Improved Chamber for the Isolation of Anaerobic Microorganisms

JOUNAL OF CLINICAL MICROBIOLOGY, July 1976, p. 40-45 Copyright ) 1976 American Society for Microbiology Vol. 4, No. 1 Printed in U.S.A. Improved Chamber for the Isolation of Anaerobic Microorganisms MARION E. COX* AND JAMES I. MANGELS International Shellfish Enterprises, Moss Landing, California 95039; Infectious Disease Laboratory, Stanford University Medical Center, Palo Alto, California 94304 Received for publication 10 December 1975 A small portable chamber for the recovery of anaerobic bacteria is described. This rigid chamrhr is constructed of clear acrylic with dimensions of 30 inches (ca. 76.2 cm) wide, 18 inches (ca. 44.7 cm) deep, and 18 inches (ca. 44.7 cm) high. Conventional bacteriological techniques can be used inside the chamber to efficiently isolate strict anaerobic organisms. An adapter allows the attachment of a standard anaerobic jar to the outside of the chamber. The jar can be used to store reduced media. Once the jar is attached to the chamber and the media is removed to the interior of the chamber, the jar is available to receive inoculated media. The anaerobic jar can then be removed from the chamber, without contaminating the jar or chamber with oxygen, and be placed in a conventional 37 C incubator. This chamber also allows the microbiologist to process cultures without wearing gloves as was necessary with previous anaerobic chambers. Air-tight latex rubber sleeves seal around the microbiologists arms and to the armport flange of the chamber to prevent the introduction of oxygen into the chamber. Anaerobic conditions are maintained by circulating a 80% N2, 10% H2, 10% CO2 gas mixture through alumina pellets coated with palladium. This study indicates that anaerobic conditions obtained in this chamber are sufficient for recovery of obligate anaerobes. The advantages of an anaerobic glove box for the recovery of strict anaerobic microorganisms have been well documented (1, 2, 4, 6, 8, 9, 11). The use of the anaerobic glove box in most clinical situations, however, has been limited by the large space required for the chamber and by its high initial cost. Another reported disadvantage has been the thick rubber gloves which can be cumbersome while handling primary isolation cultures (10). Most glove boxes described in the literature have also required elaborate procedures to set-up and to maintain anaerobiosis within the chamber (3, 9). Described below is an anaerobic chamber designed by one of us (M. Cox) which will minimize these difficulties; thus making isolation and identification of anaerobic organisms more practical for the clinical laboratory, and allowing for anaerobic investigations where space, cost, and the ability to use conventional microbiological techniques are essential factors. Fundamental to this chamber is the fact that it permits the microbiologist to use conventional microbiological techniques in a small portable work area, without the inconvenience of gloved hands, and with the added advantage of more efficient methods for introduction of materials into and out of the chamber. (Certain parts of the described chamber are covered by U.S. patent no. 3907389; other patents are pending.) Rigid glove boxes contain a finite volume. The introduction of an additional volume such as the operator's arms cannot be accommodated by an increase in volume and results in an increase in pressure. This causes displacement resistance or glove "fight-back" (2). This chamber provides a means of adjusting the internal pressure to overcome displacement resistance. MATERIALS AND METHODS The chamber consists of an air-tight acrylic chamber 30 inches (ca. 76.20 cm) wide and 18 inches (ca. 44.7 cm) high and deep (Fig. 1). Adaptors have been designed so that conventional anaerobic jars such as GasPak jars (Baltimore Biological Laboratory [BBI], Division of Bioquest, Cockeysville, Md.) can be attached to the outside of the chamber without exposing the gaseous environment of either the chamber or of the anaerobic jar to air, as shown in Fig. 2. Anaerobic cultures can be placed into the anaerobic jar from within the chamber, and then the jar can be sealed and removed to a conventional 35 C incubator. The chamber is fitted with glove sleeves, an 8-inch (ca. 19.3 cm)-square pass box, an elec- 40 Downloaded from http://jcm.asm.org/ on May 14, 2018 by guest

VOL. 4, 1976 IMPROVED ANAEROBIC ISOLATION CHAMBER 41 1 1 FIG. 1. Front view of chamber: 1, gloveless sleeve; 2, vacuumlgas tubing to evacuate and refill sleeves; 3, vacuum/gas tubing for air lock; 5, catalystldessicant basket; 6, adaptor for dissecting microscope. A 167 Downloaded from http://jcm.asm.org/ 14 13 15 2 td t~~~ on May 14, 2018 by guest 13~~~~~~~~1 FIG. 2. (A) Anaerobic jar shown without lid: 16, an adaptor to hold the catalyst basket. (B) View facing the anaerobic jar adaptor: 7, acrylic disk; 15, 0-ring; 21, opening to the interior ofthe Lhamber covered by disk 7. (C) Anaerobic jar: 13, with lid; 14, in place shown attached to the anaerobic jar adaptor. (D) Anaerobic jar: 13, rotated away from lid; 14, to the chamber opening by disk 7.

42 COX AND MANGELS tronic internal chamber pressure regulator (Microbial Systems, Cupertino, Calif.), a foot switch to operate the armport flushing system, two airport covers, a catalyst/dessicant basket, an electrical outlet, and an adapter to allow the use of a dissecting microscope through the acrylic wall. In addition, this chamber can be mounted on a portable table to allow movement from the laboratory to a walk-in incubator. Operation of chamber. Anaerobic conditions are established initially in the chamber by the use of a displacement balloon to physically expel air from the chamber (6). Then the chamber is refilled with a gas mixture of 80% N2, 10% C02, and 10% H2 (Liquid Carbonic Corp., San Carlos, Calif.). The above procedure is repeated twice. Alumina pellets coated with palladium are present to remove trace amounts of oxygen. The catalyst basket is divided into two parts, one half for the catalyst and the other half filled with a silica dessicating agent. The catalyst is reactivated every 3 days by heating at 160 C for 2 h. The silica dessicating agent is replaced as needed. A positive pressure of 3 inches (ca. 7.6 cm) of water is provided by the electronic pressure controller to maintain the gaseous environment of the chamber. One simple operation can simultaneously remove the cover from the anaerobic jar and place the anaerobic jar over an opening to the chamber. This allows access from the interior of the chamber to the inside of the jar. This operation can be done in less than 30 s. Neither the chamber nor the anaerobic jar needs to be flushed before attachment or after removal of the jar, provided that both the chamber and the anaerobic jar are initially anaerobic. A circular opening with a diameter of 4.75 inches (ca. 11.9 cm) was made in the back wall of the chamber to allow passage of material from the anaerobic jar to the inside of the chamber. Covering this chamber opening is an acrylic disk with a diameter of 14 inches (ca. 35.6 cm). This disk is attached at its center to a pivot point located 0.5 inch (ca. 1.27 cm) to the side of the chamber opening. An opening was made in this disk with a diameter of 6 inches (ca. 15.2 cm) to J. CLIN. MICROBIOL. receive the rim portion of an anaerobic jar. Figure 2B shows the disk covering the chamber opening and Fig. 2C and D show the disk rotated to communicate the anaerobic jar to the chamber opening. Guide rails are provided to define the path of the anaerobic jar during connection or removal. Two clamps, one at the disk center and the other at the periphery are provided to maintain proper sealing of the covering disk or an anaerobic jar to the 0-ring of the chamber opening. The positive pressure of 3 inches (ca. 76.2 cm) of water maintained inside the chamber prevents oxygen contamination during connection or removal of an anaerobic jar. This also serves as a source of new gas to maintain anaerobic conditions. A second method for the introduction of material into the chamber is through an air lock. Materials are placed inside the air lock which is evacuated to 25 mm of Hg and refilled with an oxygen-free gas. This is repeated two additional times. The material is then brought into the chamber from the air lock. A third method which allows access to the interior of the chamber is a unique armport system as shown in Fig. 1, 3, and 4. The microbiologist inserts each hand into a gloveless latex sleeve which at one end comfortably seals around the arm of the microbiologist and at the other end seals to the armport flange (Fig. 4B). An inner acrylic port door separates the armport entry from the chamber interior prior to insertion of the microbiologist's hands as shown in Fig. 4A. The microbiologist can then evacuate the air from around his arms and inside the sleeves with a vacuum source and refill the space with oxygenfree gas. The inner armport cover can then be removed and the bare hands and arms of the operator can move freely within the chamber (Fig. 4C). The addition or removal of gas from the chamber via the armport flushing system once the armport covers are removed, allows adjustment of internal pressure to prevent displacement resistance. This addition or removal of gas from the chamber provides further renewal of the gaseous environment. For the purpose of this study the anaerobic jars as Downloaded from http://jcm.asm.org/ on May 14, 2018 by guest FIG. 3. Side view of chamber: 1, gloveless sleeve shown sealed to the operator's arm; 7, acrylic disk; 8, periphery disk clamp; 9, center disk clamp; 10, adaptor base; 11, guide rail to define the path ofan anaerobic jar; 21, opening to the interior of the chamber.

VOL. 4, 1976 IMPROVED ANAEROBIC ISOLATION CHAMBER 43 17-20 L-0. U, / I IA.1 1 1 i um %.1 I 20 Downloaded from http://jcm.asm.org/ FIG. 4. (A) Gloveless sleeve: 1, shown open with the chamber closed by door; 20, which is held tight with clamp 17. (B) Sleeve: 1, is shown sealed closed by the operator's arm with door; 20, closed to allow flushing of gas inside the sleeve. (C) Operator's hand shown holding door upon entering the chamber: 18, clamp bar; 19, 0-ring; 20, acrylic port door; 21, sleeve clamp. on May 14, 2018 by guest well as the chamber were incubated in a walk-in 35 C incubator. Oxidation-reduction potential. Methylene blue indicator strips (BBL) and prereduced brain heart infusion agar plates, with resazurin (Scott Laboratories Inc., Fiskeville, R.I.) were used to monitor the anaerobic condition of the atmosphere of the chamber and of the accessory anaerobic jars. Test recovery of strict anaerobes. All agar plates were poured in room air and placed in an anaerobic jar as soon as they had solidified. The anaerobic jar was flushed three times with 80% N2, 10o H2, 10% C02, and the plates were considered ready to use when the methylene blue strips turned colorless. Various anaerobes which differ in their oxygen sensitivity were used as biological controls to evaluate the usefulness of this chamber under operational conditions. One loop of Clostridium novyi B, Fusobacterium symbiosum, Peptostreptococcus anaerobius, Bacteroides oralis, Eubacterium lentum, Clostridium cadaveris, Clostridium haemolyticum, and Fusobacterium nucleatum grown overnight in PRAS chopped meat glucose broth at 35 C was plated on 5% (vol/vol) sheep blood agar (Brucella agar base, GIBCO Diagnostics, Madison, Wis.; vitamin k,, 10 gg/ml, Difco Laboratories, Detroit, Mich.) (10). Viability of each test organism was determined by streaking the anaerobe onto four sheep blood agar plates. Two plates were incubated in the chamber and two in an anaerobic jar at 35 C. Growth of each

44 COX AND MANGELS organism was evaluated qualitatively after 24 h of incubation. Each day for 5 days the anaerobic jars were attached to the chamber, the contents of the jar were inspected inside the chamber, placed back into the anaerobic jars, and the anaerobic jars were removed. Each of the four agar plates of each organism was restreaked for viability after 5 days. This procedure was carried out to evaluate the possibility of oxygen contamination to the interior of the chamber as well as to the anaerobic jar. RESULTS The results of this study indicate that various obligate anaerobic microorganisms can be processed in this chamber with no appreciable loss of viability. Table 1 shows a qualitative recovery of these organisms incubated inside the chamber and inside anaerobic jars. All cultures were inoculated, transferred, and inspected inside the chamber. This study also indicates that anaerobic conditions can be maintained as monitored with methylene blue strips and plates containing resazurin during all steps of this study. In addition this chamber was sealed for 7 days without additional gas input without loss of anaerobic conditions as indicated by methylene blue strip or resazurin plates. DISCUSSION Several investigations have indicated that the GasPak system is adequate for the isolation of most clinically significant anaerobes. However, the GasPak system does not allow inspection of cultures before 48 h has elapsed (3, 7, 12). This present study has indicated that the inspection of plates can be undertaken at any time without compromising the atmosphere of the culture system. This chamber allows the microbiologist to TABLE 1. Qualitative recovery of anaerobes incubated at 35 C for 24 h Organism Qualitative recovery of anaerobesa Chamber Anaero bic jar Bacteroides oralis 4+ 4+ Clostridium cadaveris 4+ 4+ Clostridium haemolyticum 4+ 4+ Clostridium novyi B 3+ 3+ Eubacterium lentum 4+ 4+ Fusobacterium nucleatum 4+ 4+ Fusobacterium symbiosum 4+ 4+ Peptostreptococcus anaerobius 4+ 4+ a 1+, Poor growth in confluent area, no isolated colonies; 2+, good growth in confluent area, no isolated colonies; 3+, good growth in confluent area, poor growth of isolated colonies; 4+, good growth in confluent area, good growth of isolated colonies. J. CLIN. MICROBIOL. maintain uncompromised anaerobic conditions at all stages of culturing anaerobic microorganisms, while still allowing the use of conventional techniques. Cultures incubated in anaerobic jars can be inspected inside the chamber at any time without compromising the atmosphere, thus allowing identification procedures and sensitivity tests to be initiated on early growers, without disturbing the slower growing organisms. This chamber has the advantage of allowing the microbiologist to work on specimens without wearing gloves as was necessary in earlier anaerobic chambers (1, 2, 4, 5, 8, 9, 11). However, if gloves are desired to prevent physical contact between the hands and an object being manipulated, they can be thin and tight fitting rather than the heavy rubber gloves associated with glove box chambers. The ability to adjust the gas volume inside the chamber has eliminated the problem of displacement resistance. Another advantage of this chamber has been shown to be its size. Less space is required for this chamber, since storage of media and incubation of cultures are done outside the chamber in standard anaerobic jars or jars especially designed for this chamber. The small size of this chamber also allows it to be fully portable and move wherever necessary in or outside the laboratory to process specimens. Recent reports have shown the advantages of a mobile anaerobic laboratory for obtaining good clinical specimens (5, 13). The experiments in this study have shown that an adequate anaerobic condition can be maintained inside the chamber and accessory anaerobic jars. In addition, various strict anaerobes were left exposed to the atmosphere of the chamber and its accessory anaerobic jars for a period of 5 days and successfully transferred with no loss in viability. This chamber allows anaerobic bacteriology to be done in a conventional manner while requiring minimal space and purchase of equipment. With this chamber, anaerobic bacteriology can be satisfactorily done in the smallest clinical laboratory. The microbiologist can use standard procedures to isolate and identify anaerobes; the only significant difference is the presence of a clear plastic shield between the microbiologist and his work. ACKNOWLEDGMENT We thank L. H. Lindberg for critical review of this manuscript. LITERATURE CITED 1. Aranki, A., S. A. Syed, E. B. Kennedy, and R. Freter. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 17:568-576. Downloaded from http://jcm.asm.org/ on May 14, 2018 by guest

VOL. 4, 1976 2. Aranki, A., and R. Freter. 1972. Use of anaerobic glove boxes for the cultivation of strictly anaerobic bacteria. Am. J. Clin. Nutr. 25:1329-1334. 3. Dowell, V. R., Jr. 1972. Comparison of techniques for isolation and identification of anaerobic bacteria. Am. J. Nutr. 25:1335-1343. 4. Drasar, B. S. 1967. Cultivation of anaerobic intestinal bacteria. J. Pathol. Bacteriol. 94:417427. 5. Fulghum, R. S. 1971. Mobile anaerobe laboratory. Appl. Microbiol. 21:769-770. 6. Gordon, J. H., and R. Dubos. 1970. The anaerobic bacterial flora of the mouse cecum. J. Exp. Med. 251-260. 7. Killgore, G. E., S. E. Starr, V. E. Del Bene, D. Niwhalen, and V. R. Dowell. 1973. Comparison of three anaerobic systems for the isolation of anaerobic bacteria from clinical specimens. Am. J. Clin. Pathol. 59:552-559. 8. Leach, P. A., J. J. Bullen, and I. D. Grant. 1971. Anaerobic CO2 cabinet for the cultivation of strict IMPROVED ANAEROBIC ISOLATION CHAMBER 45 anaerobes. Appl. Microbiol. 22:824-827. 9. Lee, A., J. H. Gordon, and R. Dubos. 1968. Enumeration of oxygen sensitive bacteria usually present in the intestine of healthy mice. Nature (London) 220:1137-1139. 10. Loesch, W. 1969. Oxygen sensitivity of various anaerobic bacteria. Appl. Microbiol. 18:723-727. 11. Rosebury, T., and J. B. Reynolds. 1964. Continuous anaerobiosis for the cultivation of spirochetes. Proc. Soc. Exp. Biol. Med. 117:813-815. 12. Rosenblatt, J. E., A. Fallon, and S. M. Finegold. 1973. Comparison of methods for isolation of anaerobic bacteria from clinical specimens. Appl. Microbiol. 25:77-85. 13. Sutter, V. L., H. R. Attebery, J. E. Rosenblatt, K. S. Bricknell, and S. M. Finegold. 1972. Anaerobic bacteriology manual. Departments of Continuing Education in Health Sciences, University Extension and the School of Medicine, UCLA. Downloaded from http://jcm.asm.org/ on May 14, 2018 by guest