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To provide the State Water Resources Control Board (SWRCB) analytical services described herein: Provide support of source analysis for the Salinas River Fecal Coliform Total Maximum Daily Load (TMDL) through laboratory analytical services of samples provided by Water Board staff. The analytical services provided by the Contractor will include E. coli density, identification of E. coli O157:H7, and genotyping of bacteria positively identified as E. coli O157:H7.
Approach: Water samples will be processed by methods developed by PSMRU for selected isolation of E. coli O157:H7 and also split for analysis by accepted methods for generic E. coli. The extended IMS protocol developed by PSMRU starts with a non-selective enrichment (2 hrs at 25°C and 6 hr at 42°C). Ten g of water will be centrifuged to pellet particles and bacteria, then add the pellet to 100 ml of TSB and enrich. Immunocaptured and washed beads will be plated on selective agar for identification of putative EcO157. EcO157 will be confirmed by multiplex PCR for virulence genes, rfb, eae, stx1, stx2, hly. EcO157 strains isolated by the extended IMS protocol will be characterized initially by multilocus variable tandem repeat analysis (MLVA). Comparisons and phylogenetic relationships between strains will be determined using PAUP 4.0 software. EcO157 strains subtyped by MLVA will be characterized by PFGE. Formerly 5325-42000-041-03R (6/06).