Manual for the determination of the clinical role of anaerobic microbiology

91 Pages·1981·10.06 MB·English

by  Gall

Manual for the
Determination of
the Clinical Role of
Anaerobic
Microbiology
Authors
Lorraine S. Gall
President
Bacti-Consult Associates
Houston, Texas
Phyllis E. Riely
Manager
Microbiological Development
Marion Laboratories, Inc.
Kansas City, Missouri
Boca Raton London New York
CRC Press, Inc.
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Taylor & FBraonccias GRrouapt,o ann ,in Ffolromrai bduasi ness
First published 1981 by CRC Press
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Reissued 2018 by CRC Press
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Library of Congress Cataloging in Publication Data
Gall, Lorraine S.
Manual for the determination of the clinical
role of anaerobic microbiology.
Bibliography: p.
Includes index.
1. Bacteria, Anaerobic—Identification. 2. Bacterial
diseases–Diagnosis. 3. Diagnosis, Laboratory.
I. Riely, Phyllis E. II. Title. [DNLM: 1. Bacteria—
Isolation and purification. 2. Bacteriological
technics. QY 100 G162m]
QR67.G35 616’.014 81-147
ISBN 0-8493-5935-X AACR2
A Library of Congress record exists under LC control number: 81000147
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ISBN 13: 978-1-315-89517-8 (hbk)
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PREFACE
Clinicians are becoming more aware and concerned about anaerobic bacterial
infections as more is learned about these anaerobic bacteria. As a result, many
clinical bacteriologists and pathologists are being asked to decide whether to add
the routine culture of anaerobes to their laboratory analysis of body fluids and tissue
specimens, and if so, in what depth should the examination be conducted. This is
not a simple decision and must be based on a consideration of all the aspects of
anaerobic culturing. An overall evaluation of the situation can be made for each
laboratory by considering the several steps in an anaerobic microbiological analysis
with respect to the techniques available and their requirements for equipment, lab-
oratory space, costs, and technical staff. The important role of the attending phy-
sician is recognized as well. An attempt will be made in this manual to provide the
information to perform this evaluation for each individual laboratory.
The first three chapters of this manual are introductory in nature, dealing with a
working concept of what comprise clinically important anaerobes and the general
requirements for a microbiological analysis of these anaerobes. The next four chap-
ters discuss several procedures for the four principal steps in the analysis-collection
and transport of the specimens, primary culturing and isolation, identification, and
antibiotic susceptibility testing of the clinically important anaerobe-in sufficient
detail to form the basis for a decision on which of the alternate methods will best
suit the individual laboratory. The next chapters contain a discussion of the re-
sponsibilities of the professional staff and a look at the future developments apt to
occur in anaerobic bacteriology. The last chapter summarizes the information from
previous chapters that is pertinent to the decision on the role of anaerobic micro-
biology in an individual laboratory. Much of this summary is presented in tabular
form and there are blank tables provided for the senior personnel of the laboratories
to fill in their choice of procedures, to aid in arriving at the overall requirements for
the total anaerobic analysis, and guiding the personnel in selecting the best com-
bination of techniques for their individual laboratory.
THE AUTHORS
Lorraine S. Gall, Ph.D., has had a long career in microbiological research with
emphasis for many years on anaerobic bacteriology. She pioneered in the isolation
and characterization of the predominant rumen bacteria, which are fastidious an-
aerobes, and related their occurrence to the diet of the ruminant. This led to her
study of the predominant anaerobes in the human intestinal tract and the influence
of diet on these fastidious anaerobes. Many of these studies were conducted in
connection with NASA projects during which an assessment was also made of
anaerobes present in or on the body of subjects held in simulated space chambers.
In isolating these bacteria, many different approaches to culturing anaerobes were
evaluated for their usefulness under various conditions including their use in clinical
laboratories.
Dr. Gall received her B.S. and Ph.D. from Cornell University and had a post-
doctorate fellowship to Yale University. She is the author of more than 100 scientific
publications, predominantly on anaerobes. Recently she has become active in the
field of automated microbiology and has CO-authoreda CRC Uniscience book on
this subject. Dr. Gall's work has been recognized internationally and she has been
included in American Men of Science and several Who's Who, such as Who's Who
of Women, Education, Commerce and Industry, Dictionary of International Biog-
raphy, International Register of Profiles, North American edition, Book of Honor
and others. She was awarded a Fulbright Senior Research A grant to New Zealand
to study rumen anaerobes and teach her techniques for isolating these bacteria.
Dr. Gall has recently retired from Baylor College of Medicine and is currently a
microbiological consultant.
Phyllis E. Riely received a degree in Medical Technology from Huron Road Hos-
pital after 3 years of Pre-med at Capital University, Columbus, Ohio. She has spent
the last 20 years in Applied Microbiology, where her practical approach has resulted
in many patented products including a Waste Management System for space explo-
ration. She has published in the field of Anaerobic Microbiology and Indigenous
Microflora. She is listed in Who's Who in Technology Today.
She is currently Manager of Microbiological Development for Marion Laboratories.
ACKNOWLEDGMENT
We wish to acknowledge the valuable assistance of Dr. Vera L. Sutter in reviewing
and commenting on a significant portion of the manuscript and Dr. Lillian V. Holde-
man for reviewing the material covering the VPI anaerobic system.
We also wish to thank Marion Laboratories, Inc. for their support and assistance
in researching the literature and in typing the manuscript.
Dedicated to Dr. Vera L. Sutter for her
many significant contributions to the field of
anaerobic microbiology
TABLE OF CONTENTS
Chapter 1
The Role of Anaerobic Bacteriology in the Clinical Laboratory ....................... 1
I . Introduction .................................................................................. 1
I1. Factors Entering Decision for an Anaerobic Bacteriology Laboratory ..... 1
A . Technical Aspects ..................................................................... 1
B . Practical Aspects ...................................................................... 1
C . Personnel Aspects ..................................................................... 2
D . Future Aspects ......................................................................... 2
E . Evaluation Process .................................................................... 2
I11 . Subject Matter of Manual ................................................................ 2
IV . Literature Sources .......................................................................... 3
Chapter 2
Clinically Important Anaerobic Bacteria ....................................................
I . Introduction ..................................................................................
I1 . What is an Anaerobe? .....................................................................
A . Cause of Anaerobiosis ...............................................................
B . Definition of an Anaerobe ..........................................................
C . Degrees of Anaerobiosis .............................................................
I11 . Which are the Clinically Important Anaerobes? ...................................
A . Clinically Important Anaerobes ...................................................
B . How are These Clinically Important Anaerobes Selected? ................
IV . Classification and Taxonomy of Anaerobes ........................................
References ..........................................................................................
Chapter 3
Requirements for Anaerobic Microbiological Analysis ................................... 9
I . Introduction .................................................................................. 9
I1. Requirements for Anaerobic Microbiological Analysis .......................... 9
A . Collection and Transport of Anaerobic Specimens .......................... 9
1. Selection of Appropriate Specimens ......................................... 9
2 . Selection of Collection Method ........................................ 9
3 . Protection from Oxygen ......................................................... 10
4 . Precautions during Transport .................................................. 10
5 . Summary ............................................................................. 10
B . Culturing of Anaerobic Specimens ............................................... 10
l . Protection from Oxygen ......................................................... 11
2 . Media ................................................................................. 11
3 . Summary ............................................................................. 11
C . Identification ............................................................................ 11
1. Methods of Identification ....................................................... 11
2 . Levels of Identification .......................................................... 12
3 . Anaerobic Conditions during Growth ....................................... 12
4 . Media ................................................................................. 12
5. Pure Cultures ....................................................................... 13
6 . Summary ............................................................................. 13
D . Susceptibility Testing ................................................................. 13
1 . Protection from Oxygen ......................................................... 13
2 . Phase of Growth and Concentration of Inoculum ........................ 14
3 . Media ................................................................................. 14
4 . Pure Cultures ....................................................................... 14
5 . Summary ............................................................................. 14
References ....................................................................................... 14
Chapter 4
Collection and Transport of Specimens for Anaerobic Culturing ..................... 15
I . Introduction ................................................................................. 15
I1 . Collection of Specimen ................................................................... 15
A . Indications for Anaerobic Culturing .............................................. 15
1. Diagnostic Indications ........................................................... 15
2. Type of Infection .................................................................. 15
3. Suitable Body Sites ............................................................... 16
B . Methods of Collection ................................................................ 16
1. Specific Methods .................................................................. 16
2 . General Comments ................................................................ 16
111 . Transport of the Specimen ............................................................... 17
A . Simple Laboratory-Prepared Transport Devices ............................. 17
B . Commercial Devices for Anaerobic Transport ................................ 17
l . Anaerobic Culturette @ (Marion Scientific) ............................... 18
2. Vacutainer @ Anaerobic Specimen Collector (B.D.) ................... 18
3. Transwab @ (Medical Wire and Equipment Co.) ........................ 20
4 . Anaport @ and Anaswab @ (Scott Laboratories, Inc.) ................. 20
5. Anacult @ (Lab-Tek) ............................................................. 20
C . Evaluation of Transport Device Summary of Relevant Features ........ 21
References .......................................................................................... 21
Chapter 5
Commonly Used Techniques for the Primary Culturing and Isolation of Clinically
Important Anaerobic Bacteria ................................................................. 23
I . Introduction .................................................................................. 23
I1 . Preparation of Specimens and Media ................................................. 23
A . Preparation of the Specimen ...................................................... 23
B . Media for Primary Cultures ........................................................ 24
I . Freshly Prepared Enriched Blood Agar (BAP) ........................... 24
2 . Kanamycin-Vancomycin Laked Blood Agar (KVLB) .................. 24
3 . Bacteroides Bile Esculin (BBE) ............................................... 24
4 . Phenylethyl Alcohol Blood Agar (PEA) .................................... 24
5. Media for Microaerophilic and Aerobic Bacteria ........................ 24
6 . Supplemented Thioglycollate .................................................. 24
7 . Other Media ........................................................................ 24
8 . Gram Stain .......................................................................... 24
I11 . Methods of Isolation of Anaerobes .................................................... 25
A . Roll Tube or Streak Tube Technique for Isolation of Anaerobes with
Prereduced Anaerobically Sterilized Media (PRAS) ........................ 25
1. Overview of Technique ........................................................ 25
2 . Prereduced Anaerobically Sterilized Media (PRAS) ................... 25
3 . Inoculation of PRAS in Streak Tubes with the Specimen ........... 26
. Preparation of Specimen for Inoculation .............................. 26
ri
4 . Inoculation of the Media ....................................................... 26
5 . Isolation of Colonies ............................................................ 26
6 . Special Techniques .............................................................. 28
7 . Time Schedule of Analysis .................................................... 28
8 . Special Requirements for Roll or Steak Tube Technique ............ 28
a . Special Requirements for Prereduced Media Preparation ....... 28
b . Unique Equipment for Roll or Streak Tube Technique .......... 28
c . General Anaerobic Culturing Requirements Used with Roll or
Streak Tube Method ........................................................ 30
9 . Features and Benefits of Roll or Streak Tube Technique ............ 30
10. Disadvantages and Limitations of Roll or Streak Tube Method ... 30
11 . Other Relevant Factors Influencing Decision ........................... 30
B . Anaerobic Chamber Technique .................................................... 31
1. Preparation of the Specimen and Inoculation ............................. 31
2 . Incubation ........................................................................... 31
3 . Isolation .............................................................................. 31
4 . Special Equipment for Anaerobic Chamber Technique ................ 31
5 . Features and Benefits ............................................................ 33
6 . Limitations ........................................................................ 33
7 . Other Relevant Factors Influencing Decision ............................. 33
C . Anaerobic Jar Technique ............................................................ 33
I . Description of Technique ....................................................... 33
2 . Specimen Preparation, Media, and Inoculation .......................... 35
3 . Incubation ........................................................................... 35
4 . Isolation .............................................................................. 35
5 . Special Equipment and Reagents Needed for Anaerobic Jar
Techniques .......................................................................... 35
6 . Features and Benefits ............................................................ 35
7 . Limitations .......................................................................... 36
8. Other Relevant Factors Influencing Decision ............................. 36
D . Individual Plate Pack (BioIBag Q) ................................................ 36
1. Description of Technique ....................................................... 36
2 . Specimen Preparation, Media. and Inoculation .......................... 36
3 . Incubation ........................................................................... 36
4 . Isolation .............................................................................. 36
5 . Special Equipment and Reagents Needed for Bio-Bag @ Technique 37
6 . Features and Benefits ............................................................ 37
7 . Limitations .......................................................................... 39
8 . Other Relevant Factors Influencing Decision ............................. 39
IV . Summary ...................................................................................... 39
References .......................................................................................... 39
Chapter 6
Methods of Identification of Clinically Important Anaerobes .......................... 41
I . Introduction .................................................................................. 41
I1 . Methods of Identification ................................................................. 41
A . Preliminary Common Steps ......................................................... 41
B . Presumptive or Group Identification ............................................. 41
1. Tests Included ...................................................................... 41
2 . Information Developed .......................................................... 42
C . Definitive Identification .............................................................. 42
1. Tests Included ...................................................................... 42
2 . Commercial Kits ................................................................... 42
3 . Additional Tests ................................................................... 43