1media for Enumerating Enterococci in Frozen Vegetables

SPLITTSTOESSER, D. F. (Cornell University, Geneva, N. Y.), R. WRIGHT, AND G. J. HUCKER. Studies on media for enumerating enterococci in frozen vegetables. Appl. Microbiol. 9:303-308. 1961.-A study was made of the relative sensitivity and specificity of presumptive and confirmatory media for the most probable number enumeration of enterococci in frozen vegetables. Azide dextrose broth yielded the highest numbers of confirmable enterococci and its sensitivity was shown to be comparable to nonselective media. The use of ethyl violet azide broth as a confirmatory medium resulted in a significant number of false positive tests. Growth in broth containing 6.5% sodium chloride incubated at 45 C for 2 days was found to be a more specific confirmatory test for enterococci. There is a divergence of opinion with regard to the significance of the presence of enterococci in frozen I Approved by the Director of the New York State Agricultutral Experiment Station for publication as Journal paper no. 1237, October 4, 1960. foods. To clarify the question of their sanitary implications there is need for further information on the accuracy of the various methods used for their enumeration. A number of procedures have been used for determining the most probable number (MPN) of enterococci in frozen foods. In general, the methods are based on the same principle as those used to demonstrate the presence of the coliform types, i.e., growth in a presumptive medium followed by confirmatory tests. For the presumptive tests most workers have used a medium containing sodium azide as the selective agent. Confirmation procedures have been based either on the so-called "Sherman tests" (Sherman, 1937) or growth in ethyl violet azide (EVA) broth (Litsky, Mallmann, and Fifield, 1955). The suitability of the various media for determining the MPN of enterococci in precooked frozen foods has been evaluated by Zaborowski, Huber, and Rayman (1958), and Fanelli and Ayres (1959). Zaborowski et al. (1958) found that azide dextrose (AD) broth as a presumptive medium yielded the highest most probable number of counts. Also they reported 100 % confirma19611 303 on O cber 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom D. F. SPLITTSTOESSER, R. WRIGHT, AND G. J. HUCKER tion using EVA when one-tenth dilutions of the original samples were inoculated into this medium. The use of these two media for presumptive and confirmatory tests has been recommended by the Subcommittee on Methods for the Microbiological Examination of Foods (1958). Extensive studies on media for the enumeration of enterococci in fresh frozen vegetables have not been made. Inasmuch as the microflora of precooked preparations may differ significantly from that of frozen vegetables this investigation was initiated to determine the most satisfactory presumptive and confirmatory method for enumerating enterococci in frozen vegetables. MATERIALS AND METHODS The most effective probable number method for enumerating enterococci must be both sensitive and specific. Thus the medium should select for enterococci or permit their differentiation from other types, and at the same time, should be sufficiently sensitive that the introduction of only one cell would result in a positive test. Comparative sensitivity of presumptive media was determined by inoculating a series of tubes of each medium with a decreasing inoculum of pure strains of Streptococcus faecalis. The medium producing growth from the smallest inoculum was considered the most