Laboratory Procedures in Clinical Microbiology

869 Pages·1981·22.33 MB·English

by  John A. Washington II M.D. (auth.), John A. Washington II (eds.)

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 e-ISBN-13: 978-1-4684-0118-9
DOl: 10.1007/978-1-4684-0118-9
To the Staff, Fellows, Supervisors, Technologists
and Technicians, and Secretaries and Clerical Per-
sonnel 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 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 identifica-
tion 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 in-
cluded 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 par-
ticular 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
vii
viii Preface
applicable. As has been recognized in all inspection and accreditation pro-
grams, 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 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 ap-
plications 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 manu-
facturers.
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 per-
severance 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
ix
x Contents
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
xii Contents
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