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TECHNICAL REPORTS

Waste Management

EPA Worst Case Water Microcosms for Testing Phage Biocontrol of Salmonella

USDA, Agricultural Research Service, Waste Management and Forage Research Unit, POB 5367, Mississippi State, MS 39762. Journal article number J-11061 of the Mississippi Agricultural and Forestry Experiment Station. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. This work was prepared by employees of the U.S. Government as part of their official duties and is in the public domain and may be used without further permission

^* Corresponding author (mike.mclaughlin{at}ars.usda.gov).

Received for publication January 8, 2007. A microplate method was developed as a tool to test phages for their ability to control Salmonella in aqueous environments. The method used EPA (U.S. Environmental Protection Agency) worst case water (WCW) in 96-well plates. The WCW provided a consistent and relatively simple defined turbid aqueous matrix, high in total organic carbon (TOC) and total dissolved salts (TDS), to simulate swine lagoon effluent, without the inconvenience of malodor and confounding effects from other biological factors. The WCW was originally defined to simulate high turbidity and organic matter in water for testing point-of-use filtration devices. Use of WCW to simulate lagoon effluent for phage testing is a new and innovative application of this matrix. Control of physical and chemical parameters (TOC, TDS, turbidity, temperature, and pH) allowed precise evaluation of microbiological parameters (Salmonella and phages). In a typical application, wells containing WCW were loaded with Salmonella enterica susp. enterica serovar Typhimurium (ATCC14028) and treated with phages alone and in cocktail combinations. Mean Salmonella inactivation rates (k, where the lower the value, the greater the inactivation) of phage treatments ranged from –0.32 to –1.60 versus –0.004 for Salmonella controls. Mean log₁₀ reductions (the lower the value, the greater the reduction) of Salmonella phage treatments were –1.60 for phage PR04-1, –2.14 for phage PR37-96, and –2.14 for both phages in a sequential cocktail, versus –0.08 for Salmonella controls. The WCW microcosm system was an effective tool for evaluating the biocontrol potential of Salmonella phages.

Abbreviations: ATCC, American Type Culture Collection • BA, blood agar • EPA, (US) Environmental Protection Agency • ISO, International Organization for Standardization • MOI, multiplicity of infection • MPN, most probable number • NTU, nephelometric turbidity units • PBS, phosphate buffered saline • phage, bacteriophage • SM, suspension medium • TDS, total dissolved salts • TOC, total organic carbon • TSA, trypticase soy agar • TSB, trypticase soy broth • WCW, worst case water