JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1976, p. 128-132 Copyright C 1976 American Society for Microbiology

Vol. 3, No. 2 Printed in U.S.A.

Survival of Anaerobic and Aerobic Bacteria in Nonsupportive Gassed Transport System

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ANTHONY W. CHOW,* PATRICK J. CUNNINGHAM, AND LUCIEN B. GUZE Department of Medicine, Harbor General Hospital, Torrance, California 90509*; Research and Medical Services, Wadsworth Veterans Administration Hospital, Los Angeles, California 90073, and University of California at Los Angeles School of Medicine, Los Angeles, California 90024 Received for publication 8 August 1975

Survival of anaerobic and aerobic bacteria in a commercially available, nonsupportive, gassed (oxygen-free) transport container (Anaport) was evaluated quantitatively. Saline-suspended obligate anaerobes survived significantly better in the gassed container than in aerobic control tubes (P < 0.025, t test), and counts were virtually unchanged after 8 h of holding. Similarly, initial counts and relative proportions of a mixture of Bacteroides fragilis and Staphylococcus aureus were maintained for 72 h. The value of the gassed transport system was less apparent when microorganisms were suspended in nutrient broth. The major advantage of the gassed transport system appears to be for holding of specimens collected by saline irrigation.

Appropriate collection and transport of specimens is an important prerequisite for the successful isolation of fastidious anaerobes in the diagnosis of anaerobic infections (1). For this purpose, the gassed (oxygen-free) non-nutrient container has been considered an optimal transport system (2, 3) and is commercially available for general use. Despite this, there have been little published data to examine the survival of various anaerobic and aerobic bacteria in such a system and the maximum interval during which storage of specimens in such a system may be beneficial. This study was undertaken to evaluate quantitatively the survival of various anaerobes and aerobes in a commercially available gassed transport system (Anaport, Scott Laboratories, Fiskeville, R.I.) as compared to holding under aerobic conditions.

transport system evaluated (Anaport, Scott Laboratories) consisted of stoppered 10-ml vials evacuated and replaced with oxygen-free CO2, moistened for autoclave sterilization with 0.1 ml of Virginia Polytechnic Institute (VPI) salts solution (3), and containing a resazurin indicator (Eh = -42 mV). All gassed containers studied were from the same com-

mercial batch. Sterile, aerobic, screw-cap glass tubes (13 by 100 mm) (Kimax, Kimble, Toledo, Ohio) were used for controls. Culture, dilution, and quantitation. Stock cultures of obligate anaerobes were grown in prereduced, anaerobically sterilized, thioglycolate broth (Difco), streaked on brain heart infusion agar (Difco) enriched with 5% sheep blood, and incubated in a GasPak anaerobic jar evacuated and replaced with a gas mixture of 80% N2, 10% H2, and 10% CO2. Colonies were washed off, suspended in sterile, physiological saline without bacteriostatic preservative, and adjusted to a turbidity corresponding to a MacFarland no. 1/2 nephalometer standard. Aliqucts (5 ml) of this saline suspension were transferred by needle and syringe into a gassed vial and a correMATERIALS AND METHODS sponding aerobic control tube and kept at room temTest organisms. Bacteria chosen in this study perature. Immediately (0 h) and serially at 1, 2, 4, 8, were representative of clinical isolates with varying 24, 48, and 72 h, aliquots of 0.5 ml were removed degrees of fastidiousness and aerotolerance. These from each system with a needle and syringe. Colony included six obligate anaerobes (Bacteroides fra- counts of viable organisms were determined anaerogilis, Peptostreptococcus intermedius, Veillonella bically and in triplicate in roll tubes containing parvula, Clostridium perfringens, Propionibacte- prereduced, anaerobically sterilized brain heart inrium acnes, Eubacterium lentum), six facultative fusion agar (Difco) supplemented with cysteine, bacteria (Escherichia coli, Staphylococcus aureus, yeast extract, and menadione according to the VPI Streptococcus faecalis, group A Streptococcus, Hae- Anaerobe Laboratory Manual (3). mophilus influenzae, Neisseria gonorrhoeae), and Stock cultures of facultative and aerobic bacteria one aerobe (Pseudomonas aeruginosa). All were were grown in Mueller-Hinton broth (BBL) and stock cultures of clinical isolates maintained either streaked aerobically on brain heart infusion blood by lyophilization or in 20% skim milk at -70 C until agar. Colonies were washed off, suspended, and diluted in sterile physiological saline without bacteriready for study. Transport systems. The gassed non-nutrient ostatic preservative and transferred to the gassed 128

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SURVIVAL OF ANAEROBIC AND AEROBIC BACTERIA

vials and aerobic control tubes in the same manner as with obligate anaerobes. Colony counts of facultative and aerobic bacteria were likewise determined serially and in triplicate on brain heart infusion blood agar plates, using a 0.01-ml platinum quantitative loop (Scientific Products, McGaw Park, Ill.). In a separate experiment, survival curves were similarly determined in gassed vials and aerobic control tubes, with the test organisms suspended either in thioglycolate broth (obligate anaerobes) or Mueller-Hinton broth (aerobic and facultative bacteria) rather than in physiological saline. Finally, to determine the survival of anaerobic and aerobic components of a mixed flora, saline and

broth suspensions of B. fragilis and S. aureus were admixtured in equal proportions (vol/vol) prior to instillation in the gassed transport system. Serial colony counts of both organisms were carried out in triplicate in roll tubes containing prereduced, anaerobically sterilized brain heart infuision agar as described earlier.

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the finding that both aerobes suspended in saline also survived better in the gassed container (P < 0.025, t test) by 1 h and thereafter. Survival of anaerobic and aerobic bacteria suspended in nutrient broth in gassed containers and their corresponding aerobic control tubes is shown in Table 2. Surprisingly, when suspended in broth, both-P. intermedius and B. fragilis survived well under aerobic conditions even at 72 h, and their colony counts were virtually identical to those in gassed containers. Among the aerobic organisms, with the exception of N. gonorrhoeae and H. influenzae, bacterial counts increased serially in both transport systems. N. gonorrhoeae and H. influenzae maintained their initial counts for 24 h, but rapidly declined in number thereafter. Both B. fragilis and S. aureus, mixed in saline suspension and held in the nonsupportive gassed transport systeri, maintained their initial counts and relative proportions and survived significantly better than in aerobic control tubes (P < 0.0005, t test) at 72 h (Table 3). In contrast, colony counts of B. fragilis and S. aureus increased steadily in both gassed and control containers when mixed in nutrient broth and decreased steadily in aerobic control tubes when mixed in saline. Surprisingly, a saline suspension of B. fragilis mixed with S. aureus was more aerotolerant under aerobic conditions than a saline suspension of B. fragilis in pure culture, and significant viable counts remained at 72 h in the aerobic control tube.

RESULTS Colony counts of saline-suspended anaerobic and aerobic bacteria surviving at various intervals in gassed containers and their corresponding aerobic control tubes are summarized in Table 1. Obligate anaerobes held under aerobic conditions displayed varying degrees of aerotolerance. A 3-log decrease in colony counts occurred after a mean interval of 8.7 h. E. lentum was most sensitive, with a 3-log decrease in 1.5 h, and none could be recovered in 4 h. P. intermedius, V. parvula, and B. fragilis were intermediately sensitive, with a 3-log decreasc in 3, 5, and 4 h, respectively, and none could be recovered between 8 and 24 h. PropionibacteDISCUSSION rium acnes and C. perfringens were least sensiAppropriate transportation and minimal detive, with a 3-log decrease in 10 and 29 h, respectively, and none could be recovered be- lay in processing of clinical specimens are imtween 48 and 72 h. All obligate anaerobes sur- portant prerequisites for the successful isolavived better in gassed containers than in aero- tion of fastidious obligate anaerobes (1, 2). An bic control tubes, with significant differences in ideal transport system should be nonselective counts (P < 0.025, t test) by 2 h and thereafter. as well as nonsupportive, capable of maintainExcept for E. lentum, colony counts of oligate ing viability without promoting growth or apanaerobes in gassed containers were virtually preciably altering the relative proportion of unchanged after 8 h. The mean interval for a 3- various organisms initially present in the clinilog decrease with these organisms was 53 h, cal specimen. Our data of survival of salinecompared to 8.7 h in aerobic control tubes (P < suspended obligate anaerobes clearly demon0.0025, t test). E. lentum survived better in the strate the detrimental effect of oxygen and the gassed container (P < 0.0025, t test) at 2 and 4 protective effect of the gassed transport system. h; none could be recovered by 8 h. All obligate anaerobes tested survived better in Similar to obligate anaerobes, both Esche- the gassed transport system, and, except for E. richia coli and Pseudomonas aeruginosa de- lentum, colony counts were virtually unclined in colony counts under aerobic condi- changed after 8 h. In addition, the gassed contions, with a 3-log decrease after 4 and 8 h, tainer maintained the initial counts and their respectively (Table 1). However, in contrast to relative proportions of both B. fragilis and S. obligate anaerobes, significant counts of these aureus after 72 h when mixed in saline suspenaerobes remained after 48 h. Of interest was sion. A surprising finding was that saline sus-

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CHOW, CUNNINGHAM, AND GUZE

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Survival of anaerobic and aerobic bacteria in a nonsupportive gassed transport system.

Survival of anaerobic and aerobic bacteria in a commercially available, non-supportive, gassed (oxygen-free) transport container (Anaport) was evaluat...
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