Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters.

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.

Survival and detection of Bacteroides spp., prospective indicator bacteria.

Preliminary experiments were performed to assess the use of intestinal Bacteroides spp. as indicators of fecal contamination of water. Viable counts of Bacteroides fragilis, an anaerobic bacterium, declined more rapidly than those of Escherichia coli and Streptococcus faecalis. However, a fluorescent antiserum prepared against B. fragilis successfully detected high proportions (18 to greater than 50%) of B. fragilis cells suspended for 8 days in aerobic water in dialysis bags at the ambient temperature. These percentages were higher than the percent viable recoveries of the two indicator bacteria used for comparison. Thus, the fluorescent antiserum test for B. fragilis might serve as a useful indicator of fecal contamination of water. An advantage of this approach over coliform analysis is the rapidity at which the test can be performed.

Membrane Filter Method for Enumeration of Acinetobacter calcoaceticus from Environmental Waters.

A membrane filter method was developed and evaluated for the quantitative recovery of Acinetobacter calcoaceticus from environmental waters. The procedure utilized a mineral medium, with sodium acetate and potassium nitrate as the carbon and nitrogen sources, respectively. Formic acid was included to enhance the recovery of A. calcoaceticus and to inhibit background growth. The medium was incubated for 46 h at 30 degrees C, after which fermentation and cytochrome oxidase tests were performed on the colonies as they appeared on the membrane. Background microbial growth decreased on the average by 1.77 orders of magnitude. An essentially quantitative recovery relative to that on nutrient agar spread plates was obtained from freshly prepared suspensions of eight A. calcoaceticus strains in filter-sterilized pond water and from suspensions of five of the strains held for up to 96 h in filter-sterilized pond water at 15 and 22 degrees C. Markedly reduced relative recoveries were obtained with the three remaining strains. However, these three strains, in contrast to the first five, not only did not grow, but also decreased in number in the eutrophic, filter-sterilized pond water. The confirmation rate of presumptive A. calcoaceticus colonies was 95%, whereas 8% of the presumptively negative colonies were A. calcoaceticus. The precision of the method did not exceed that expected from random error alone. Densities of A. calcoaceticus in freshwaters ranged from <1 to 7.9 x 10 organisms per 100 ml and were about 10 organisms per 100 ml in raw sewage.