Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 12/6/2006
Publication Date: 12/6/2006
Citation: Gerbyshak, H., O'Connor, A., Wesley, I.V. 2006. Identification of Salmonella-positive fecal samples using a 96-well microculture plate technique (RX method) [abstract]. Research Workers in Animal Diseases Conference Proceedings. p. 114.
Technical Abstract: Conventional Salmonella isolation involves multiple sample transfers to culture media performed by an experienced microbiologist. The Reaction (RX) Plate method, a modification of the RX tube 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 single well of a 96-deep well plate. Wells are inoculated with a 10% suspension of swine feces or 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 45 samples can be screened in duplicate using this 96-well format. 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.