Fecal Streptococci in Water'

ROSE, ROBERT E. (Massachusetts Department of Public Health, Amherst), AND WARREN LITSKY. Enrichment procedure for use with the membrane filter for isolation and enumeration of fecal streptococci in water. Appl. Microbiol. 13:106-108. 1965.-By the use of PYC enrichment medium, the recovery of fecal streptococci from river water has been increased more than twofold over that of M-Enterococcus Agar. Of the isolates tested, 94.6% could be classified as enterococci or enterococcus biotypes. This method seems to yield a larger number of strains which would not normally be revealed. Serological typing of atypical streptococcus strains isolated indicates that the majority of these "biotypes" can be placed in the enterococcus group. Following Sherman's (1937) comprehensive review of the streptococci, the use of the fecal streptococci as indicators of human and animal pollution in water has been advocated by a number of investigators (Mallman and Seligmann, 1950; Litsky, Mallman, and Fifield, 1953; Slanetz and Bartley, 1957; Kjellander, 1960). In the past decade, the formulation of media for the recovery of streptococci by the membrane-filter method has given even greater impetus to the importance of these organisms. WVith this technique, a direct count may be obtained efficiently and economically. Likewise, it is possible to examine larger quantities of water than were practical by the most probable number (MNP) tube or plate count methods. During a routine survey on an indoor swimming pool, minute streptococcal-like colonies were observed growing on M-Enterococcus Agar (Difco). When these colonies were transferred to Brain Heart Infusion (BHI) Broth (Difco) for subsequent identification, no growth could be noted. This indicated that the M-Enterococcus Agar was either incomplete or inhibitory for the primary isolation of all of the enterococci in the sample, or it had permitted minute nonenterococcal colonies to develop. The work reported herein was an attempt to clarify this situation by for' A contribution from the Massachusetts Agricultural Experiment Station, Amherst. 2 Present address: Millipore Filter Corp., Bedford, Mass. mulating a more efficient medium for the recovery and isolation of the enterococci from water by use of the membrane-filter technique. MATERIALS AND METHODS PYC (peptone-yeast extract-Casitone) enrichment broth was formulated on the basis of its efficiency and comparative results for enterococci recovery. The formula for PYC enrichment broth is as follows: nutrient broth (Difco), 0.8%; Proteose Peptone (Difco), 0.2%; Proteose Peptone #3 (Difco), 2.0%; yeast extract (Difco), 0.6%; Casitone (Difco), 0.2%; distilled water, 100 ml; final pH, 7.0 to 7.1. The prepared broth was dispensed into screwcapped test tubes (the caps supplied with rubber liners) and autoclaved at 121 C for 10 min. After sterilization, the test tubes were allowed to cool at room temperature, and then stored at 10 C prior to use. Essentially, the recovery procedure followed was (i) an enrichment phase in PYC broth followed by (ii) an incubation phase on M-Enterococcus Agar. To determine the efficiency of the PYC enrichment procedure, split samples were analyzed with only M-Enterococcus Agar for comparison. M-Enterococcus Agar (4 to 5 ml) was poured into 60-mm plastic petri dishes and allowed to solidify. After this agar had hardened, a filter pad was placed in the upper half of the inverted petri dish and saturated with 1.8 ml of PYC enrichment broth. The water samples were filtered through the membrane filter. (All equipment for use with the membrane-filter technique was obtained from the Millipore Filter Corp., Bedford, 106 on N ovem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom ISOLATION OF FECAL STREPTOCOCCI IN WATER TABLE 1. Comparison of recovery* of enterococci by use of M-Enterococcus Agar with and without enrichment Colonies per 100 Colonies per 100 Sample no. ml on M-Enteroml on M-Enterococcus Agar coccus Agar with enrichment