Plating methods for estimating survival of indicator organisms, such asEscherichia coli, and water-borne pathogens includingVibrio cholerae, have severe limitations when used to estimate viable populations of these organisms in the aquatic environment. By combining the methods of immunofluorescent microscopy, acridine orange direct counting, and direct viable counting, with culture methods such as indirect enumeration by most probable number (MPN) estimation and direct plating, it was shown that bothE. coli andV. cholerae undergo a "nonrecoverable" stage of existence, but remain viable. Following 2-week incubations in saltwater (5-25%o NaCl) microcosms, total counts, measured by direct microscopic examination of fluorescent antibody and acridine orange stained cells, remained unchanged, whereas MPN estimates and plate counts exhibited rapid decline. Results of direct viable counting, a procedure permitting estimate of substrate-responsive viable cells by microscopic examination, revealed that a significant proportion of the nonculturable cells were, indeed, viable. Thus, survival of pathogens in the aquatic environment must be re-assessed. The "die-off" or "decay" concept may not be completely valid. Furthermore, the usefulness of the coliform and fecal coliform indices for evaluating water quality for public health purposes may be seriously compromised, in the light of the finding reported here.

Environmental and clinical strains of Vibrio cholerae were exposed to nutrient-free artificial seawater and filtered natural seawater microcosms for selected time intervals and examined for changes in cell morphology and number. Cells observed by transmission electron and epifluorescence microscopy were found to undergo gross alterations in cell morphology with time of exposure. The vibroid cells decreased in volume by 85% and developed into small coccoid forms surrounded by remnant cell walls. The initial number of cells inoculated into nutrient-free microcosms (culturable count and direct viable count) increased 2.5 log10 within 3 days, and even after 75 days the number of viable cells was still 1 to 2 log10 higher than the initial inoculum size. Nutrient-depleted coccoid-shaped cells were restored to normal size and assumed a bacillary shape within 3 h and began to divide within 5 h after nutrient supplementation. The increase in cell number and decrease in cell volume under nutrient-depleted conditions, as well as the rapid growth response after nutrient supplementation, may describe some of the survival mechanisms of V. cholerae in the aquatic environment.

Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars 01 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (250oo0) and a temperature of 10°C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.

Extracts of the sponge Tedania ignis have been reported to contain several diketopiperazines. As part of an investigation of the commensal and symbiotic microflora of sponges, we have consistently isolated, from specimens of T. ignis, a Micrococcus sp. which produces diketopiperazines in laboratory cultural media. This is the first demonstration that a bacterium associated with a sponge produces secondary metabolites ascribed to the sponge host.

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