Multi-Laboratory Validation of Methods 1642 and 1643 for Male-Specific and Somatic Coliphage in Fresh and Marine Recreational Waters and Wastewater Samples

Oral Presentation

Prepared by Y. Chambers-Velarde1, R. Ghei2, K. Miller1
1 - CSRA, 6361 WALKER LN, STE 300, ALEXANDRIA, VA, 22310, United States
2 - CSRA, 650 Peter Jefferson Parkway, Ste 300, Charlottesville, Virginia, 22911, United States


Contact Information: yildiz.chambers@csra.com; 703-461-2165


ABSTRACT

EPA has a number of validated bacterial methods to detect and identify fecal indicators but has received requests to develop a coliphage method to detect sources of fecal contamination in fresh and marine waters and wastewater effluents. In response to stakeholders’ needs for a validated method for coliphage for monitoring recreational waters and wastewaters, EPA conducted a multi-laboratory validation (MLV) study of two methods. The first method consisted of a dead-end ultrafiltration (UF) procedure concentration procedure to assay 2 L recreational water samples and advanced treatment wastewater effluents, followed by a modified version of EPA Method 1602 (Male-specific (F+) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure) to assay for both male-specific and somatic phage. The second method was a modified version of EPA Method 1602 to assay 100 mL volumes of secondary (prior to disinfection) wastewater effluents. The SAL procedure requires the addition of host bacteria (E. coli Famp (ATCC® 700891™) as the male-specific host and E. coli CN-13 (ATCC® 700609™) as the somatic host), magnesium chloride, and double strength molten agar medium to the sample, followed by pouring the total volume of the mixture into plates. All plates from a single sample were examined for phage presence, as evidenced by plaque formation (zones of bacterial host lawn clearing). Unspiked and spiked phosphate buffered saline, fresh and marine waters, and advanced treatment and secondary (prior to disinfection) wastewater samples were analyzed during the study.

During the MLV, 18 volunteer laboratories collected and analyzed fresh and marine waters, and advanced treatment and secondary (prior to disinfection) wastewater samples. Results of the study indicate that Method 1642 is appropriate for the analyses of male-specific and somatic coliphage in fresh and marine waters and advanced wastewater effluents, and Method 1643 is appropriate for 100 mL secondary (prior to disinfection) wastewater samples.