JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1979, p. 331-333

Vol. 10, No. 3

0095-1137/79/09-0331/03$02.00/0

Practical Technique for Quantitatmg Anaerobic Bacteria in Tissue Specimens MICHAEL D. SPENGLER, GEORGE T. RODEHEAVER,* AND RICHARD F. EDLICH Department of Plastic Surgery, University of Virginia Medical Center, Charlottesville, Virginia 22908

Received for publication 19 June 1979

Quantitative bacteriology has considerable influence on the care and management of surgical wounds. Heretofore, these techniques have been limited to measurements of aerobic bacteria. A technique is reported herein which permits quantitation of obligately anaerobic bacteria in tissue specimens. This technique

is easily reproduced in any clinical laboratory and eliminates the need for expensive anaerobic chambers.

The importance of quantitative bacteriology in wound management and the monitoring of infection has, recently been reviewed by Krizek and Robson (5). Quantitative techniques have proved useful in deciding the safety of wound closure (both primary and delayed primary), determining graft bed receptivity, diagnosing the onset of burn wound sepsis, and monitoring the efficacy of antimicrobial therapy. In each of these clinical circumstances, the quantitative procedures are performed under aerobic conditions and thus do not permit measurement of obligately anaerobic organisms. This lack of quantitative information on anaerobic organisms is disturbing in light of the growing awareness of the importance of these organisms in the development of surgical infection (2, 3, 7). Major technical advances (4, 6) have evolved which permit the routine isolation and identification of obligately anaerobic organisms from clinical specimens. The use of these techniques for quantitating anaerobic organisms has primarily been limited to research studies. Even in these situations the quantitative procedures have involved only fluid specimens or fluid-dispersible samples such as feces, which can be serially diluted and processed by routine procedures. The technique reported herein is simple and practical and can be easily reproduced in any clinical laboratory to quantitate the number of obligately anaerobic bacteria in tissue biopsies. MATERIALS AND METHODS Quantitation technique. Our approach to anaerobic bacterial quantitation was patterned after our published aerobic bacterial quantitation procedure (1), except that anaerobiosis was maintained throughout the procedure. The technique was initiated by placing a weighed tissue sample into a 50-ml, modified Sorvall homogenization chamber containing 15 ml of prere-

duced salt solution (4) (Fig. 1). Carbon dioxide was continually introduced into the chamber through an external stainless-steel tube (internal diameter, 2 mm), which entered at the bottom of the chamber. The specimen was homogenized for 1 min, during which time the chamber was immersed in ice water. With a Virginia Polytechnic Institute anaerobic system (Beilco Glass Inc.), the homogenate was subjected to three serial 1:10 dilutions in prereduced salt solution. A portion (0.1 ml) from the homogenate and each serial dilution was placed in molten (45 to 50°C) brain heart infusion roll tubes (Carr/Scarborough Microbiologicals). After each tube was gassed with carbon dioxide, it was rotated along its long axis until the agar cooled in a thin film along the walls. The sealed tubes were then incubated at 37°C for 24 to 96 h. The number of colonies in the tube containing between 30 and 300 colonies was counted. Assuming that each colony is the product of a single organism, the result was reported as the number of bacteria per gram of tissue. All tubes were observed for an additional 2 weeks to determine whether any further growth developed. Evaluation. A quantitative procedure for isolation of obligately anaerobic bacteria is judged to be successful if it permits recovery of the most oxygen-sensitive organism. The purpose of the first part of the study was to identify the aerotolerance of several anaerobic pathogens that result in clinical disease in humans. The organisms included in this study were Bacteroides fragilis, Fusobacterium nucleatum, F. varium, and Peptostreptococcus anaerobius. Before each experiment, the test organism was transferred to prereduced brain heart infusion broth and incubated at 37°C. During the log phase of growth, a portion of bacteria was added to prereduced salt solution which had been oxidized by exposure to air. After an exposure of 6 h in oxidized fluid, the number of viable organisms remaining in each tube was quantitated. The species experiencing the largest kill after the exposure was judged to be the most oxygen sensitive. In the second part of the study, the influence of homogenization under aerobic conditions on the survival of the four test organisms was assessed. In this series of experiments, a suspension of each organism

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J. CLIN. MICROBIOL.

FIG. 1. Technique for quantitating anaerobic bacteria. TABLE 1. Aerotolerance as measured by the recovery rate of obligately anaerobic organisms after a 6-h exposure in oxidized fluid Viable bacteriaa P Organism

was separately homogenized for 1 min in air. In another study, a suspension of the least aerotolerant organism was homogenized for 1 min under anaerobic conditions. The number of viable bacteria was measured before and after each homogenization.

RESULTS Surprisingly, suspensions of three of the four obligately anaerobic bacteria were not adversely affected by 6 h of exposure to oxidized fluid (Table 1). P. anaerobius was extremely sensitive to air, with no viable organisms recovered after 6 h. This loss of viable organisms occurred rapidly in an aerobic environment, with a significant decrease in viable bacteria being evident within 30 min (Fig. 2). As expected, the more aerotolerant organisms survived homogenization in an aerobic environment (Table 2). In contrast, the average number of organisms of the oxygen-sensitive P. anaerobius was decreased by homogenization in air. The deleterious effects of aerobic homogenization on the recovery of this organism were eliminated by performing the procedure under anaerobic conditions (Table 3). DISCUSSION The quantitation of anaerobic bacteria requires maintenance of an anaerobic environment. With fluid samples, maintenance of anaerobiosis is relatively simple, being accomplished in anaerobic jars or roll tubes (6). Quantitation of these organisms in tissue specimens is a more challenging problem, since the procedure involves tissue homogenization with potential exposure to oxygen. This problem can be obviated by performing the homogenization in a flexible plastic anaerobic chamber equipped

Initial

6h

6.3 ± 0.1

Practical technique for quantitating anaerobic bacteria in tissue specimens.

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1979, p. 331-333 Vol. 10, No. 3 0095-1137/79/09-0331/03$02.00/0 Practical Technique for Quantitatmg Anaerob...
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