Anaerobic Bacteria. adequate growth of nonsporeforming as well as. medium. the lyophilized or frozen collections of the CDC Anaerobe

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1982, p /82/ $02.00/0 Vol. 16, No. 2 Gelatin Agar Medium for Detecting Gelatinase Production by Anaerobic Bacteria D. N. WHALEY, V. R. DOWELL, JR.,* L. M. WANDERLINDER,t AND G. L. LOMBARD Hospital Infections Program, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia Received 20 January 1982/Accepted 30 April 1982 A new medium, Lombard-Dowell gelatin agar, was developed for detecting gelatinase activity by anaerobic bacteria. The medium contained: Trypticase (BBL Microbiology Systems), 5.0 g; yeast extract (Difco Laboratories), 5 g; sodium chloride, 2.5 g; sodium sulfite, 0.1 g; L-tryptophan, 0.2 g; L-cystine, 0.4 g; hemin, 10.0 mg; vitamin Kl, 10.0 mg; agar, 20.0 g; D-glucose, 1.0 g; gelatin, 4.0 g; and distilled water to 1 liter. The ph was adjusted to 7.5. The medium was dispensed in 100- by 15-mm quadrant plastic dishes (5 ml per quadrant). To test for gelatinase activity, we inoculated the medium with a young enriched thioglycolate or chopped meat glucose broth culture or a turbid cell suspension in Lombard- Dowell broth, using a sterile cotton swab, and incubated it under anaerobic conditions for 48 h at 35 C. The quadrants were then flooded with Frazier solution, and clear zones around the bacterial growth were recorded as positive for gelatinase activity. The new medium was tested with a variety of anaerobic bacteria, and the results were compared with data obtained with the conventional technique for detecting gelatinase activity. Overall, there was satisfactory agreement between the two tests in the detection of gelatinase activity, but the Lombard-Dowell gelatin agar tests was more rapid and somewhat more sensitive than the conventional test. The conventional technique for detecting gelatinase activity of anaerobes used by the Anaerobe Reference Laboratory at the Centers for Disease Control (CDC) utilizes Thiogel (BBL Microbiology Systems) medium in screwcapped tubes which has been conditioned in an anaerobic atmosphere after autoclaving (8). After the tubes of Thiogel are inoculated with active cultures, they are incubated in an anaerobic atmosphere for as long as 2 weeks at 35 C, depending on the rapidity of the bacterial growth. Routinely, tests for gelatin liquefaction are made after 48 h, 72 h, and 5 to 7 days of incubation by refrigerating the tubes and comparing with an uninoculated control tube of medium (6). If the gelatin is liquefied as the result of gelatinase activity, the medium no longer solidifies when refrigerated. In this report, we describe the development and evaluation of a technique for detecting gelatinase activity which is based on the use of a gelatin agar medium in plates as described by Frazier in 1926 (9). The new medium (Lombard-Dowell [LD] gelatin agar) consists of LD agar (7, 8) supplemented with 0.1% glucose and 0.4% gelatin. This medium is convenient to use, supports t Present address: University of Zulia, Maracaibo, Venezuela. adequate growth of nonsporeforming as well as sporeforming anaerobes, and allows detection of gelatinase activity more rapidly than Thiogel medium. MATERIALS AND METHODS Organisms. Initially, 291 strains of anaerobes (26 species of Clostridium and 12 species of nonsporeforming gram-positive bacilli and cocci) were used in developing and evaluating the medium. All were from the lyophilized or frozen collections of the CDC Anaerobe Reference Laboratory and had been identified with techniques described previously (6). Before use in the study, the strains were rechecked for relation to oxygen, purity, colony characteristics on anaerobe blood agar, microscopic features, and biochemical characteristics. A single isolated colony of each was subcultured to a tube of enriched thioglycolate (THIO) medium and incubated anaerobically to provide inoculum for other media. Working cultures of the microorganisms were maintained in chopped meat glucose medium at ambient temperature after incubation anaerobically in an anaerobic glove box containing 5% C02, 10%o H2, and 85% N2 at 35 C for 18 to 48 h. Some of the strains were lyophilized or frozen for long-term storage as described previously (6). In addition, after the LD gelatin agar medium was evaluated, we used it to test strains of various anaerobes received for identification by the Anaerobe Reference Laboratory. Media. All media used in the study except the gelatin agar have been described previously (8) and 224

2 VOL. 16, 1982 MEDIUM FOR DETECTING GELATINASE 225 were purchased from the Nolan Biological Laboratories, Tucker, Ga. Formulation of LD gelatin agar medium. The formulation for gelatin agar was developed by supplementing LD agar (7) with glucose (1.0 g/liter) and various concentrations of gelatin. Media containing final concentrations of 0.3 to 0.5% gelatin were tested with a battery of microorganisms to determine the optimal concentration of gelatin. On the basis of these results, the following formulation was selected: Trypticase (BBL Microbiology Systems) 5.0 g; yeast extract (Difco Laboratories), 5.0 g; sodium chloride, 2.5 g; sodium sulfite, 0.1 g; L-tryptophan, 0.2 g; L-cystine, 0.4 g; hemin, 10.0 mg; vitamin Kl (3-phytylmenadione), 10.0 mg; D-glucose, 1.0 g; gelatin (BBL Microbiology Systems), 4.0 g; agar, 20.0 g; distilled water, 1,000.0 ml. The hemin and L-cystine were dissolved in 5 ml of 1 N sodium hydroxide and mixed with the other ingredients after they were dissolved. The vitamin Kl was added from a stock solution prepared by dissolving 1 g of vitamin Kl (ICN) in 99 ml of absolute ethanol. Preparation of LD gelatin agar. All of the ingredients except the gelatin and agar were dissolved in 750 ml of water. With a separate flask, 4.0 g of gelatin was added to 10 ml of water, and the mixture was heated gently until the gelatin was dissolved. The dissolved gelatin was then added to the other ingredients. The flask was then heated, and 20 g of agar was added with continued stirring to prevent the agar from burning. After all ingredients were dissolved, the remaining water was added, and the ph was adjusted to 7.5. Next, the medium was autoclaved for 15 min at 121 C. The medium was then cooled to 50 C and dispensed in 100- by 15-mm quadrant plastic plates (5 ml per quadrant). After the medium cooled and solidified, the plates were stored in sealed plastic bags at 4 C. Before use, the quadrant plates were held in an anaerobic glove box for 4 to 18 h to expose the medium to anaerobic conditions. Procedure for inoculating LD gelatin agar and determining gelatinase activity of cultures. The LD gelatin agar was inoculated with young (12 to 24 h at 35 C) THIO cultures, using sterile cotton swabs. A swab was dipped in a THIO culture, and excess fluid was removed by pressing gently against the wall of the tube. The medium was then inoculated with a single streak across the middle of the quadrant beginning near the middle of the plate and streaking toward the periphery. Within 15 min after inoculation, the plates were placed in an anaerobic glove box and then incubated at 35 C for 24 or 48 h. After incubation, the plates were removed from the glove box, and the surface of the medium in each quadrant was flooded with 0.5 to 1.0 ml of Frazier solution (mercuric chloride, 15.0 g; concentrated hydrochloric acid, 20.0 ml; distilled water, ml) (9). After addition of the solution, gelatin was precipitated, and the medium became cloudy. When the gelatin was hydrolyzed as the result of gelatinase activity, there was a clear zone around the bacterial growth (Fig. 1). This is best demonstrated by viewing the plates with transmitted light over a black surface. Comparison of LD gelatin Agar with Thiogel medium. The LD gelatin agar was tested in parallel with Thiogel in tubes (8), using a variety of anaerobes. After the purity and identity of the cultures were FIl. 1. Gelatinase reactions of anaerobe cultures on LD gelatin agar. Quadrant c, uninoculated medium (after addition of Frazier reagent); quadrant a, negative reaction; quadrant b, moderate positive reaction; quadrant d, strong positive reaction. rechecked, each strain was subcultured in THIO broth and incubated for 12 h anaerobically in a glove box. Two quadrants of LD gelatin agar (separate quadrant plates) and three tubes of Thiogel medium were inoculated with each strain. The LD gelatin agar was inoculated with a swab as described above, and the Thiogel was inoculated as described by Dowell and Hawkins (6) with a capillary pipette. All of the inoculated media (quadrant plates and tubes) were incubated anaerobically in a glove box at 35 C. The plates were incubated for 24 or 48 h, and the tubes were incubated for up to 7 days. One gelatin agar plate was read after 24 h, and the other was read after 48 h of incubation. The Thiogel tubes were removed from the glove box at 24 h (one tube), 48 h (one tube), and 7 days (one tube) and checked for liquefaction by immersing the tubes in a cold (4 C) water bath for 2 h and comparing them with an uninoculated tube of Thiogel (6). Gelatinase activity was indicated by failure of the Thiogel culture to solidify after cooling at 4 C. RESULTS Optimal concentration of gelatin. A gelatin concentration of 0.4% was found to be most effective. Gelatin concentrations below 0.4% gave reactions which were difficult to interpret because of lack of contrast between positive and negative zones after Frazier solution was added. A gelatin concentration of 0.5% gave a cup effect with positive strains; i.e., the gelatin was cleared immediately around and under the growth of the bacteria, but deeper in the medium it was still cloudy. Comparison of LD gelatin agar with Thiogel medium. Gelatinase reactions exhibited by 68 strains of various obligately anaerobic and facultatively anaerobic bacteria in LD gelatin agar and Thiogel medium are presented in Table 1.

3 226 WHALEY ET AL. J. CLIN. MICROBIOL. TABLE 1. Gelatinase reactions exhibited by 68 strains of various obligately anaerobic and facultatively anaerobic bacteria in LD gelatin agar and Thiogel medium No. of positive reactions Species No. of strains LD h gelatin 48_h_24_h_48_h_ Thiogel 7_days 24 h 48 h 24 h 48 h 7 days Clostridium absonum Clostridium beijerinckii Clostridium bifermentans Clostridium botulinum type A Clostridium botulinum type B (proteolytic) Clostridium butyricum Clostridium cadaveris Clostridium capitovale Clostridium celatum Clostridium chauvoei Clostridium clostridiiforme Clostridium difficile Clostridium durum Clostridium fallax Clostridium hastiforme Clostridium innocuum Clostridium limosum Clostridium paraperfringens Clostridium perfringens Clostridium septicum Clostridium sordellii Clostridium sphenoides Clostridium sporogenes Clostridium subterminale Clostridium symbiosum Clostridium tertium Clostridium tetani Bifidobacterium eriksonii Eubacterium limosum Propionibacterium acnes Propionibacterium granulosum Peptococcus asaccharolyticus Peptococcus magnus Peptococcus prevotii Peptococcus saccharolyticus Peptostreptococcus anaerobius Streptococcus intermedius Streptococcus durans a Weak positive reactions are shown within parentheses (1) ~ (1) (1) (1) The largest number of positive reactions exhibited by these cultures was 37 (54%) in LD gelatin agar after 48 h of incubation. Only 27 positive reactions (40%) were obtained with the cultures in Thiogel medium after 48 h of incubation. The overall agreement between the LD gelatin agar and Thiogel medium on the basis of negative as well as positive reactions was 88.2% after 24 h and 80.9% after 48 h of incubation. When the reactions obtained with the LD gelatin agar at 48 h were compared with those with Thiogel at 7 days, the agreement was somewhat higher, 61 of 68 strains (89.7%). The majority of the reactions in the two media which did not agree were due to weak gelatinase producers which gave delayed positive reactions (Table 1). More of the weak gelatinase producers were detected in the LD gelatin agar at 48 h than in Thiogel medium at 48 h or 7 days of incubation (Table 1). To check the repeatability of the gelatinase reactions, we tested the same 68 strains of bacteria with LD gelatin agar and Thiogel medium, prepared on different days, as described above. Overall, the results obtained in the second trial agreed closely with those obtained in the first experiment. There were 10 strains which showed different reactions in the two trials. Some Clostridium strains (C. cadaveris, C. capitovale, C. chauvoei, and C. symbiosum) gave positive reactions in the LD gelatin agar at 24 and 48 h of incubation in the second trial, but the reactions with the organisms remained the

4 VOL. 16, 1982 MEDIUM FOR DETECTING GELATINASE 227 same in Thiogel. Two of the C. tetani cultures gave different reactions in the second trial. One strain was positive only in the LD gelatin (24 h and 48 h), and another strain was positive in LD gelatin (24 h and 48 h) and Thiogel (24 h, 48 h, and 7 days). Propionibacterium granulosum was positive in LD gelatin after 48 h (second trial) but remained negative in Thiogel. On the other hand, the Propionibacterium acnes strain was negative in LD gelatin as well as Thiogel in the second trial. Two of the anaerobic cocci (Peptococcus prevotii and Peptococcus anaerobius) gave unexpected positive reactions in the second trial. The P. prevotii strain was weakly positive only in Thiogel after 7 days of incubation, and one strain of P. anaerobius showed positive reactions in LD gelatin after 48 h and in Thiogel after 7 days of incubation. The overall gelatinase reactions obtained with the 68 strains in two trials are summarized in Table 2. The LD gelatin agar gave a higher percentage of the total positive reactions after 24 h of incubation (98.6%) than the Thiogel medium after 48 h (75.0%). In another experiment, we tested larger numbers of Clostridium difficile, Clostridium perfringens, and P. acnes strains. These data are summarized in Table 3. The LD gelatin agar consistently gave more positive reactions than the Thiogel medium in tubes. The results obtained with the C. difficile and P. acnes strains, which usually give weak or negative reactions in conventional media (6, 10), were particularly impressive. All of the 34 C. difficile strains and 14 of the 16 P. acnes strains were positive on LD gelatin agar after 48 h of incubation. DISCUSSION In this paper, we have described the development and evaluation of a new medium, LD gelatin agar, for detecting gelatinase activity of anaerobic bacteria. The LD agar was chosen as the basal medium because it supports adequate growth of a variety of nonsporeforming as well as sporeforming anaerobes and has been used in formulating various other differential agar media for characterization and identification of anaerobes (7, 8). Use of 0.4% gelatin in LD gelatin agar is in agreement with Frazier's recommendation for a gelatin agar (9). Although the Frazier technique for detecting gelatinase is well known and has been used by numerous investigators to test aerobic and facultatively anaerobic bacteria (1, 5), it has seldom been used to test obligately anaerobic bacteria. A modification of the Frazier technique was used by Jackson et al. (11) in their study of anaerobic "corroding bacilli," but the reactions were read after 5 days of incubation only and compared with the Virginia Polytechnic Institute gelatin stab method (10) read after 5 days and 2 weeks of incubation. One of the obvious disadvantages (although only a minor one) of the Frazier technique is the necessity for adding acidified mercuric chloride solution to the agar after growth has occurred to visualize gelatinase activity. To avoid contamination of other materials and equipment with the caustic solution, the quadrant plates are placed in a plastic bag and autoclaved before they are discarded. We are presently investigating the possibility of using another solution instead of Frazier reagent for demonstrating zones of gelatinase activity. Tannic acid (9), trichloroacetic acid (5), and saturated ammonium sulfate (3) have been tried by others. Although we have not systematically studied the stability and shelf life of the LD gelatin agar, we have had considerable experience with it during the last several months (Lombard, Whaley, and Dowell, Jr., unpublished data). On the basis of this experience, we have concluded that the shelf life of the medium is at least two months if the quadrant plates are stored in sealed plastic bags at 4 C and used as described in this report. The Frazier reagent is stable indefinitely at ambient temperature if kept in a tightly sealed bottle. It should be mentioned that the gelling capacity of gelatin may vary from lot to lot, and overheating a gelatin medium can affect its gelling capacity as pointed out by Cowan (5). For this reason, it is imperative to carefully test the performance of LD gelatin agar-each batch that is prepared-with appropriate strains of bacteria. Strains which are known to give nega- TABLE 2. Composite gelatinase reactions obtained with 68 strains of obligately anaerobic and facultatively anaerobic bacteria in LD gelatin agar and Thiogel medium Total no. positive in LD No. of positive reactionsa gelatin agar or Thiogel % positive reactions Incubation mdu medium period (h) LD gelatin Thiogel in LD gelatin Thiogel in LD gelatin Thiogel in agar tubes agar tubes agar tubes a Average of two independent trials.

5 228 WHALEY ET AL. J. CLIN. MICROBIOL. TABLE 3. Gelatinase reactions exhibited by selected anaerobes in LD gelatin agar and Thiogel medium No. of No. of positive reactions Species strains LD gelatin Thiogel tested 24 h 48 h 24 h 48 h 7 days Clostridium difficile Clostridium perfringens Propionibacterium acnes tive, weak positive, and strong positive gelatinase reactions should be used (8). The use of LD gelatin agar in plastic quadrant plates as described in this report is a convenient method to test anaerobes for gelatinase activity. The method requires a maximum of 48 h to obtain results if a suitable pure culture isolate is available. Since the gelatin is contained in an agar matrix, the medium does not liquefy at 35 to 37 C. This is a distinct advantage since tubed gelatin media incubated at these temperatures require cooling (e.g., in an ice bath) at intervals during the incubation period to determine gelatin hydrolysis. Some bacteria may require more than 1 week of incubation to liquefy gelatin media in tubes (4-6, 10, 12-15). A differential test with such a long incubation period is not practical for use in a clinical microbiology laboratory. The data obtained in our study showed that LD gelatin agar was consistently better in detecting gelatinase activity by anaerobes than Thiogel medium. The strongly proteolytic Clostridium strains (e.g., C. bifermentans, C. botulinum, C. limosum, C. sordellii, and C. sporogenes) and nonproteolytic Clostridium strains (e.g., C. butyricum, C. clostridiiforme, C. fallax, C. sphenoides, and C. tertium) tested gave identical reactions in the two media after 48 h of incubation. However, the sensitivity of LD gelatin agar in detecting the gelatinase activity of weakly proteolytic strains (e.g., C. cadaveris, C. difficile, and P. acnes) was greater than that of Thiogel medium (Tables 1 and 3). The gelatin content of media commonly used for detecting gelatinase production by bacteria is quite variable. The concentrations of gelatin in Thiogel (4.85%) and prereduced anaerobically sterilized gelatin (12%) (10) are approximately 12 and 30 times, respectively, that of LD gelatin agar. It is not surprising that it takes longer to detect gelatinase activity in the tubed media with higher gelatin concentrations than in LD gelatin agar plates. In a recent CDC publication (7a), we reported gelatinase reactions of more than 1,100 strains of various sporeforming and nonsporeforming anaerobes. When the composite reactions obtained with LD gelatin agar at 48 h were compared with composite gelatinase reactions for the same species published by others (2, 6, 10), it was found that overall there was good agreement. The main discrepancies were in the reactions of the nonsporeforming gram-negative rods. There was good agreement between the reactions in LD gelatin agar and Thiogel as used by CDC, but a number of the species showed different reactions from those in other manuals (2, 10). These variations could have been due to differences in gelatin concentration, incubation period, method for interpreting gelatinase activity, and other factors. For instance, the technique used by the Virginia Polytechnic Institute laboratory (10) for judging liquefaction of gelatin in tubed media is different from that used by CDC (6). In summary, a new gelatin agar medium for use in detecting gelatinase activity of anaerobic bacteria was developed. When used in quadrant plates, the medium gave excellent results with a variety of obligately anaerobic and facultatively anaerobic bacteria. Since the use of the LD gelatin agar is convenient and requires only 48 h of incubation for detecting gelatinase activity, the method should be useful as an aid for identification and anaerobe isolates in clinical microbiology laboratories. LITERATURE CITED 1. Blazevic, D. J., and G. M. Ederer Principles of biochemical tests in diagnostic microbiology, p John Wiley and Sons, New York. 2. Buchanan, R. E., and N. E. Gibbons (ed.) Bergey's manual of determinative bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore, Md. 3. Burnett, G. W., M. J. Pelczar, Jr., and H. J. Conn Preparation of media, p In H. J. Conn (ed.), Manual of microbiological methods. McGraw-Hill, New York. 4. Clark, P. H. and S. T. Cowan Biochemical methods for bacteriology. J. Gen. Microbiol. 6: Cowan, S. T Cowan and Steel's manual for the identification of medical bacteria, 2nd ed. Cambridge University Press, London. 6. Dowell, V. R., Jr., and T. M. Hawkins Laboratory methods in anaerobic bacteriology. CDC laboratory manual. Center for Disease Control, Atlanta, Ga. 7. Dowell, V. R., Jr., and G. L. Lombard Presumptive identification of anaerobic, nonsporeforming, gram-negative bacilli. Center for Disease Control, Atlanta, Ga. 7a.Dowell, V. R., Jr., and G. L. Lombard Reactions of anaerobic bacteria in differential agar media. Centers for Disease Control, Atlanta, Ga. 8. Dowell, V. R., Jr., G. L. Lombard, F. S. Thompson, and A. Y. Armfield Media for isolation, characteriza-

6 VOL. 16, 1982 MEDIUM FOR DETECTING GELATINASE 229 tion, and identification of obligately anaerobic bacteria. Center for Disease Control, Atlanta, Ga. 9. Frazier, W. C A method for the detection of changes in gelatin due to bacteria. J. Infect. Dis. 39: Holdeman, L. V., E. P. Cato, and W. E. C. Moore Anaerobe laboratory manual, 4th ed. Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va. 11. Jackson, F. L., Y. E. Goodman, F. R. Bel, P. C. Wong, and R. L. S. Whitehouse Taxonomic status offacultative and strictly anaerobic "corroding bacilli" that have been classified as Bacteroides corrodens. J. Med. Microbiol. 4: Kohn, J A preliminary report of a new gelatin liquefaction method. J. Clin. Pathol. 6: Koneman, E. W., S. D. Allen, V. R. Dowell, Jr.. and H. M. Sommers Color atlas and textbook of diagnostic microbiology, p J. B. Lippincott Co., Philadelphia. 14. Lautrop, H A modified Kohn's test for the demonstration of bacterial gelatin liquefaction. Acta Pathol. Microbiol. Scand. 39: Le Minor, L., and M. Piechaud Note techniques une methode rapide de recherche de la proteolyse de le gelatine. Ann. Inst. Pasteur. 105: