Laboratory Procedures in Clinical Microbiology

892 Pages·1985·19.12 MB·English

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

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