#08-120

Complete

Date Full Report Received

02/17/2010

Date Abstract Report Received

02/17/2010

Investigation

Institution: , ,
Primary Investigator:

The goal of this study was to develop a single-step assay which combined all of the steps of conventional culture of Salmonella into a single well of a 48-deep well microtiter plate. The second goal was to screen hog carcass samples and ground pork to continuously refine the assay as it was being developed. Finally, the specificity and sensitivity of the assay was compared to conventional microbiological Salmonella testing methods. Conventional culture begins with pre-enrichment, followed by transfer of an aliquot of this nonselective enrichment to a selective broth containing novobiocin, and then plating to a selective agar, such as XLT-4, on which Salmonella appear as black colonies. The components used for conventional Salmonella isolation were consolidated into each well (5 ml/well capacity): XLT-4 was first added to each plate and allowed to solidify after which Modified Semisolid Rappaport-Vassiliadis (MSRV) with novobiocin was added followed by the selective broth. The 10% suspension of the sample to be screened was the final upper layer. Plates were sealed, incubated (42C, 48 hrs), and scored as positive if the lower XLT-4 indicator agar layer turned black. The assay is called the RX plate because it incorporates Rappaport-Vassiliadis modified semisolid agar (R) and for XLT-4 agar (X). Pure cultures were initially screened to ensure that the assay could detect Salmonella. Next, hog carcass lymph nodes harvested after the deep chill were screened (n=264). This was done to ensure that non-specific blackening of the wells due to the tissue itself did not occur. Swine lymph nodes harvested prior to the chiller, hog fecal samples (n=80) and retail pork sausage (n=240) were also evaluated. The reliability of the RX plate was based on specificity and sensitivity estimates, which compared RX plate results with those obtained by conventional bacteriological isolation. The RX format, while theoretically attractive, failed to achieve the either the specificity or sensitivity of conventional microbiological culture.
Contact Information: Irene.wesley@ars.usda.gov.