reagents 810 III. Equipment 837 References 840

Index 841

List of Contributors

John P. Anhalt, Ph.D., M.D. Consultant, Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation; Assistant Professor of Micro­biology and of Laboratory Medicine, Mayo Medical School.

Nelson S. Brewer, M.D. Consultant, Division of Infectious Diseases, Department of Internal Medi­cine, and Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation; Assistant Professor of In­fectious Diseases and Internal Medicine, Mayo Medical School.

Glenn D. Roberts, Ph.D. Consultant, Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation; Assistant Professor of Micro­biology and of Laboratory Medicine, Mayo Medical School.

Jon E. Rosenblatt, M.D. Consultant, Section of Clinical Microbiology, Department of Laboratory Medicine, and Division of Infectious Diseases, Department of Internal Medicine, Mayo Clinic and Mayo Foundation; Associate Professor of Micro­biology and of Internal Medicine, Mayo Medical School.

Thomas F. Smith, Ph.D. Consultant, Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation; Assistant Professor of Micro­biology and of Laboratory Medicine, Mayo Medical School.

John H. Thompson, Ph.D. Consultant, Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation; Professor of Laboratory Medicine, Mayo Medical School.

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xiv Contributors

Edward Warren Administrative Coordinator, Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation.

John A. Washington II, M.D. Head, Section of Clinical Microbiology and Consultant, Section of Clinical Microbiology, Department of Laboratory Medicine; Professor of Micro­biology and of Laboratory Medicine, Mayo Medical School; Professor of Microbiology, University of Minnesota.

Pauline K. W. Yu, M.S. Associate, Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation; Assistant Professor of Micro­biology and of Laboratory Medicine, Mayo Medical School.