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 BUTTERWORTHS LONDON - BOSTON Sydney - Wellington - Durban - Toronto THE BUTTERWORTH GROUP United Kingdom Butterworth & Co (Publishers) Ltd London: 88 Kingsway, WC2B 6AB Australia Butterworth Pty Ltd Sydney: 586 Pacific Highway, Chatswood NSW 2067 Also at Melbourne, Adelaide and Perth Canada Butterworth & Co (Canada) Ltd Toronto: 2265 Midland Avenue, Scarborough, Ontario, M1P 4SI New Zealand Butterworths of New Zealand Ltd Wellington: 26-28 Waring Taylor Street, 1 South Africa Butterworth & Co (South Africa) (Pty) Ltd Durban: 152-154 Gale Street USA Butterworth (Publishers) Ine Boston: 19 Cummings Park, Woburn, Mass. 01801 All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, including photocopying and recording, without the written permission of the copyright holder, application for which should be addressed to the publisher. Such written permission must also be obtained before any part of this publication is stored in a retrieval system of any nature. This book is sold subject to the Standard Conditions of Sale of Net Books and may not be re-sold in the UK below the net price given by the Publishers in their current price list. 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  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).