Byron Brehm-Stecher - Combination of adhesive-tape-based sampling and fluorescence in situ hybridization for rapid detection of Salmonella on fresh produce.

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  Publication Details (including relevant citation   information):

  J Vis Exp. 2010   Oct 18;(44). pii: 2308. doi: 10.3791/2308.


  This protocol describes a simple approach for adhesive-tape-based   sampling of tomato and other fresh produce surfaces, followed by   on-tape fluorescence in situ hybridization (FISH) for rapid   culture-independent detection of Salmonella spp. Cell-charged   tapes can also be placed face-down on selective agar for   solid-phase enrichment prior to detection. Alternatively,   low-volume liquid enrichments (liquid surface miniculture) can be   performed on the surface of the tape in non-selective broth,   followed by FISH and analysis via flow cytometry. To begin,   sterile adhesive tape is brought into contact with fresh produce,   gentle pressure is applied, and the tape is removed, physically   extracting microbes present on these surfaces. Tapes are mounted   sticky-side up onto glass microscope slides and the sampled cells   are fixed with 10% formalin (30 min) and dehydrated using a   graded ethanol series (50, 80, and 95%; 3 min each   concentration). Next, cell-charged tapes are spotted with buffer   containing a Salmonella-targeted DNA probe cocktail and   hybridized for 15 - 30 min at 55°C, followed by a brief rinse in   a washing buffer to remove unbound probe. Adherent, FISH-labeled   cells are then counterstained with the DNA dye   4',6-diamidino-2-phenylindole (DAPI) and results are viewed using   fluorescence microscopy. For solid-phase enrichment, cell-charged   tapes are placed face-down on a suitable selective agar surface   and incubated to allow in situ growth of Salmonella   microcolonies, followed by FISH and microscopy as described   above. For liquid surface miniculture, cell-charged tapes are   placed sticky side up and a silicone perfusion chamber is applied   so that the tape and microscope slide form the bottom of a   water-tight chamber into which a small volume (≤ 500 μL) of   Trypticase Soy Broth (TSB) is introduced. The inlet ports are   sealed and the chambers are incubated at 35 - 37°C, allowing   growth-based amplification of tape-extracted microbes. Following   incubation, inlet ports are unsealed, cells are detached and   mixed with vigorous back and forth pipetting, harvested via   centrifugation and fixed in 10% neutral buffered formalin.   Finally, samples are hybridized and examined via flow cytometry   to reveal the presence of Salmonella spp. As described here, our   "tape-FISH" approach can provide simple and rapid sampling and   detection of Salmonella on tomato surfaces. We have also used   this approach for sampling other types of fresh produce,   including spinach and jalapeño peppers.

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