Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/1/2006
Publication Date: 10/1/2006
Citation: Gerbyshak, H., Wesley, I.V. 2006. Identification of Salmonella-positive fecal samples using a 96-well microculture plate technique (RX method)[abstract]. Food Safety Consortium 2006 Symposium. October 1-3, 2006, Fayetteville, Arkansas. 2006 CDROM.
Technical Abstract: Conventional Salmonella isolation involves multiple sample transfers to culture media performed by an experienced microbiologist. The modified semi-solid RV and XLT (RX) Plate method, a modification of the RX tube format designed by Gailey et al. (2004), consolidates pre-enrichment (buffered peptone water or GN Hajna), enrichments (modified semisolid RV; MSRV), and plating to a selective agar (XLT-4) into a singlewell of a 96-deep well plate. Wells are inoculated with a 10% suspension of swine fecesor poultry cecal contents (100 ul) and incubated (48-96 hrs, 42C). Salmonella-positive samples are identified by a blackening of the XLT-4 agar in the butt of the well. Up to 45samples can be screened in duplicate using this 96-well format. We optimized the RX plate using pure cultures of Salmonella as well as Proteus and Citrobacter, which could potentially cause false positive reactions (blackening of XLT4 agar). Pure cultures of motile Salmonella enterica strains Abaetetuba and typhimurium were detected to 3 cfu/ well; nonmotile Salmonella (pullorum and gallinarum), which cannot swim through to the XLT4 layer, were not detected. Addition of novobiocin (20 ug/ml) to the MSRV layer inhibited Proteus mirabilis; Citrobacter freundii produced a slight positive reaction at concentrations >104 cfu/ well. Since the assay is dependent upon blackening of the XLT4 layer, H2S negative Salmonella (~5% of Salmonella) will not be detected. The assay was then used to screen swine fecal (n= 30) and avian cecal (n=50) samples. Conventional culture detected Salmonella in 16 of 30 swine fecal samples. The RX plate technique identified Salmonella in 14 of these 16-positive samples. Positive reactions can be verified by restreaking a loop of growth from the MSRV/XLT4 interface of the culture well for Salmonella isolation, or by PCR amplification of the invA Salmonella gene using a 2 ul aliquot as the template. For avian samples, conventional isolation detected Salmonella in 35 of 50 turkey ceca whereas the RX plate technique detected Salmonella in 26 of 50 samples. Efforts are ongoing to optimize the system for poultry. Initial results suggest that the RX plate requires little technical experience to prepare, inoculate and read.