12015 LABORATORY METHODS IN ANAEROBIC BACTERIOLOGY CDC LABORATORY MANUAL V. R. Dowell, Jr., Chief Enterobacteriology Branch Bacteriology Division Bureau of Laboratories And T. M. Hawkins, Chief Bacteriology Training Branch Laboratory Training and Consultation Division Bureau of Laboratories Reprinted January 1974 November 1976 January 1978 April 1979 February 1981 January 1983 September 1987 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES PUBLIC HEALTH SERVICE CENTERS FOR DISEASE CONTROL ATLANTA, GEORGIA 30333 CDC INFORMATION CENTER HHS Publication No. (CDC) 87-8272 CENTERS FOR DISEASE CONTROL ATLANTA, GEORGIA 30333 TABLE OF CONTENTS Page I. Introduction..................................................................................................................................... 1 II. Isolation of anerobic bacteria from clinical material ................................................................. 3 III. Determination of cultural and biochemical characteristics........................................................ 7 IV. Detection of clostridial toxin, toxin neutralization tests, and pathogenicity tests............................................................................................................. ^ V. Toxin typing of Clostridium perfringens...................................................................................... 17 VI. Identification of anaerobic bacteria............................................................................................... 21 VII. Detection of Clostridium botulinum and botulinal toxin ........................................................ 41 VIII. Examination of foods and feces for Clostridium perfringens .................................................. 45 IX. Storage and shipment of anaerobes............................................................................................... 49 X. Media for isolation and characterization of anaerobic bacteria Introduction ...................................................................................................................... 53 Plating media...................................................................................................................... 53 Differential media ............................................................................................................. 55 Storage media...................................................................................................................... 59 XI. Reagents........................................................................................................................................... 61 XII. Staining procedures Gram stain ......................................................................................................................... 63 Spore stains .......................................................................................................................... 63 Flagella stain ...................................................................................................................... 64 XIII. Anaerobic systems Use of the anaerobe culture ja r......................................................................................... 67 Roll-streak (PRAS medium) tube technique ................................................................. 70 Anaerobic glove box techniques ...................................................................................... 73 XIV. Identification of acid metabolic products by gas liquid chromatography .............................. 77 XV. Bibliography References cited ................................................................................................................ 83 Supplemental references..................................................................................................... 84 XVI. Subject Index.................................................................................................................................. 91 Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education, and Welfare. LIST OF TABLES Table No. Page I. Interpretation of reactions given in differential media for identification of anaerobes............................................................................................................. 11 II. Mouse toxin neutralization tests of C. perfringens centrifuged culture fluids .................................................................................................................................. 19 III. Clinical isolates of anaerobic bacteria most frequently submitted to the CDC anaerobe unit ............................................................................................................. 22 IV. List of changes in the nomenclature of anaerobic bacteria........................................................ 23 V. Characteristics of commonly encountered Clostridium species which are particularly useful for identification...................................................................................... 24 VI. Differential characteristics of commonly encountered Clostridium species .............................................................................................................................................. 25 VII. Differential characteristics of Clostridium species infrequently isolated from clinical materials ..................................................................................................... 26 VIII. Differential characteristics of Bacteroides and Fusobacterium species commonly isolated from clinical materials .................................................................... 29 IX. Characteristics of anaerobic, nonsporeforming, gram-negative bacilli infrequently isolated from clinical materials................................................................................ 30 X. Key characteristics for subspeciation of Bacteroides fragilis..................................................... 31 XI. Characteristics of anaerobic, nonsporeforming, gram-positive bacilli commonly isolated from clinical materials................................................................................... 32 XII. Characteristics of anaerobic, nonsporeforming, gram-positive bacilli infrequently isolated from clinical materials................................................................................ 33 XIII. Differential characteristics of anaerobic cocci commonly isolated from clinical materials .................................................................................................................... 34 XIV. Detection of C. botulinum toxin in food extracts or culture fluids......................................... 43 LIST OF FIGURES Figure No. Page 1 Colony characteristics and description of bacterial colonies............................................... 9 2 Differentiation of gram-negative, nonsporeforming, anaerobic bacteria to the genus level .................................................................................................................... 27 3 Differentiation of gram-positive, nonsporeforming, anaerobic bacteria to the genus level ................................................................................................................... 31 4 Evacuation-replacement system ............................................................................................ 69 ii LIST OF PLATES Plate No. Page 1—2 Representative colonies of anaerobic bacteria ..........................................................................35-36 3—5 Representative photomicrographs of anaerobic bacteria ........................................................37-39 6 GasPak anaerobic systems ..........................................................................................................71 7 VPI anaerobic culture system and anaerobic glove box system..............................................75 iii I. INTRODUCTION The clinical importance of the anaerobic bacteria, particularly the toxigenic clostridia and some of the nonsporulating anaerobes is well recognized, however, our overall knowledge of these bacteria and their role in human and animal infections is quite limited. Fortunately, interest in the anaerobes has increased in recent years, and anaerobic methodology is now used routinely by most clinical and public health laboratories. This increase in interest has created an immediate demand for microbiologists and technologists who are familiar with the techniques of anaerobic bacteriology. Since the establishment of the anaerobic bacteriology laboratory at the Center for Disease Control, a course in laboratory methods in anaerobe bacteriology has been offered for interested and qualified laboratory personnel. This manual was designed primarily as a guide for the laboratory portion of the course, and the material presented is supplemented with lectures. The manual can also be used as a laboratory reference for anaerobe techniques. The media and techniques described are used routinely by the CDC Anaerobe Unit, and the reactions for the various organisms presented in the tables are based on data obtained with cultures studied by these methods. For comparative purposes it is necessary to study cultures under uniform conditions, and an effort should be made to standardize the media and techniques used as closely as possible. Since this manual has been revised regularly, a number of people have contributed to its contents and organization. The authors would like to express their gratitude to Mrs. Frances Thompson, Mrs. Ann Armfield, Mr. David Whaley, and Mrs. Loretta McCroskey of the Anaerobe Unit staff and to Miss Gilda Jones and Mr. Bobby Strong of the Bacteriology Training Unit staff. Our particular appreciation goes to Dr. George Lombard, in charge of the CDC Anaerobe Unit, and to Dr. Lillian Holdeman, formerly in charge of the Anaerobe Laboratory at CDC. 1 ' II. ISOLATION OF ANAEROBIC BACTERIA FROM CLINICAL MATERIAL A. The anaerobic bacteria can be isolated and studied quite readily provided certain cardinal principles of anaerobic bacteriology are rigidly applied. Four of the most important considerations in the cultivation of anaerobic bacteria are: 1. Proper collection and transport of the material to be examined. 2. Culture of the material as soon as possible after collection. 3. Use of freshly prepared and properly reduced media. 4. Proper anaerobic conditions. Proper collection and transport of clinical specimens is of primary importance in recovery of anaerobes. The sample should be collected from the active site of infection and precautions should be taken to exclude surface contaminants and aeration of the sample. Whenever possible tissue samples or fluid aspirates should be collected rather than swab samples. The material on swabs should never be allowed to dry out. Specimens should be placed under anaerobic conditions immediately after collection for transport to the laboratory since some anaerobes are quite oxygen sensitive and will die rapidly in an aerobic environment. Sterile rubber stoppered transport vials and tubes containing an oxygen free C02 atmosphere are available commercially. Specimens aspirated with a needle and syringe can be injected directly into the transport bottles; care must be taken to exclude any air. If necessary, a specimen tube can be opened in an upright position, the specimen or swab added, and the tube closed for transport to the laboratory. Since CO2 is heavier than air, the CO2 atmosphere is maintained in the transport tube. As a very minimum procedure, the material can be placed in a medium containing a reducing agent such as cysteine or thioglycollate at room temperature for a period not exceeding 2 hours. Samples should not be refrigerated since chilling is detrimental to some anaerobes, and oxygen absorption is greater at lower temperatures. 3 All clinical material except specimens likely to be contaminated with normal flora should be routinely cultured for anaerobes. Specimens that should not be cultured include nasal swabs, throat swabs, sputum, gastric contents, skin, feces, voided or catheterized urine, and vaginal swabs. For isolation of anaerobes from blood specimens, 5-10 ml of blood should be inoculated into 50-100 ml of liquid media (10% V/V) and the blood cultures incubated up to 14 days. Broth media containing 0.025% sodium polyanethol sulfonate (liquoid) and an anaerobic or partial CO2 atmosphere are commercially available. Tryptic soy broth, trypticase soy broth, thioglycollate medium and pre-reduced brain heart infusion broth designed for anaerobic blood culture all appear to be equally satisfactory.1 Liquoid may prevent the growth of some anaerobic cocci and slow the growth of some strains of Bacteroides melaninogenicus.2 Blood cultures should be subcultured to plating media whenever there is any obvious growth and blind subcultures made at least after 48 hours incubation and at the end of 14 days. In addition to plating on blood agar plates, it has been shown that subculturing to a selective plating medium will allow detection of anaerobes mixed with aerobic organisms in bacteremic infections.3 Ideally, specimens should be cultured as soon as possible after collection and every effort should be made to prevent exposure of culture media to molecular oxygen. Plating media for primary isolation should be prepared on the day it is used, or freshly prepared media should be placed under anaerobic conditions for a period no longer than 2 weeks. Plating media can be stored in an anaerobe jar, glove box, or in an air-tight cabinet containing an oxygen free CO2 atmosphere such as the one used in the Mayo Clinic1 anaerobe laboratory.4 Liquid media containing reducing agents should be stored in the dark at room temperature in tightly capped tubes for not longer than 2 weeks. Provided the media is fresh and properly reduced, successful cultivation of anaerobes can be obtained by use of the GasPak anaerobe jar or by use of an anaerobe jar with a gas replacement method.5,6,7 At the CDC, an anaerobe glove box system8 is used routinely for primary isolation of the anaerobes from clinical specimens and for subculture of colony isolates. Two excellent methods for the cultivation of anaerobes are the glove box system and the roll-streak tube system in which prereduced anaerobically sterilized (PRAS) media are used as recommended by the VPI anaerobe laboratory.9 A section covering the use of various anaerobe systems is included in this manual. All specimens except blood should be Gram stained and cultured by both direct plating and enrichment procedures. All liquid or semi-solid media stored in an aerobic environment should be prereduced by heating the media for 10 minutes in a boiling water bath and cooling before inoculation. Since most clinical laboratories are not set up for the glove box or roll-streak tube systems, the following procedures are designed for use with the anaerobe culture jar. Prepare and stain direct smears from each specimen. In order to gain some insight into the quantity and type of organisms in the specimen, examine a gram stained smear. Examination of wet mounts of unstained material, acid fast stained smears, and Giemsa stained smears may also be helpful. Use capillary pipettes to prepare smears from liquid specimens or use swabs directly. Observe and record: 1. The gram reaction, size, shape and relative numbers of organisms present. 2. The presence of spores and their shape and position in the cell. 3. Any distinctive morphological features such as branching, pseudo-branching, chaining, filaments, spherical bodies, or minute granular forms.