Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 1/12/2007
Publication Date: 5/21/2007
Citation: Jenkins, M., Endale, D.M. 2007. A most probable number method for enumerating Salmonella in environmental waters. Annual Meeting of the American Society for Microbiology, May 21-25, 2007, Toronto, Ontario, Canada.
Technical Abstract: Background Most agricultural animals such as beef and dairy cattle, swine, and poultry are a source of Salmonella. Watersheds with animal agriculture can thus adversely impact recreational waters and threaten public health especially at the expanding interface of urban and agricultural lands. To understand better and manage the fate and transport of this zoonotic pathogen in agricultural watersheds a most probable number (MPN) method for enumerating Salmonella in environmental waters was developed. Methods The first step is to filter, for example, 20 l of environmental water through a FALP 293 mm membrane filter with a 1 µm pore size, and centrifuge material removed from filter. Centrifuged pellet is resuspended in PBS and used to inoculate tubes of Tetrathionate broth arranged in a 5-tube three or more 10-fold dilution scheme. Positive tubes of Tetrathionate broth are inoculated onto Brilliant Green agar plates for isolating colonies. At least three pinkish-white colonies on a red background are transferred to Beef Heart Infusion (BHI) agar plates and streaked for isolation and purity. Fresh colonies from the BHI plates are stabbed into slants of Lysine Iron agar (LIA) and Triple Sugar Iron (TSI) agar. If LIA and TSI slants are positive, confirmation is made by performing a TaqMan assay on a colony from the respective BHI plate. Primers and probe for the real-time PCR assay are specific for a 250-bp fragment of the junction between the two virulent genes SipB and SipC. MPN of cells is calculated using Microsoft Excel spread sheet and its Solver program. Results This method has determined the density of Salmonella in a 20 l sample of pond water from a watershed with cattle to be 0.1 cells/100 ml. Conclusions This represents a culture-based method that can detect small numbers of Salmonella in environmental waters impacted by animal agriculture and to which real-time PCR methods can be compared.