Anaerobic Bacteriology. Clinical and Laboratory Practice

365 Pages·1977·17.96 MB·English

by  A. Trevor Willis (Auth.)

To Sir James Howie
Anaerobic Bacteriology:
Clinical and Laboratory Practice
Third edition
A. TREVOR WILLIS
DSc, MD, FRACP, PhD, FRCPath, FRCPA
Director, Public Health Laboratory, Luton
Consultant Microbiologist, Luton and Dunstable Hospital
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First published 1960
Second edition 1964
Third edition 1977
ISBN 0 407 00081 X
© Butterworths and Co. (Publishers) Ltd, 1977
British Library Cataloguing in Publication Data
Willis, Allan Trevor
Anaerobic bacteriology. - 3rd ed.
1. Bacteriology, Medical 2. Bacteria,
Anaerobic
I. Title
616.0Γ4 QR46 77-30019
ISBN0-407-00081-X
Type set by Butterworths Litho Preparation Department
Printed in Great Britain by Butler & Tanner Ltd, London & Frome
Preface
In the twelve years since the second edition appeared there has been a
great resurgence of interest in anaerobic bacteriology among both
medical microbiologists and their clinical colleagues. This is due largely
to an increasing recognition of the importance of the non-sporing
anaerobic bacteria as significant causes of infection in man. Although
it has been suggested by some that the non-clostridial anaerobes, like
the mycoplasmas and 'L-forms' before them, are simply another band­
wagon that commands fashionable attention, it seems unlikely that the
importance attached to their role in human and animal pathology
will be so evanescent.
These developments have led to major additions of detail and to
changes in emphasis in our knowledge on many subjects, especially
in anaerobic methodology and in the systematics and the ecological
and pathogenetic associations of the non-sporing anaerobes. These
additions and changes are reflected in the present text, which now
includes separate chapters devoted to the descriptive bacteriology of
the non-clostridial anaerobes, and to the clinical syndromes produced
by them in man. The remainder of the text has been thoroughly revised
and much of it rewritten, and appropriate additions made to the
bibliography.
My grateful thanks are due to Professor R. A. Willis for literary
criticism and advice, to Miss B. H. Why te and her staff at the Central
Public Health Laboratory Library at Colindale for their help in obtaining
many of the original publications, and to my colleagues at the Luton
Public Health Laboratory and at the Luton and Dunstable Hospital
for many helpful and stimulating discussions. During the preparation
of this new edition I was fortunate in working first under Sir James
Howie and subsequently under Sir Robert Williams in the Public Health
Laboratory Service, to both of whom I owe a considerable debt of
gratitude for their interest and support.
v
vi Preface
I am deeply grateful to my wife for her constant encouragement and
for her help in preparing the manuscript, in reading proofs and in
checking references.
Finally, my thanks are due to my Publishers for their help during the
production of this book.
Public Health Laboratory, and A. Trevor Willis
Lut on and Dunst able Hospital,
Lu ton
1
Methods of Growing Anaerobes
A variety of methods is available for the culture of anaerobic organisms
in the clinical laboratory. Exclusion of oxygen from part of the medium
is the simplest method, and is effected by growing the organism within
the culture medium as a shake or fluid culture.
When an oxygen-free or anaerobic atmosphere is required for obtain­
ing surface growths of anaerobes, anaerobic jars provide the method of
choice. Some of the methods for obtaining anaerobiosis in ajar are also
applicable to single tube or plate cultures, but for laboratories that
intend to undertake anaerobic bacteriology seriously, anaerobic jars are
essential.
More sophisticated methods for the surface culture of anaerobes
are the pre-reduced anaerobically sterilized (PRAS) roll-tube technique
of Hungate (1950), and use of the anaerobic cabinet or glove box.
These two techniques utilize complex and expensive equipment, are
time consuming, and require specially trained staff and specialized
medium preparation facilities. There is no doubt that these complex
techniques provide the most meticulous anaerobic conditions, and
are appropriately used for the isolation and study of exacting anaerobic
species that are highly sensitive to oxygen (Spears and Fréter, 1967;
Barnes and Impey, 1970). They are, however, too demanding in time,
space and expense for routine use in the clinical laboratory where
anaerobic work is but one facet of the diagnostic routine. Moreover,
comparative studies have shown that these methods are not superior
to the anaerobic jar for the recovery of clinically significant anaerobes
from pathological specimens (Killgore et al., 1972, 1973; Rosenblatt
et al, 1972, 1973, 1974; Dowell, 1972; Cadogan-Cowper and Wilkinson,
1
2 Methods of growing anaerobes
1974; Spaulding et al, 1974; Starr, 1974; Watt, Hoare and Brown
1974). Although Peach and Hayek (1974) experienced a three-fold
increase in the isolation rate of anaerobes from clinical specimens
when an anaerobic cabinet was used instead of an anaerobic jar, it seems
likely from the data they published that their conventional anaerobic
technique was faulty.
THE ANAEROBIC JAR
The modern anaerobic jar is based, both in design and in operation, on
that originally described by Mclntosh and Fildes (1916). In their
anaerobic jar, hot platinum was used to catalyse the combination of
oxygen with hydrogen to form water—a principle first applied to the
culture of anaerobes by Laidlaw (1915). In the original jar, the catalyst
capsule was composed of asbestos wool impregnated with finely divided
palladium and enclosed in a fine-mesh brass or copper gauze envelope.
The palladium was activated by heating it in a bunsen burner flame
immediately before setting up the jar, and the hydrogen was supplied
to the jar from a Kipp's apparatus or from a cylinder of the gas.
Electrical heating of the catalyst was introduced by Smülie (1917),
and was adopted by Fildes and Mclntosh (1921) and Brown (1921,
1922). Subsequently various minor modifications to jar design were
made by Brewer (1938-39) and Evans, Carlquist and Brewer (1948).
These early forms of anaerobic jar have now been replaced by cold
catalyst jars; indeed, electrically operated jars are no longer manu­
factured. The room temperature catalyst (Heller, 1954), which is
manufactured under patent as Deoxo pellets by Engelhard Industries
Ltd, London, consists of pellets of alumina coated with finely divided
palladium. Baird and Tatlock (London) developed the first commercially
available jar (the BTL or Torbal jar) which utilized this principle of
catalysis (Laboratory Equipment Test Report, 1958). Subsequently
Becton, Dickinson, in association with Baltimore Biological Laboratories
in America introduced the Gaspak anaerobic system (Brewer and
Allgeier, 1966), and more recently Don Whitley Scientific developed
the Gaskit system for use with their Schrader valve vented jars.
Figure 1.1 depicts the BTL jar. Anaerobic jars are cylindrical vessels
made of metal, glass or plastic, flanged at the top to carry an air-tight
lid which is held firmly in place by a clamp or lug closure. The lid
carries on its undersurface the room temperature catalyst capsule. The
essential difference between the BTL and the Gaspak jars is that in the
former the lid is provided with two valves through which air can be
The anaerobic jar 3
withdrawn and hydrogen introduced. The lid of the standard Gaspak
jar is not vented, because the jar is specifically designed for use with an
internal disposable hydrogen—carbon dioxide generator. Important
Screw Clamp
Hydrogen valve
Vacuum valve
Lid O-ring
Side arm
Sachet containing
catalyst
Rubber tubing
Indicator capsule
Figure 1.1. BTL anaerobic jar. (Reproduced by courtesy of
Baird and Tatlock (London) Ltd)
features of the Whitley anaerobic jar (Burt and Whitley, in press)
include venting by special Schrader valves, and a quick release 4-g cold
catalyst capsule of large surface area.
The BTL and Whitley anaerobic jars
The hydrogen source
Hydrogen is obtained from a cylinder of the compressed gas. The use of
coal gas as a source of hydrogen should be avoided since it contains
carbon monoxide which is inhibitory to some organisms. The use of
illuminating gas or natural gas is inappropriate, since it consists mainly
of methane, and is usually deficient in hydrogen. Since it is important
that the hydrogen should be supplied to the jar at low pressure, the
hydrogen cylinder should be fitted with a reducing valve and the gas
delivered at a pressure of not more than 0.5 psi. If a reducing valve is
Â
To hydrogen-
carbon dioxide
cylinder
Figure 1.2. Low pressure
hydrogen source using a
football bladder
To anaerobic jar
To hydrogen-
carbon dioxide
cylinder
Figure 1.3. Low pressure
hydrogen source using an
aspirator bottle system
The anaerobic jar 5
not available, a convenient low pressure source is either a football
bladder or anaesthetic bag {Figure 1.2), or an aspirator bottle system
{Figure 1.3) filled from the cylinder. The pressure reducing mechanism
is attached to a gas washing bottle, which acts as a flow meter, and this
in turn is connected to the anaerobic jar.
The carbon dioxide source
Since the growth of many anaerobes is improved by the addition to the
jar of 10% carbon dioxide, and since no anaerobes are adversely affected
by this concentration of the gas, it is important to have available a
carbon dioxide source. This may be provided from a separate cylinder
of the compressed gas, but it is much more convenient to use a hydrogen-
carbon dioxide mixture. A suitable mixture is 90% hydrogen with 10%
carbon dioxide, and is available from BOC Ltd. Do well and Hawkins
(1968) recommended a gas mixture containing 80% nitrogen, 10%
hydrogen and 10% carbon dioxide.
In the interests of safety, it is important that compressed gases, and
especially explosive gases, should be housed outside the building
(Department of Health and Social Security, 1972), and the gases led
into the laboratory through copper tubing. Cylinders containing mixtures
of hydrogen with carbon dioxide must be stored horizontally, since the
gases tend to 'layer off. When this happens the cylinder is likely to
supply mainly hydrogen early in its life, and mainly carbon dioxide
as it becomes empty.
The catalyst
The Deoxo pellet catalyst (grade 0.5% Pd) is contained in a wire gauze
capsule attached to the underside of the lid of the jar. The sachet con­
tains pellets of alumina coated with finely divided palladium; the
sachet in the BTLjar contains about 1 g of catalyst, that in the Whitley
jar about 4 g. Certain gases such as chlorine, sulphur dioxide, carbon
monoxide and hydrogen sulphide poison the catalyst, as does oil, the
vapour of some organic solvents and strong acids. Inactivation by
hydrogen sulphide is especially relevant to the microbiologist, since
many anaerobes give off appreciable amounts of this gas, especially
from fluid cultures. The catalyst is also inactivated by moisture, which
is plentiful in the anaerobic jar, but it is readily reactivated by heating
in a hot air oven {see below).