MINIATURIZED TECHNIQUES for Imvic TESTS1 DANIEL Y

328 ]. Milk Food Technol., Vol. 35, No. 6 (1972) MINIATURIZED TECHNIQUES FOR IMViC TESTS1 DANIEL Y. c. FuNG AND RICHARD D. MILLER Department of Microbiology The Pennsylvania State University University Park 16802 ( Received for publication January 24, 1972) ABsTRACT Indole test Sterile tryptone broth ( Difco) was introduced into the A miniaturized method for performing IMViC tests is pro wells ( 0.2 ml per well) of a series of Microtiter plates; the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/35/6/328/2399137/0022-2747-35_6_328.pdf by guest on 27 September 2021 posed. Twenty-four gram-negative bacterial species and plates were then inoculated, covered, and incubated at 37 C. strains were tested by use of miniaturized and conventional At intervals of 8, 12, and 24 hr, Microtiter plates were re methods; the results were comparable. The miniaturized moved from the incubator for testing. To detect indole pro method effects savingG of time, space, materials, and effort. duction, 2 drops of Kovac reagent were transferred by a The advantages and applications of microbiological Pasteur pipette to each well of the Microtiter plate. A red layer formed on top of the broth in the well indicated a posi~ tests using small volumes of media have been dis tive reaction. Parallel conventional tests for indole produc cussed in detail by Hartman (6). The combination tion, as well as other IMViC tests, were performed according of small volumes of media in Microtiter plates and to the Difm Manual (3), in each species and strain in four multiple inoculation techniques has been used by replicates. Fung and Miller ( 4) for rapid carbohydrate fermen Methyl red-Voges-Proskauer tests tation tests. Davies et al. (2) have also proposed a Sterile MR-VP medium ( Difco), in 0.2 ml aliquots, was Microliter method for carbohydrate fermentation. placed in the wells of a series of Microtiter plates. Inocula tion and incubation were performed as in the indole test. IMViC (indole, methyl red, Voges-Proskauer, ci At 24-bx intervals ( up to 5 days for the MR test) Microtiter trate) tests have been miniaturized (1, 6), hut a com culture plates were removed from the incubator to perform bined multiple inoculation Microtiter test system has the MR test on one set of plates and the VP test on another not been proposed. This paper describes the use of set. The multipoint inoculation device was used as the both miniaturized techniques and multiple inocula "instrument" for simultaneously transferring methyl red to all 96 micro-cultures. A large petri dish ( 150 X 15 rom) tion procedures in performing IMViC tests. was filled with methyl red; 4 transfers with the multipoint inoculation device carried about 0,003 ml of methyl red to MATERIALS AND METHODS the wells containing the cultures. This amount of methyl red was optimum for observation for mlor change. Pink Bacterial cultures or red color development in the wells indicated a positive Twenty-four gram-negative bacterial species and strains reaction. For the VP test, Microtiter loops ( 0.05 ml) were (Table 1), representing a variety of metabolic patterns, were used to transfer a-napthol and KOH to the cultures to detect tested. The inocula were grown in trypticase soy broth ( Dif the presence of acetyl-methyl-carbinol (pink or red color co) for 24 bx at 37 C. reactions); 1 loop of a-napthol followed by 1 loop of KOH Vessels and multipoint inoculator gave the best results. Twelve loops were operated simul Sterile, individually wrapped, flat or round bottomed Mic taneously to increase efficiency. rotiter plates and plastic covers and Microtiter loops ( 0.05 ml) were obtained from Cooke Engineering Co., Alexandria, Va. Citrate test Koser citrate ( Difco) was used for citrate utilization test. The multipoint inoculator was constructed by using pins Addition of medium to the Microtiter plates, mass inoculation, fixed on the bottom of the wells of a Microtiter plate, as pre and incubation were the same as for the indole test. After 8, viously reported ( 4), or nailed on a wood block patterned to 12, and 24 bx incubation, growth of cultures in the Microtiter fit into the wells of a Microtiter plate. Sterilization of the multipoint inoculator was by almhol flaming. wells was observed. Mass inoculation procedure REsULTS AND DISCUSSION A master plate was prepared by placing four drops of a bacterial culture into each of the 96 wells of a Microtiter Results of the IMViC tests on 24 bacterial speeies plate. Twenty-four cultures were tested in quadruplicate and strains, comparing the miniaturized methods on each plate. These cultures were transferred from the master with the conventional methods, are recorded in Table plate to other Microtiter plate containing liquid test media with the sterilized multipoint inoculator. Each pin head of 1. Results obtained with the miniaturized tests the inoculator carries ca. 0.0008 ml of liquid. correspond directly to results obtained with the con ventional tests indicating the reliability of the min iaturized tests (1, 6). The incubation periods neces 1Portion of the paper was presented at the 71st Annual Meet ing of the American Society for Microbiology in Minneapolis, sary for obtaining definite results were shorter for Minn., Z-7, May 1971. the miniaturized tests than for the conventional MINIATURIZED TECHNIQUES 329 Test organism Ac,nromobm:;ter parvulus Agrobm:;terlum radiobacter Alcaligenes faecalis + + Corynebm:;terlum xerosis Enterobm:;ter aerogenes 11 + + + Enterobm:;ter aerogenes lla + + Enterobacter cloacae + + &cherichw coli A-1 + + Escherlchw coli B + + + + Escherichw coU C-30 + + Proteus vulgaris 9'434 + + Downloaded from http://meridian.allenpress.com/jfp/article-pdf/35/6/328/2399137/0022-2747-35_6_328.pdf by guest on 27 September 2021 Proteus~ vulgaris X19 + + Pseudomonas aeruglnosa + Salmonella: paratypki + + . + - Salmonella pullarum + + Salmonella thompsvn + + + + + + Salmonella typhimurium I + + + Salmonella typhosa + + Serratia marcescens + Shigella alko.lescens + + Shigella flexneri V I + Shigella flexnerl W + + Shlgella flexneri: 9'7 48 + + Shigella· sonnei + + 1The indole, M-R, V-P, and citrate tests were made as describoo in the text. Mini :::: miniaturized method; Conv :::: con ventional method. The times given are the minimum incuhation periods necessary for all positive cultures to respond to the miniaturized test or the recommended incubation period for the conventional test. + = positive test; negative test. tests. .. Positive indole and citrate miniaturized tests and conventional methods. The multiple inocula could be observed as early as 8 hr of incubation for tion device used in these procedures may also be most cultures although 12 hr incubation time pro utilized for conducting simplified .~tests su-ch as oxi vided more definite results. "Overnigh( incubation dase production, phenylpyruvic .acid reaction, oxi time would be a convenient schedule for these two dation of gluconates (51 and various other routine tests. The miniaturized MR and VP tests were re microbiological tests using liquid media (6). duced to 48 hr and 24 hr, respectively, compared to 120 hr and 48 hr stipulated in the conventional pro ACKNOWLEDGEMENTS cedures (3). The authors thank Dr. Paul A. Hartman of the Iowa State The amount of reagent to be added to the culture University for comments and suggestions. after growth proved to be .rather critical in ·a studY. of this nature. Because of the small volume of cul REFEI.U!!NCES ture, excess reagent will cause erroneous readings. 1. Barry, A. L., K. L. Bemsohn, A. P. Adams, and LY.. ~· The authors tested many different "instruments" such Thrupp. 1970. Improved 18-hour methyl red test. Appl. as pin-heads, pin-tails, Pasteur pipettes, tooth picks, Microbial. 20:866-870. · ' and loops to add reagents to these cultures. The 2. Davies, J. A., J. R. Mitzel, and W. E. Beam, Jr. 1971. Carbohydrate fermentation patterns of N eisserio meningttidis amounts added by these "instruments" was also criti determined by a Microtiter method. Appl. Microbial. 21:1072- cally tested. The data presented in Table 1 were 1074. obtained using the optimum procedures and quanti 3. Difco Laboratories. 1953.. Difco manual, 9th ed.~:Difco ties of reagents that provided results identical to those Laboratories. Detroit._ 35(}~ p. obtained using the conventional methods. 4. Fung, D. Y. C., and R. D. Miller. 19'70, Rapid pro The advantages of the miniaturized methods in cedure for the detection of acid and gas productiori by bac terial cultures. Appl. -Microbial. 20: · clude savings of time in operation, time in data col 527-~28~~ lection, materials, and effort. The reliability of the 5. Greaves; P. W~ 1970. A sifnplified technique for :;the identification of coliform bacilli: J. -Med. I,a}); TeChribt 27: miniaturized methods for the IMViG tests was dem ).90-200~ '~z • onstrated by direct eomparison of 24 bacterial species 6. Hartman, P. A. 1968. Miniaturized mfcrobiologlcal 1nd strains grown and tested by both the miniaturized methods. Academic Press Inc.,- New York• .227. p. .

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