Evaluation of the Oxyrase OxyPlate Anaerobe Incubation System

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2000, p Vol. 38, No /00/$ Evaluation of the Oxyrase OxyPlate Anaerobe Incubation System LOIS S. WIGGS, JOSEPH J. CAVALLARO,* AND J. MICHAEL MILLER Diagnostic Microbiology Section, Hospital Infections Program, Centers for Disease Control and Prevention, Atlanta, Georgia Received 9 August 1999/Returned for modification 27 September 1999/Accepted 8 November 1999 The Oxyrase OxyPlate anaerobe incubation system was evaluated for its ability to support the growth of clinically significant anaerobic bacteria previously identified by the Anaerobe Reference Laboratory at the Centers for Disease Control and Prevention. The results were compared with those obtained with conventional anaerobe blood agar plates incubated in an anaerobe chamber. We tested 251 anaerobic bacterial strains. Plates were read at 24, 48, and 72 h; growth was scored by a numerical coding system that combines the degree of growth and the colony size. Organisms (number of strains tested) used in this study were Actinomyces (32), Anaerobiospirillum (8), Bacteroides (39), Campylobacter (8), Clostridium (96), Fusobacterium (12), Leptotrichia (8), Mobiluncus (8), Peptostreptococcus (16), and Propionibacterium (24). At 24 h, 101 (40.2%) of the 251 strains tested showed better growth with the anaerobe chamber than with the OxyPlate system, 10 (4.1%) showed better growth with the OxyPlate system, and the remaining 140 (55.8%) showed equal growth with both systems. At 48 h, 173 (68.9%) showed equal growth with both systems, while 78 (31.1%) showed better growth with the anaerobe chamber. At 72 h, 176 (70.1%) showed equal growth with both systems, while 75 (29.9%) showed better growth with the anaerobe chamber. The OxyPlate system performed well for the most commonly isolated anaerobes but was inadequate for some strains. These results indicate that the Oxyrase OxyPlate system was effective in creating an anaerobic atmosphere and supporting the growth of anaerobic bacteria within 72 h. OxyPlates would be a useful addition to the clinical microbiology laboratory lacking resources for traditional anaerobic culturing techniques. Conventional methods for isolating and identifying anaerobes from clinical specimens are often costly and time-consuming (2, 3). The gold standard procedure of using an anaerobe chamber in conjunction with selective and enriched anaerobe culture media for isolation and identification of anaerobes is not practical for many clinical microbiology laboratories. One means of reducing costs is by using an alternate method for the production of an anaerobic environment. In recent years, several alternative anaerobic generation systems have become available; these include the Oxoid Anaero- Gen System (Unipath, Inc., Nepean, Ontario, Canada), BBL GasPak Anaerobic System (Beckon Dickinson Microbiology Systems, Cockeysville, Md.), Anaerobic Pouch System (Difco Laboratories, Detroit, Mich.), and AnaeroPack System (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan). Recently, Oxyrase, Inc., Mansfield, Ohio, introduced the Oxyrase OxyPlate anaerobe incubation system. OxyPlates combine the anaerobic generation property of Oxyrase into the culture medium itself and thus serve as self-contained anaerobe chambers (1). In this study, we evaluated the Oxyrase OxyPlate system for its ability to support the growth of clinically significant anaerobic bacteria previously identified by the anaerobe reference laboratory at the Centers for Disease Control and Prevention (). (This study was presented in part at the 98th General Meeting of the American Society for Microbiology, Atlanta, Ga., 17 to 21 May 1998.) MATERIALS AND METHODS Bacterial strains. A total of 251 clinically significant anaerobic and facultatively anaerobic strains (Table 1) from the culture collection of the anaerobic reference laboratory at the were used to evaluate the efficacy of the * Corresponding author. Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Rd., N.E., Mail Stop C-16, Atlanta, GA Phone: (404) Fax: (404) OxyPlate anaerobe incubation system for culturing anaerobic bacteria. Results were compared with those obtained with conventional anaerobe blood agar plates (BAP) incubated in an anaerobe chamber. The anaerobe chamber used in this study (Coy Laboratory Products, Inc., Grass Lake, Mich.) is a flexible glove box kept at 35 to 37 C and filled with a gas mixture of 5% carbon dioxide, 10% hydrogen, and 85% nitrogen. Before the chamber is entered, the port is automatically flushed twice with nitrogen and once with the anaerobic gas mixture. Anaerobic conditions within the chamber are indicated by a colorless methylene blue solution with chamber air bubbled slowly through it (8). The solution turns blue in the presence of air. All bacteria tested were either clinical isolates obtained from specimens submitted to the anaerobe reference laboratory for isolation and characterization or strains from the American Type Culture Collection. In general, eight strains of each species were tested. The species chosen were the anaerobes isolated most frequently from clinical specimens. One American Type Culture Collection reference strain of each organism tested was included in the evaluation (Tables 2 to 4). All strains tested had been identified previously to genus and species levels by conventional reference identification methods and gas-liquid chromatography (4 6, 8, 9, 11). Test organisms were stored lyophilized at 4 C. All lyophilized strains were subcultured a minimum of two times on anaerobe BAP (Carr-Scarborough Microbiologicals, Stone Mountain, Ga.) before being used in the study. As recommended by the OxyPlate manufacturer, Bacteroides fragilis ATCC was used for the quality control testing of each new lot of test plates. Oxyrase OxyPlates. Each OxyPlate is a self-contained anaerobe chamber in which a sealing ring in the lid of the plate rests on the agar surface to create an airtight seal. The anaerobic brucella blood agar (BBA) Oxyrase OxyPlate was evaluated in this study. BBA OxyPlates are similar to BAP; both contain an enriched medium that, when incubated anaerobically, allows the growth of both obligate and facultative anaerobes. Both BBA OxyPlates and BAP contain 5% (vol/vol) sheep blood for enrichment and detection of hemolysis, vitamin K 1 (required by some Porphyromonas spp.), and hemin, which enhances the growth of members of the B. fragilis group and certain other Bacteroides spp. In a closed and properly sealed plate, the Oxyrase enzyme removes oxygen from the agar medium and the confined space above the agar. Inoculated OxyPlates are inverted and incubated at 35 C in a standard, humidified, aerobic incubator. OxyPlates remain reduced during storage and normal handling. After opening and reclosing of the plate, the time required to achieve anaerobiosis inside the plate is temperature dependent. For this reason, the manufacturer recommends that OxyPlates be kept at room temperature or prewarmed to 37 C prior to being inoculated. Anaerobiosis inside an OxyPlate can be monitored by the use of OxyBlue indicator. Following inoculation of the OxyPlate and prior to incubation, a small drop of the indicator is applied to the inside of the OxyPlate lid. The plate is inverted and incubated as usual. The change in color of the OxyBlue indicator during incubation is used as a guide to the state of anaerobiosis inside the 499

2 500 WIGGS ET AL. J. CLIN. MICROBIOL. TABLE 1. Taxa used in the evaluation of Oxyrase OxyPlates Category Description Group or genus (n) Species Gram-negative bacilli Bile tolerant B. fragilis group (31) B. distasonis, B. fragilis, B. thetaiotaomicron, B. vulgatus Bile tolerant, nonpigmented Campylobacter (8) C. curvus, C. rectus Nonpigmented, pitting Anaerobiospirillum (8) A. succiniproducens Bacteroides (8) B. ureolyticus Fusobacterium (12) F. necrophorum, F. nucleatum Leptotrichia (8) L. buccalis Clostridia Clostridium (96) C. beijerinckii, C. bifermentans, C. butyricum, C. clostridiiforme, C. difficile, C. innocuum, C. perfringens, C. sordellii, C. sphenoides, C. septicum, C. sordellii, C. sporogenes, C. tertium Non-spore-forming gram-positive bacilli OxyPlate. White or pale blue indicates an anaerobic condition, while blue indicates an aerobic condition. Test procedure. Lyophilized strains were reconstituted and passed a minimum of two times on anaerobe BAP in an anaerobic chamber at 35 C. After 24 to 48 h of incubation (up to 72 h for some slowly growing cocci and Actinomyces spp.), colonies from pure isolates were harvested aseptically with cotton-tipped applicator swabs and suspended in a labeled tube of Lombard-Dowell broth to a turbidity equivalent to a McFarland no. 4 standard. Three anaerobe BAP and one OxyPlate were inoculated at the bench with the suspension using a sterile 0.01-ml loop. One OxyPlate and one anaerobe BAP were incubated aerobically at 35 C; as required, one anaerobe BAP was incubated in CO 2 at 35 C, and one anaerobe BAP was incubated in an anaerobe chamber at 35 C to serve as the growth control. Plates were read at 24, 48, and 72 h. Growth was scored by a numerical coding system used at the that combines the degree of growth and the colony size. Criteria for recording the degree of growth and the colony size on each agar plate were as follows: 0, no growth; 1, sparse ( 30) and tiny ( 1 mm) colonies; 2, sparse and small to medium (1 to 5 mm) colonies; 3, sparse and large ( 5 mm) colonies; 4, moderate (30 to 300) and tiny ( 1 mm) colonies; 5, moderate and small to medium (1 to 5 mm) colonies; 6, moderate and large ( 5 mm) colonies; 7, abundant ( 300) and tiny ( 1 mm) colonies; 8, abundant and small to medium (1 to 5 mm) colonies; and 9, abundant and large ( 5 mm) colonies. RESULTS Actinomyces (32) Mobiluncus (8) Propionibacterium (24) Gram-positive cocci Peptostreptococcus (16) P. anaerobius, P. magnus We evaluated the degree of growth and the colony size of 251 anaerobic bacterial strains grown in the BBA Oxyrase OxyPlate anaerobe incubation system and compared the results with those obtained for the same organisms grown on BAP incubated in a conventional anaerobe chamber. Performance characteristics for individual strains varied. With few exceptions, after 72 h of incubation, most strains tested exhibited adequate growth in both systems to allow for further workup. At 72 h, 176 (70.1%) of the 251 strains showed abundant growth (a rating of 7 to 9) in both systems; 37 of the remaining 75 strains showed moderate growth (a rating of 4 to 6) and 38 strains showed sparse growth (a rating of 1 to 3) or no growth (a rating of 0) in the OxyPlate system. In contrast, at 72 h, for the organisms grown on BAP in a conventional anaerobe chamber, 62 strains showed abundant growth and 13 strains showed moderate growth. Table 2 shows the results for the anaerobic gram-negative bacilli. Of the 31 B. fragilis group strains tested, 19 (61.3%) showed equal growth in both systems after 24 h. At 48 and 72 h, 28 (90.3%) of the B. fragilis group strains showed equal (abundant) growth in both systems, while 1 strain (3.2%) showed sparse growth and 2 strains (6.5%) showed moderate growth in A. israelii, A. meyeri, A. naeslundii, A. odontolyticus M. curtisii subsp. holmesii P. acnes, P. avidum, P. granulosum the OxyPlate system but abundant growth on BAP. Of the eight nonpigmented Bacteroides group strains (B. ureolyticus) tested, after 72 h of incubation, three (37.5%) showed moderate growth, one (12.5%) showed sparse growth, and four (50%) showed no growth in the OxyPlate system; all strains showed abundant growth on BAP. Repeat testing produced similar results. Likewise, only one of eight Campylobacter rectus strains and five of eight Leptotrichia buccalis strains tested showed any degree of growth on OxyPlates after 72 h of incubation. Repeat testing produced similar results. Of the Fusobacterium strains tested, two (50.0%) of the four Fusobacterium necrophorum strains showed equal (abundant) growth in both systems after 72 h of incubation; the remaining two strains did not grow on OxyPlates. Likewise, two (25.0%) of the eight F. nucleatum strains showed equal (abundant) growth in both systems after 72 h of incubation; two of the remaining six strains showed moderate growth on OxyPlates after 72 h of incubation. In summary, after 72 h of incubation in the OxyPlate system, 35 (46.7%) of 75 gram-negative bacilli tested exhibited abundant growth equal to that on anaerobe BAP grown in the anaerobe chamber, 11 (14.7%) of 75 showed moderate growth, and the remaining 29 (38.7%) of 75 showed no growth. Table 3 shows the results for the anaerobic gram-positive bacilli. A total of 32 Actinomyces strains were tested; of these, 23 (71.9%) showed equal (abundant) growth in both systems after 48 to 72 h of incubation. A total of five (62.5%) of eight Actinomyces israelii strains tested showed only sparse growth on OxyPlates after 72 h of incubation; all eight strains showed moderate growth on BAP incubated in the anaerobe chamber. Of the 96 Clostridium strains tested, 61 (63.5%) showed equal (abundant) growth in both systems after 48 to 72 h incubation; 13 (13.5%) showed moderate growth on OxyPlates after 72 h of incubation but abundant growth on anaerobe BAP. The growth of Clostridium innocuum and C. sordellii in the OxyPlate system was variable. Of the seven C. innocuum strains tested, after 72 h of incubation, two of seven (28.6%) showed abundant growth, four of seven (59.1%) showed moderate growth, and one of seven (14.3%) showed no growth on OxyPlates; seven of seven (100%) showed abundant growth on anaerobe BAP. A total of nine C. sordellii strains were tested; of these, after 72 h of incubation, two of nine (22.2%)

3 VOL. 38, 2000 OXYRASE OXYPLATE ANAEROBE INCUBATION SYSTEM 501 TABLE 2. Growth differences for anaerobic gram-negative bacilli with Oxyrase OxyPlates and anaerobe BAP Growth score b at the following time (h): Test organism a Anaerobiospirillum succiniciproducens A. succiniciproducens ATCC A. succiniciproducens A. succiniciproducens A. succiniciproducens A. succiniciproducens A. succiniciproducens A. succiniciproducens Bacteroides distasonis B. distasonis B. distasonis B. distasonis B. distasonis ATCC B. distasonis B. distasonis B. distasonis B. fragilis ATCC B. fragilis B. fragilis B. fragilis B. fragilis B. fragilis B. fragilis B. thetaiotaomicron ATCC B. thetaiotaomicron B. thetaiotaomicron B. thetaiotaomicron B. thetaiotaomicron B. thetaiotaomicron B. thetaiotaomicron B. thetaiotaomicron B. vulgatus ATCC B. vulgatus B. vulgatus B. vulgatus B. vulgatus B. vulgatus B. vulgatus B. vulgatus B. ureolyticus ATCC B. ureolyticus B. ureolyticus B. ureolyticus B. ureolyticus B. ureolyticus B. ureolyticus B. ureolyticus A7116 Campylobacter rectus ATCC A8201 C. rectus A8202 C. rectus A8204 C. rectus C. rectus C. rectus C. rectus C. rectus Fusobacterium necrophorum F. necrophorum ATCC F. necrophorum F. necrophorum Continued on following page

4 502 WIGGS ET AL. J. CLIN. MICROBIOL. TABLE 2 Continued Growth score b at the following time (h): Test organism a F. nucleatum F. nucleatum F. nucleatum ATCC F. nucleatum F. nucleatum F. nucleatum F. nucleatum F. nucleatum Leptotrichia buccalis ATCC L. buccalis L. buccalis L. buccalis L. buccalis L. buccalis L. buccalis L. buccalis a B. distasonis, B. fragilis, B. thetaiotaomicron, and B. vulgatus are members of the B. fragilis group. B. ureolyticus is a nonpigmented Bacteroides. b 0, no growth; 1 to 3, sparse ( 30/plate) and tiny ( 1 mm), small to medium (1 to 5 mm), or large ( 5 mm) colonies; 4 to 6, moderate (30 to 300/plate) and tiny, small to medium, or large colonies; 7 to 9, abundant ( 300/plate) and tiny, small to medium, or large colonies. showed abundant growth, two of nine (22.2%) showed moderate growth, four of nine (44.4%) showed sparse growth, and one of nine (11.1%) showed no growth on OxyPlates; all nine strains of C. sordellii showed abundant growth on anaerobe BAP. None of the eight strains of C. beijerinckii grew on OxyPlates after 72 h of incubation; however, all eight strains showed abundant growth on anaerobe BAP. Among the anaerobic non-spore-forming gram-positive bacilli tested, seven of eight (87.5%) Mobiluncus curtisii subsp. holmesii strains showed equal (abundant) growth in both systems after 48 h of incubation; one strain showed moderate growth on OxyPlates after 72 h of incubation. Of 24 Propionibacterium spp. tested, after 72 h of incubation, 16 (66.6%) showed equal (abundant) growth in both systems, 5 (20.8%) showed moderate growth on OxyPlates, and 3 (12.5%) showed better growth (abundant) on OxyPlates than on anaerobe BAP. In summary, after 72 h of incubation in the BBA OxyPlate system, 110 (68.8%) of 160 gram-positive bacilli tested showed abundant growth equal to that obtained on anaerobe BAP incubated in the anaerobe chamber; 25 of 160 (15.6%) showed moderate growth on OxyPlates and abundant growth on anaerobe BAP, and 25 of 160 (15.6%) showed sparse growth on OxyPlates and abundant growth on anaerobe BAP. Table 4 summarizes the results for the anaerobic grampositive cocci. After 72 h of incubation in the BBA OxyPlate system, 14 (87.5%) of 16 Peptostreptococcus strains tested showed abundant growth equal to that obtained on anaerobe BAP grown in the anaerobe chamber; 2 strains (12.5%) showed moderate growth in the BBA OxyPlate system and abundant growth on anaerobe BAP. DISCUSSION In this study, we evaluated the BBA Oxyrase OxyPlate anaerobe incubation system to determine its ability to support the growth of a wide variety of anaerobic bacteria and to compare results obtained with this system to those obtained with the reference method of conventional anaerobe BAP incubated in an anaerobe chamber. Each OxyPlate contains an agar medium (BBA) incorporating the Oxyrase enzyme. The agar medium is reduced at the time of use and remains reduced when exposed to air due to the catalytic action of Oxyrase. OxyPlates can be opened several times throughout the culture workup, and the enzyme will continue to reduce the oxygen in the medium and the space between the lid and the agar surface. In effect, each OxyPlate, properly closed and sealed, serves as a self-contained anaerobe chamber. Whereas the anaerobic environment existing in a conventional anaerobe chamber includes a mixture of carbon dioxide, hydrogen, and nitrogen, the anaerobic environment within a properly closed OxyPlate is initially simply one devoid of oxygen. The enzyme Oxyrase specifically removes oxygen from the microenvironment but does not generate any other gas. After an initial period of familiarization with the handling and use of OxyPlates, the reading of inoculated plates and the interpretation of test results were not difficult. A numerical coding system used at the that combines the degree of growth and the colony size was used to record comparative growth and colony size obtained with the two systems. It is generally recognized that while many different species of anaerobes can potentially be isolated from clinical specimens, the actual number of species routinely isolated is relatively small. It has been reported (7) that only five anaerobes or groups account for two-thirds of clinically significant, anaerobic infectious processes: anaerobic cocci, members of the B. fragilis group, pigmented species of Bacteroides (recently reclassified as Porphyromonas and Prevotella spp.), F. nucleatum, and C. perfringens. In our study, the Oxyrase OxyPlate system performed well in supporting the growth of these critical anaerobes as well as that of a variety of other clinically significant anaerobic bacteria. At this point, it should be emphasized that the numerical coding system used in this evaluation to indicate the level of growth of the anaerobes tested combines both the degree of growth and the colony size. Therefore, a classification of growth as abundant (a rating of 7 to 9) indicates a degree of

5 VOL. 38, 2000 OXYRASE OXYPLATE ANAEROBE INCUBATION SYSTEM 503 TABLE 3. Growth differences for anaerobic gram-positive bacilli with Oxyrase OxyPlates and anaerobe BAP Growth score a at the following time (h): Test organism Actinomyces israelii A. israelii ATCC A. israelii A. israelii A. israelii A. israelii A. israelii A. israelii A. meyeri A. meyeri A. meyeri ATCC A. meyeri A. meyeri A. meyeri A. meyeri A. meyeri W1946 A. naeslundii A. naeslundii ATCC A. naeslundii A. naeslundii A. naeslundii A. naeslundii A. naeslundii A. naeslundii A. odontolyticus ATCC A. odontolyticus A. odontolyticus A. odontolyticus A. odontolyticus A. odontolyticus A. odontolyticus A. odontolyticus Clostridium beijerinckii C. beijerinckii C. beijerinckii C. beijerinckii C. beijerinckii C. beijerinckii ATCC C. beijerinckii C. beijerinckii C. bifermentans ATCC C. bifermentans C. bifermentans C. bifermentans C. bifermentans C. bifermentans C. bifermentans C. bifermentans C. butyricum C. butyricum C. butyricum C. butyricum C. butyricum C. butyricum C. butyricum C. butyricum ATCC C. clostridiiforme ATCC C. clostridiiforme Continued on following page

6 504 WIGGS ET AL. J. CLIN. MICROBIOL. TABLE 3 Continued Growth score a at the following time (h): Test organism C. clostridiiforme C. clostridiiforme C. clostridiiforme C. clostridiiforme C. clostridiiforme C. clostridiiforme A4897 C. difficile ATCC C. difficile C. difficile C. difficile C. difficile C. difficile C. difficile C. difficile C. difficile C. difficile C. innocuum ATCC C. innocuum C. innocuum C. innocuum C. innocuum C. innocuum C. innocuum C. perfringens ATCC C. perfringens C. perfringens C. perfringens C. perfringens C. perfringens C. perfringens C. perfringens A1599 C. ramosum ATCC C. ramosum C. ramosum C. ramosum C. ramosum C. ramosum C. septicum C. septicum ATCC C. septicum C. septicum C. septicum C. septicum C. septicum C. septicum C. sordellii ATCC C. sordellii C. sordellii C. sordellii C. sordellii C. sordellii C. sordellii C. sordellii C. sordellii C. sporogenes ATCC C. sporogenes C. sporogenes C. sporogenes Continued on following page

7 VOL. 38, 2000 OXYRASE OXYPLATE ANAEROBE INCUBATION SYSTEM 505 TABLE 3 Continued Growth score a at the following time (h): Test organism C. sporogenes C. sporogenes C. sporogenes C. sporogenes C. tertium ATCC C. tertium C. tertium C. tertium C. tertium C. tertium C. tertium C. tertium Mobiluncus curtisii subsp. holmesii M. curtisii subsp. holmesii M. curtisii subsp. holmesii M. curtisii subsp. holmesii M. curtisii subsp. holmesii M. curtisii subsp. holmesii M. curtisii subsp. holmesii M. curtisii subsp. holmesii ATCC Propionibacterium acnes ATCC P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. avidum P. avidum P. avidum P. avidum ATCC P. avidum P. avidum P. avidum P. avidum P. granulosum ATCC P. granulosum P. granulosum P. granulosum P. granulosum P. granulosum P. granulosum P. granulosum a 0, no growth; 1 to 3, sparse ( 30/plate) and tiny ( 1 mm), small to medium (1 to 5 mm), or large ( 5 mm) colonies; 4 to 6, moderate (30 to 300/plate) and tiny, small to medium, or large colonies; 7 to 9, abundant ( 300/plate) and tiny, small to medium, or large colonies. growth of 300 colonies per plate ranging in size from tiny ( 1 mm) to large ( 5 mm); a classification of growth as moderate (a rating of 4 to 6) indicates a degree of growth of 30 to 300 colonies ranging in size from 1 mmto 5 mm. In the clinical laboratory, this amount of growth would be sufficient to Gram stain, subculture, and otherwise characterize and identify the organisms isolated. Most anaerobic gram-positive cocci encountered in clinical specimens are Peptostreptococcus spp. As previously noted, the Peptostreptococcus strains tested grew well in the OxyPlate system; 14 of 16 (87.5%) showed abundant growth and 2 of 16 (12.5%) showed moderate growth on OxyPlates after 72 h of incubation, and all 16 strains showed abundant growth on anaerobe BAP. Similarly, the OxyPlate system performed well in supporting the growth of 31 strains of the B. fragilis group. Members of the B. fragilis group are the most commonly recovered anaerobes found in clinical specimens. Members of this group are more resistant to antimicrobial agents than most other anaerobes and exhibit species-to-species variability in both virulence and drug resistance. Therefore, it is important to be able to recover and identify these organisms. In our evaluation, 28 (90.3%) of the 31 strains of the B. fragilis group that we tested showed abundant growth, 2 (6.5%) showed moderate growth, and 1

8 506 WIGGS ET AL. J. CLIN. MICROBIOL. TABLE 4. Growth differences for anaerobic gram-positive cocci with Oxyrase OxyPlates and anaerobe BAP Growth score a at the following time (h): Test organism A993 Peptostreptococcus anaerobius A1738 P. anaerobius A1739 P. anaerobius A1996 P. anaerobius P. anaerobius ATCC P. anaerobius P. anaerobius P. anaerobius A795 P. magnus A1164 P. magnus A2104 P. magnus ATCC A2541 P. magnus P. magnus P. magnus P. magnus P. magnus a 0, no growth; 1 to 3, sparse ( 30/plate) and tiny ( 1 mm), small to medium (1 to 5 mm), or large ( 5 mm) colonies; 4 to 6, moderate (30 to 300/plate) and tiny, small to medium, or large colonies; 7 to 9, abundant ( 300/plate) and tiny, small to medium, or large colonies. (3.2%) showed sparse growth after 72 h of incubation on Oxy- Plates; 31 (100%) showed abundant growth on BAP incubated in the anaerobe chamber. So, essentially both systems were comparable in supporting the growth of B. fragilis group organisms. Conversely, Oxyrase OxyPlates did not adequately support the growth of the nonpigmented Bacteroides strains tested. Only three (37.5%) of the eight strains of B. ureolyticus tested showed moderate growth, one (12.5%) showed sparse growth, and four (50%) showed no growth after 72 h of incubation. In contrast, all eight strains (100%) showed abundant growth on BAP. Repeat testing of these strains produced the same results. In the clinical laboratory, growth and identification of this organism are important because B. ureolyticus has been isolated from infectious processes of the respiratory and intestinal tracts and from blood following tooth extraction. The results of the evaluation of the Porphyromonas and Prevotella spp. are not included here because the conventional media used in their growth failed to meet acceptable quality control standards. The Fusobacterium species most commonly isolated from human clinical specimens is F. nucleatum. This organism is associated with severe odontogenic infections and is an important pathogen in head, neck, and lower-respiratory-tract infections. In our evaluation, the growth of F. nucleatum in the OxyPlate system was variable: of the eight strains tested, two (25%) showed abundant growth, two (25%) showed moderate growth, and four (50%) showed no growth after 72 h of incubation; however, all eight strains showed abundant growth on anaerobe BAP. F. necrophorum is a much more virulent Fusobacterium species than F. nucleatum; this organism can cause necrotizing ulcerative gingivitis as a complication of Vincent s angina. It may also cause postanginal septicemia in children or young adults. Of the F. necrophorum strains tested, two of four (50%) showed abundant growth and two (50%) failed to grow on OxyPlates after 72 h of incubation; all four strains (100%) showed abundant growth on anaerobe BAP after 72 h of incubation. Repeat testing of these strains produced the same results. The Clostridium strains presented the Oxyrase OxyPlate system with its greatest challenge. Of the 96 Clostridium strains tested, 61 (63.5%) showed abundant growth and 13 (13.5%) showed moderate growth on OxyPlates. These results are skewed due to the inadequate growth of C. sordellii and C. innocuum and no growth of C. beijerincki on OxyPlates. Likewise, after 72 h, 3 of 10 (30%) OxyPlate-grown C. difficile strains showed abundant growth, 6 (60%) showed moderate growth, and 1 (10%) showed sparse growth; all 10 strains (100%) showed abundant growth on anaerobe BAP. In addition, colonies of C. difficile grown on OxyPlates failed to exhibit a characteristic chartreuse fluorescence when placed under long-wave UV light. As this characteristic is of value in making a presumptive identification of C. difficile, this problem could hamper the ability of a laboratory to expeditiously identify C. difficile. The above notwithstanding, the OxyPlate system did perform well in supporting abundant growth of other clinically encountered Clostridium species, i.e., C. perfingens (eight of eight; 100%), C. septicum (seven of eight; 87.5%), C. sporogenes (seven of eight; 87.5%), and C. tertium (seven of eight; 87.5%). C. perfingens is the most commonly isolated clostridial species and is the most common etiologic agent of gas gangrene. C. septicum is clinically associated with bacteremia, neutropenia, necrotizing enterocolitis, suppurative infectious processes, and gas gangrene. C. sporogenes has been isolated from bacteremia, endocarditis, pleuropulmonary infectious processes, gas gangrene, infected war wounds, and other pyogenic infectious processes. C. tertium has been isolated from appendicitis, brain abscesses, infectious processes related to the intestinal tract and soft tissue, infected wounds, bacteremia, and gas gangrene. Our experience with the Clostridium strains showed that this system has some limitations. Since the OxyPlate anaerobe system uses a modified microenvironment, growth results may differ from those previously established with conventional anaerobe chambers. In addition, it is generally recognized that minor variations may exist in strains within species. The isolates that we tested included a mixture of commonly isolated anaerobic species and others that had been phenotypically unusual and/or difficult to grow and identify; this fact provided a challenge to the sensitivity of the system. Thus, the performance of the system in this study may be a reflection of both the difficulty in working with the latter group of organisms and the limitations of the microenvironment of the system.

9 VOL. 38, 2000 OXYRASE OXYPLATE ANAEROBE INCUBATION SYSTEM 507 One aspect of the microenvironment of the system was addressed in 1997 by Gannon and Thurston (Abstr. 97th Gen. Meet. Am. Soc. Microbiol., abstr. C-212, p. 155, 1997), who compared anaerobic growth on duplicate OxyPlates incubated at 35 C in ambient air and in a 6% humidified CO 2 atmosphere. From 382 clinical wound specimens inoculated onto duplicate OxyPlates, they were able to recover 170 anaerobic bacteria, of which 29 (17%) were recovered from the 6% CO 2 - incubated OxyPlates and not from those incubated in ambient air. Our results disagree with these findings in that four of the nine organisms listed as not having been recovered from the cultures incubated in ambient air (for example, C. innocuum, B. fragilis, B. distasonis, and Mobiluncus) in fact grew well in ambient air in our evaluation; also, A. israelii showed some growth (sparse) in our study. We are of the opinion that the question of the most efficacious atmosphere for the incubation of OxyPlates warrants further controlled studies. Users of the OxyPlate anaerobe system should recognize the limitations discussed above. As with all anaerobic bacteriology work, identification of organisms grown in this system requires the application of basic microbiologic knowledge and recognition of certain key characteristics of anaerobic bacteria encountered in the clinical microbiology laboratory. When warranted, the final identification of isolates should take into consideration the possible need for additional conventional anaerobic testing. In this respect, we recommend as excellent reference sources the texts by Engelkirk et al. (6) and Summanen et al. (11) and the laboratory manual by Holdeman et al. (8). In our study, the Oxyrase OxyPlate anaerobe incubation system was shown to be an acceptable alternative to the conventional anaerobe glove chamber for the growth of most, but not all, clinically significant anaerobic bacteria. However, we wish to emphasize that the results presented reflect the growth of anaerobic strains obtained from laboratory stocks subcultured on BAP; the recovery of anaerobic strains directly from clinical specimens may differ. The list price for the Oxyrase OxyPlate anaerobe identification system varies, depending on the type of order, from $2.50 to $3.00 per plate. This cost is similar to that for the Anaero- Gen and GasPak systems (10). The OxyPlate system was easy to use, required no extraneous apparatus to create an anaerobic environment, and was as fast to use as conventional techniques. This system would be a useful addition to the microbiology laboratories of hospitals seeking an economical approach to providing anaerobic bacteriology services. REFERENCES 1. Adler, H. I The use of microbial membranes to achieve anaerobiosis. Crit. Rev. Biotechnol. 10: Ballows, A., W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.) Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 3. Baron, E. J., and S. M. Finegold Bailey and Scott s diagnostic microbiology, 8th ed. The C.V. Mosby Co., St. Louis, Mo. 4. Dowell, V. R., Jr., G. L. Lombard, F. S. Thompson, and A. Y. Armfield Media for isolation, characterization, and identification of obligately anaerobic bacteria. U.S. Department of Health and Human Services, Center for Disease Control, Atlanta. 5. Dowell, V. R. Jr., and T. M. Hawkins Laboratory methods in anaerobic bacteriology. Center for Disease Control, Atlanta, Ga. 6. Engelkirk, P. G., J. Duben-Engelkirk, and V. R. Dowell, Jr. (ed.) Principles and practice of clinical anaerobic bacteriology. Star Publishing Co., Belmont, Calif. 7. Finegold, S. M Anaerobic bacteria: their role in infection and their management. Postgrad. Med. J. 81: Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.) Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg. 9. Lombard, G. L., and V. R. Dowell, Jr Gas-liquid chromatography analysis of the acid products of bacteria. Centers for Disease Control, Atlanta, Ga. 10. Miller, P. H., L. S. Wiggs, and J. M. Miller Evaluation of AnaeroGen system for growth of anaerobic bacteria. J. Clin. Microbiol. 33: Summanen, P., E. J. Baron, D. M. Citron, C. Strong, H. M. Wexler, and S. M. Finegold (ed.) Wadsworth anaerobic bacteriology manual. Star Publishing Co., Belmont, Calif.