Fecal Coliform Organism Enumeration in Chlorinated Wastewaters

The accepted method for determining disinfection efficiency of wastewater treatment systems is routinely to perform fecal coliform bacteria analyses on the final effluent (5). Standard Methods for the Examination of Water and Wastewater outlines two procedures for fecal coliform analysis: the membrane filtration technique and the most-probable-number (MPN) method (1). For chlorinated effluents, Standard Methods specifies that only the MPN method be used. However, the U.S. Environmental Protection Agency now accepts data on chlorinated effluents obtained by the membrane filtration technique but still requires any legal questions concerning fecal coliform data to be answered by using the MPN technique (5). Several objections to the MPN technique can be raised. Among them are: test length (48 to 72 h), the extensive record keeping for inexperienced laboratory personnel, reagent quantities, glassware needs, and the number of extra steps involved. In 1972, a simplified 24-h MPN technique was introduced for determining fecal coliforms in estuarine waters (2). The method used a new medium formulation called A-1. The medium differs from lauryl tryptose in that tryptose is replaced by tryptone, the phosphate buffer system is eliminated, and salicin and Triton X-100 are added. The A-1 technique gave results similar to the Standard Methods two-step fecal coliform MPN procedure. In 1978, two reports indicated that the A1 medium gave satisfactory fecal coliform results for marine waters when used with a 3-h, 35°C preincubation (6, 7). The A-1 medium was also used without preincubation in a study by Dutka et al. (4). Many of the disadvantages of traditional MPN testing were eliminated with this new procedure, i.e., record keeping was simplified, the test could be completed within 24 h, and the confirmation step, including the use of extra media and glassware, was eliminated. Theoretically, all of the methods for bacteria enumeration in Standard Methods should work on all types of waters (1). The purpose of our study was to determine the suitability of the 24h A-1 medium technique for testing chlorinated secondary sewage treatment plant effluents and to assess its equivalency to the Standard Methods technique currently in use. Samples were collected from 12 Wisconsin sewage treatment facilities in phase 1 and from 13 facilities in phase 2. Facilities were chosen that provide various levels of secondary (biological) treatment. Since all of the plants practice chlorination to disinfect the final effluent, samples were collected in sterile, wide-mouthed polyethylene bottles containing a sufficient amount of sodium thiosulfate to neutralize any excess chlorine (1). Samples were immediately chilled to less than 4'C and tested within 24 h of collection time. Although Standard Methods mandates a maximum 6to 8-h holding time for this type of sample, we chose the 24-h limit since the original A-1 medium work by Andrews and Presnell (2) used a 24-h limit, and Standridge and Lesar (8) showed that carefully iced samples from chlorinated domestic sewage effluents, similar to those used in this study, showed little difference in fecal coliform counts between 8 and 24 h of storage. In the first phase of the study, five replicates of each of 12 effluent samples were tested for fecal coliform organisms using the standard lauryl tryptose broth (LTB)/EC medium MPN technique (1). Five replicates of each sample were also tested by the A-1 medium technique described by Miescier et al. (7) with the exception that a 2-h preincubation at 22°C was used rather than 3 h at 35°C. In the second phase of the study, five repli-