Simplified 48-Hour IMVic Test: an Agar Plate Method

APPLIED AND ENVIONMENTAL MICROBIOLOGY, Sept. 1977, p. 274-279 Copyright C 1977.American Society for Microbiology Vol. 34, No. 3 Printed in U.S.A. Simplified 48-Hour IMVic Test: an Agar Plate Method EDMUND M. POWERS* AND THOMAS G. LATT U.S. Army Natick Research and Development Command, Natick, Massachusetts 01760 Received for publication 14 March 1977 An agar plate method was developed for the performance of the IMVic (indole, methyl red, Voges-Proskauer, and citrate) tests in lieu of the conventional tubed liquid media. By modifying the composition of the media and adding agar, a single "X"-compartmented petri dish was prepared containing all four IMVic test media. Ease of performance and simplification of the test were achieved by inoculating all four media simultaneously from a single colony (single inoculum) on eosin-methylene blue agar. Tests with 87 cultures, representing 7 genera in the family Enterobacteriaceae, were completed with typical (correct) IMVic patterns for all cultures within 48 h. Parallel tests with conventional media showed that the agar plate method was superior, more sensitive, faster, and simpler to perform, and less time was required to identify Escherichia coli by 72 h. Confirmation of the presence of Escherichia devise an IMVic agar testing procedure that coli in foods by the IMVic (indole, methyl red, would reduce the time required to identify E. Voges-Proskauer, and citrate) tests is a timeconsuming and laborious procedure as pre- accomplish these goals, we planned to inoculate coli isolated from foods by at least 24 h. To scribed by standard methods. At the present all four IMVic agar media directly from a single time, most authorities (1, 13) recommend 4 or 5 colony instead of delaying the tests by transferring a colony to a nutrient agar slant and wait- days to complete these tests, because, although the indole test is usually completed within 24 h, ing an additional 24 h for the slant to incubate the Voges-Proskauer (VP) test requires 48 h or before inoculation of the IMVic media could more, and the methyl red (MR) and citrate tests proceed. This paper presents the developmental require a minimum of 96 h. Consequently, phase of this work. many laboratories limit testing to coliform and MATERIALS AND METHODS fecal coliform determinations. Despite these obvious shortcomings, the IMVic tests are probably the most important and useful tests for Media. All media were purchased from Difco Laboratories, Detroit, Mich. Indole agar test. Medium for the indole test consisted of 1.5% tryptone (Difco) containing 1% agar. differentiation of coliforms into species and varieties. Furthermore, primary differentiation The medium was dissolved by steaming and sterilized at 121 C for 15 min. Kovacs reagent for the between E. coli and Enterobacter aerogenes is usually made on the basis of IMVic tests (1, 13). detection of indole was modified by substituting isopentyl (isoamyl) alcohol (Eastman Co.) for n-amyl Techniques requiring shorter incubation periods have been proposed for both the VP and alcohol (8) and was stored at 3 C for a maximum of MR tests, but apparently have not found wide 30 days. Since Kovacs reagent should be straw (yellow) colored, one must be cautious when substitut- acceptance. Coblentz (5) in 1943, for example, reduced the time required for the VP test to ing isoamyl alcohol, because the isoamyl alcohol received from three different distributors turned the only 6 h by using a massive inoculum in 2 ml of reagent dark red, which was unsuitable for the test. MR-VP broth. Test reagents he used were a Bacterial growth on the agar surface was flooded combination of two other methods: a-naphthol with 0.3 ml of the reagent. (Barritt, 1936, as cited by Eddy [7]) followed by Methyl red agar test. The standard buffered glucose MR-VP broth (1, 6, 13) used for the MR and VP 0.3% creatine in 40% potassium hydroxide (9). Benjaminson et al. (4) and Barry et al. (2) also tests was modified by substituting sodium chloride successfully reduced the time required for the (NaCl) for dipotassium phosphate (12, 13) and adding 1% agar. The composition of the medium per VP and MR tests, respectively, to less than 18 h by using only 0.5-ml volumes of MR-VP broth. liter was as follows: proteose peptone, 7 g; glucose, 5 g; The NaCl, 5 g; agar, 10 g. The medium was dissolved purpose of this study was twofold: (i) to by steaming and sterilized at 121 C for 15 min. Final explore the possibility that, like the indole and ph was 6.3. For performance of the MR test, 5 drops citrate tests (11), the MR and VP tests could of MR indicator solution (1, 6, 13) were added to also be performed on agar media; and (ii) to bacterial growth on the agar surface. 274

VOL. 34, 1977 VP agar test. The VP test for the production of acetylmethylcarbinol was performed on the same modified (unbuffered) medium used for the MR test. Growth on the agar surface was flooded with 0.6 ml of a-naphthol (5% in absolute ethyl alcohol) followed by 0.2 ml of creatine-koh reagent (5, 13). The creatine-koh reagent was stored at 3 C for a maximum of 21 days. Citrate agar test. Citrate utilization was determined on conventional Simmons citrate agar (6). The medium was dissolved by steaming and sterilized at 121 C for 15 min. No reagents were used for this test. IMVic agar plate method. The IMVic agar plate was prepared by pouring 4 ml of each IMVic agar medium into separate compartments of a "X"-compartmented (100 by 15 mm) petri dish (Falcon #10009). The IMVic agar test was performed by inoculating all four media simultaneously from a single colony as follows. A well-isolated colony was picked from Levine eosin-methylene blue agar with an inoculating needle. A single streak was made on the surface of each agar medium in succession without returning to the colony. Simmons citrate agar was streaked first, followed by inoculation of indole agar and then both quadrants of modified MR-VP agar. This arrangement facilitated inoculation of the four quadrants from one edge of the X plate to the opposite edge in a straight line. The order of inoculation was not important except that Simmons citrate agar was inoculated first to avoid introduction of extraneous sources ofcarbon or nitrogen from the other media. Plates were incubated at 35C. Conventional IMVic test. Conventional procedures and liquid media (1, 13) were used, with the exception of the citrate test, in which a Simmons citrate agar slant was used. Tryptone broth (Difco) was used for the indole test, and MR-VP broth was used for the MR and VP tests. Conventional tests were performed in parallel with the agar tests, and reagents were the same used for agar media. However, each conventional tubed medium was inoculated with a separate inoculum. Incubation was at 350C. RESULTS Table 1 shows the indole reactions of five E. coli strains on agar medium containing 1 and 1.5% tryptone. All cultures were indole positive (red) on both media within 24 h, and reactions remained strong for 72 h. However, after 96 h, SIMPLIFIED 48-h IMVic TEST 275 three cultures were falsely negative (yellow) on 1% tryptone, whereas only one was falsely negative on 1.5% tryptone. On the basis of these results, 1.5% tryptone was selected as the medium of choice, even though the indole test is usually performed within 48 h. Reaction scores, although arbitrary and subjective, were compared with reactions in conventional tryptone broth. Although limited trials in our laboratory did not demonstrate its superiority, the modified Kovacs reagent was selected for the indole test because, in addition to being more stable at room temperature and producing a more intense red color with minimal amounts of indole, the isoamyl reagent was reported to be more sensitive and faster reacting with organisms that were slow in producing indole (8). Tables 2 and 3 show that modified VP medium (12, 13) with added agar can be used for both the MR and VP tests. Buffered MR-VP broth medium, on the other hand, was not suitable for either test when agar was added. In modified MR-VP agar (Table 2), all E. coli cultures were strongly MR+ (red) within 24 h and remained positive for 96 h. Enterobacter and Klebsiella cultures required only 48 h to give the typical MR- response. However, in MR- VP medium with added agar, the MR test was falsely negative (yellow) for all E. coli cultures tested at 24 h, for two E. coli cultures at 48 through 96 h, for six out of nine E. coli cultures at 72 h, and eight out of nine E. coli cultures at 96 h. Enterobacter and Klebsiella cultures gave negative MR reactions on MR-VP medium with added agar at 24 through 96 h. Table 3 shows that all 15 coliform cultures tested gave correct (typical) VP reactions in modified MR-VP agar within 24 h. In the MR- VP medium with added agar, however, the VP response of all E. coli cultures was equivocal (slightly pink) at 24 through 96 h. Two Klebsiella cultures in MR-VP medium with added agar gave weak (pink) VP reactions at 24 h, two out of three were falsely negative (colorless) at 48 h, and all three were falsely negative at 72 TABLz 1. Comparison of1% tryptone with 1.5% tryptone for production of indole by E. coli Indole agar containing: Culture 1% Tryptone for (h): 1.5% Tryptone for (h): 24 48 72 96 24 48 72 96 E. coli (NARADCOM) +++a +++ +++ +++ +++ +++ ++++ +++ E.coli B2C +++ +++ +++ +++ +++ +++ ++++ +++ E. coli FDA 4 +++ +++ +++ - +++ +++ ++++ + E. coli FDA 4 +++ +++ +++ - +++ +++ +++ +++ E. coli Rockefeller +++ +++ +++ - +++ +++ +++ a + + + +, Very strong red reaction; + + +, strong; + +, weak; +, positive, but very weak; -, negative (yellow) reaction.

276 POWERS AND LATT TABLz 2. APPL. ENVRON. MICROBIOL. Comparison of MR test for coliforms in modified MR-VP agar and MR-VP medium with agar MR agar test Culture Modified MR-VP agar (h) MR-VP medium with agar" (h) 24 48 72 96 24 48 72 96 E.coli FDA1 ++++C ++++ ++++ + - - - _ E. coli FDA2 ++++ ++++ ++++ ++++ - +++ +++ +++ E. coli FDA3 ++++ ++++ ++++ ++++ - + - - E. coli FDA 4 ++++ ++++ ++++ ++++ - + ++ - E.coli FDA5 ++++ ++++ +++ +++ - + - - E.coli Rockefeller ++++ ++++ ++++ ++++ - + E.coli ATCC 11840 ++++ ++++ ++++ ++++ - + - - E. coli B2C ++++ ++++ ++++ ++++ - - - - E.coli NARADCOM ++++ ++++ ++++ ++++ - + ++ - E. aerogenes 1 E. aerogenes 2 ++ - - - - - - - Enterobacter 3 +- - - - - - - K. pneumoniae DMV2 +++ - - - - - - - K. pneumoniae DM591 +++ - - - - - - - K. pneumoniae DM602 +++ - - - - - - - Uninoculated medium - - - - - - - a Modified (unbuffered) VP broth plus 1% agar. b Difco MR-VP medium plus 1% agar. c (+) Red; (-) yellow. See footnote to Table 1. TABLE 3. Comparison of VP test for coliforms in modified MR-VP agar and MR-VP medium with agar VP agar test Culture Modified MR-VP agara (h) MR-VP medium with agar" (h) 24 48 72 96 24 48 72 96 E. coli FDA 1 -c - - - +c + E. coli FDA 2 - - - - + + E. coli FDA 3 - - - - + + + + E. coli FDA 4 - - - - + + + + E. coli FDA 5 - - - - + + + E. coli Rockefeller - - - - + + + E. coli 11840 - - - - ++ E. coli B2C - - - - + + + E. coli NARADCOM - - - - + + + E.aerogenes 1 ++++C ++++ ++++ ++++ +++ ++++ ++++ ++++ E.aerogenes 2 ++++ ++++ ++++ ++++ ++++ +++ + +++ Enterobacter 3 ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ K. pneumoniae DMV2 ++++ ++++ ++++ + +++ K. pneumoniae DM591 ++++ ++++ ++ ++ + + K. pneumoniae DM602 ++++ ++++ +++ ++ + Uninoculated medium NR NR NR NR NR NR NR NR a Modified (unbuffered) VP broth plus 1% agar. b Difco MR-VP medium plus 1% agar. c (-) No reaction; (+) red; (+) slightly pink and doubtful after 4 h. See footnote to Table 1. and 96 h. This was not unexpected, since it was known that some coliforms were capable of decomposing acetoin to give weak or negative reactions (7, 14). Apparently this does not happen in modified (unbuffered) MR-VP agar. E. aerogenes gave correct (positive or red) VP reactions in both agar media at 24 through 96 h. Table 4 shows the IMVic reactions of87 members of the Enterobacteriaceae on the IMVic agar plate tested in parallel with conventional liquid media (citrate test was on Simmons citrate agar slants). Anticipated and correct IM- Vic reactions were obtained within 24 h on the IMVic agar plate with all cultures except Enterobacter and Klebsiella cultures, which required 48 h to give a correct (negative or yellow)

VOL. 34, 1977 SIMPLIFIED 48-h IMVic TEST 277 TABLz 4. IMVic reactions of Enterobacteriaceae on IMVic agar plate and in conventional tubed medium No. of strains giving typical reactions on: Culture strains Typical (correct) IVcaa IMVic agar plate lt (h) h Conventionala method Culture ~ tstrang IMVic reactionsi 24 48 24 48 E. coli type 1 24 + + - - 24 24 22b 24 E. coli type 2 4 - + - - 4 4 4 4 Enterobacter species 12 - - + + 3c 12 4c 8c,d E. agglomerans (14/73) 1 - + - + 1 1 1 1 K. pneumoniae 9 - - + + 1 9 3c 6c.d K. pneumoniae 4 + - + + 2c 4 3c 3d K. ozaenae 2 - + - - 2 2 2 2 K. rhinoscleromatis 2 - + - - 2 2 2 2 C. freundii 3 - + - + 3 3 2b 3 C. diversus 1 + + - + 0 1 0 1 Salmonella species 23 - + - + 23 23 21e 2le Proteus species 2 + + - - 2 2 lb lb a All media were liquid except Simmons citrate agar slant. b Remainder of cultures gave false-negative MR tests in MR-VP broth. c Remainder of cultures gave false-positive MR tests. d One culture gave a false-negative citrate test in 48 h but was positive after 96 h. e Two cultures gave equivocal MR reactions (orange). (h) MR response, and C. diversus, which required 48 h to give a correct (positive or blue) citrate response. Species or strains that differed from other members of a genus were listed separately. For example, four E. coli cultures (type II) were indole negative. One strain of E. agglomerans (formerly Pectobacterium and currently Erwinia herbicola in Bergey's Manual, 8th ed., 1974) was an MR+ and VP- strain. Four Klebsiella pneumoniae were indole+ (types I and II), and K. ozaenae and K. rhinoscleromatis had IMVic patterns identical to those of E. coli type II (- + - - ). Citrobacter diversus differs from other citrobacter species by being indole+ (Encise II, Roche Diagnostics, Nutley, N. J., 1974). Parallel tests in conventional tubed media confirmed the IMVic reactions of all 87 cultures on the IMVic agar plate. However, confirmation sometimes required 96 h because of delayed MR and citrate reactions on conventional media. This was not unexpected since 96 h is usually recommended for the MR and citrate tests (1, 13). Table 4 shows, for example, that in conventional tubed media only 8 of 12 Enterobacter and 9 of 13 K. pneumoniae cultures gave typical (correct) IMVic patterns after 48 h because of false-positive (red) MR reactions by three Enterobacter and two Klebsiella cultures and false-negative (green) citrate reactions by one Enterobacter and two K. pneumoniae cultures. The MR reactions of these cultures remained falsely positive even after 96 h in MR- VP broth. Of the Salmonella cultures tested, only 21 out of 23 gave correct IMVic patterns in conventional media because of equivocal MR reactions of two cultures. One Proteus culture required 72 h to give a correct (positive or red) MR reaction in MR-VP broth. On the IMVic agar plate (not shown), indole reactions of cultures examined were instantaneous. A positive reaction was indicated by a dark red color that originated from the growth streak and spread rapidly across the agar surface. Although the color was readily apparent on the agar surface, it appeared more intense when viewed through the side of the dish. In negative reactions, the original yellow color of the Kovacs reagent remained unchanged. Positive MR reactions, showing the presence of acid, were also instantaneous and were indicated by a distinct red color, which originated along the line of growth. Negative (yellow) MR reactions developed in 1 to 5 min after adding the reagent. A positive VP reaction was indicated by development of a pink or red color throughout the entire agar medium within 1 to 5 min that intensified to a dark red color within 1 h. A few Klebsiella and Enterobacter cultures did not

278 POWERS AND LArT produce coloration for 20 or 30 min, but these cultures also had delayed reactions in MR-VP broth medium. A positive citrate reaction in the IMVic agar plate was clearly indicated by growth along the streak accompanied by a color change of the medium from its initial green to deep blue, indicating production of alkali. The color change was usually apparent within 24 h and spread over the entire agar surface with prolonged incubation, but some cultures required 48 h for this reaction. Citrate-negative cultures either did not grow or left the medium unchanged. DISCUSSION The extremely long time (8 or 9 days) required to identify E. coli isolated from food is not conducive to routine testing for the organism. As a result, many busy laboratories limit their testing to coliform and fecal coliform determinations. The most time-consuming procedures are the IMVic tests, which require 4 or 5 days by conventional methods (1, 13). The method presented in this paper can reduce the time required to identify E. coli by 72 h (Fig. 1). A savings of 24 h can be accomplished by transferring a colony from eosinmethylene blue agar directly to IMVic agar media in the IMVic plate. An additional 48 h can be saved by completing all reactions within 48 h instead of the 96 h required by conventional methods. This savings in time allows one to complete the testing procedure in 120 h or less, which is not only more practical but less costly. Other advantages of the IMVic agar plates are: (i) time to perform the IMVic tests is reduced by eliminating the handling, labeling, inoculation, and incubation of four separate test tubes; (ii) all media are in a single petri PL METHOD Food Suspension Elapsed T,me(h) I LST Broth 24 124 h EC Broth (45.5C) 48 124h EMB agar 72 124h IMViC agar plate I48h + or- + 120 h (Completed) CONVENTIONAL METHOD Food Suspension Elapsed Time(h) I LST Broth 24 24 h EC Broth (45.5C) 48 1 24 h EMB ogar 72 1 24h Nutrient agar 96 1 24 h broth media IMViC 96 h ++ --or +-- 192 h Completed FIG. 1. Scheme for isolation and identification of E. coli from foods. APPL. ENVIltON. MICROBIOL. dish and can be inoculated with a single inoculum from a single colony; (iii) addition of all reagents to a single plate and reading of the reactions is more convenient; and (iv) less space is occupied in the incubator. More rapid MR and citrate reactions in the IMVic agar plate were attributed to greater surface area of the medium 'exposed to atmospheric oxygen and, consequently, greater aeration of surface growth, which enhanced both reactions. Enhancement of VP reactions by aeration (shaking) is well known (3, 5, 8), and, indeed, some broth cultures only became positive by vigorously shaking the tube after the reagents were added. Reagents used in the VP test are actually oxidizing agents that promote the oxidation of acetylmethylcarbinol, in the presence of alkali and air, to diacetyl. Diacetyl reacts with creatine to form the pink or red compound (10). This was a very strong reaction on the surface of modified MR-VP agar. The IMVic agar plate will simplify the IMVic test considerably and hopefully will encourage increased use of these tests. This paper merely presents the developmental phase of the new method. Testing of the IMVic agar plate with hundreds of cultures, isolated from foods and clinical specimens as well as with laboratory cultures, is planned. We hope to obtain the cooperation and collaboration of other laboratories to accomplish this. LITERATURE CITED 1. Association of Official Analytical Chemists. 1975. Official methods of analysis, 12th ed. Association of Official Analytical Chemists, Washington, D.C. 2. Barry, A. L., K. L. Bernsohn, A. P. Adams, and L. D. Thrupp. 1970. Improved 18-hour methyl red test. Appl. Microbiol. 20:866-870. 3. Batty-Smith, C. G. 1941. The detection of acetyl-methyl-carbinol in bacterial cultures. J. Hyg. 41:521-529. 4. Benjaminson, M. A., B. C. de Guzman, and A. J. Weil. 1964. Voges-Proskauer test: expeditious techniques for routine use. J. Bacteriol. 87:234-235. 5. Coblentz, L. M. 1943. Rapid detection of the production of acetyl-methyl-carbinol. Am. J. Public Health 33:815-817. 6. Difco Laboratories. 1953. Difco manual, 9th ed. Difco Laboratories, Inc., Detroit, Mich. 7. Eddy, B. P. 1961. The Voges-Proskauer reaction and its significance: a review. J. Appl. Bacteriol. 24:27-41. 8. Gadebusch, H. H., and S. Gabriel. 1956. Modified stable Kovac's reagent for the detection of indol. Am. J. Clin. Pathol. 26:1373-1375. 9. Levine, M., S. S. Epstein, and R. H. Vaughn. 1934. Differential reactions in the colon group of bacteria. Am. J. Public Health 24:505-510. 10. Park, G., and M. T. Suggs. 1974. Reagents, stains, and miscellaneous test procedures, p. 937. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 11. Roche Diagnostics. 1974. Improved enterotube. Roche Diagnostics, Division Hoffman-La Roche Inc., Nutley, N.J.

VOL. 34, 1977 12. Smith, N. R., R. E. Gordon, and F. E. Clark. 1952. Aerobic sporeforming bacteria. Agriculture Monograph no. 16. Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 13. Speck, M. L. 1976. Compendium of methods for the SIMPLIFIED 48-h IMVic TEST 279 microbiological examination of foods. American Public Health Association, Washington, D.C. 14. Vaughn, R. H., G. T. Wedding, and J. Tabachnick. 1948. The Voges-Proekauer reaction and differentiation of the coliform bacteria. Hilgardia 18:367-367.