Optimization of EPA Method 1601 for Rapid Detection of Male-specific (F+) Coliphages in Seawater

Male-specific or F+ coliphages are candidate microbial indicators of fecal contamination and enteric viruses in water. As indicators, their utility depends on the ability to efficiently and reproducibly detect them in various water matrices. EPA Method 1601 is an overnight two step-enrichment spot-plate procedure used to detect low concentrations of coliphages in water. To date, the method has only been validated for use in groundwater. The objective of this study was to optimize EPA Method 1601 as rapid detection method for coliphages in seawater. This study examined potential modifications to the initial sample enrichment and to the spot-plate protocol for the variables of sample salinity (0 ppt, 8 ppt, 16 ppt, 32 ppt), nutrient medium (tryptic soy broth, TSB vs. Luria broth, LB) and incubation time (3, 5, 7, 24 hrs) on the growth of F+ coliphage prototype strains (GA, QB, SP, FD) representing different families and sub-groups. In general, coliphage concentrations increased with longer incubation time and decreasing sample salinity. The effect of media was less consistent, depending on both coliphage strain and sample salinity. After 24 hours of enrichment all four coliphages trains reached their highest observable concentrations and were readily detectable in enrichments of every salinity and media combination. This observation indicates that EPA Method 1601 is a valid method for the overnight detection of F+ coliphages in seawater. After incubation times of five hours and seven hours, the variations in salinity and media were observed to differentially affect the growth and detection of the four representative coliphage strains. Therefore, the growth of each coliphage was assessed across all salinity and media combinations for five and seven hours of incubation to determine which combinations of time, salinity, and media produced their maximum concentrations. Proposed optimum conditions for rapid enrichment of F+ coliphages by EPA Method 1601 were found to be: (1) sea water sample dilution to a final salinity of 8 or 16 ppt, (2) use of LB instead of TSB enrichment medium, and (3) enrichment incubation a minimum of seven hours. The slow growth kinetics of coliphage SP was the limiting one for optimization of conditions. Additionally, strain GA of F+ RNA coliphage group II and unidentified F+ coliphage strains in seawater samples were found to require added Mg^2+ ions in culture media for effective detection.Master of Science in Public Healt