Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Summary

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Abstract

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.

Protocol

1. Surface Sampling with Sterile Adhesive Tape

1. Select a tape to use for sampling. The commercially available Fungi-Tape or Con-Tact-It sampling tapes are sterile and specially packaged for ease of use. However, we have found that transparent (optically clear) generic office tape can also be used.
2. Use a permanent marker to draw 1 cm² squares on the non-sticky side of a 10 cm piece of adhesive tape (a paper template can be used). This will serve as a visual guide for noting which portion of the tape has been used to sample the food or environmental surface.
3. Form a "C"-shaped loop of tape, with the sticky side facing the sur....

Discussion

Simple and rapid methods for detection of pathogens on produce surfaces may help mitigate foodborne disease by providing timely and actionable data. Adhesive tape-based sampling methods have been used in environmental, clinical and food microbiology since the 1950's and involve pressing of "Scotch"-style tape to surfaces for removal of microorganisms, followed by direct microscopic examination or transfer of adherent microorganisms to solid media for growth (Barnetson & Milne, 1973; Edwards & Hartman, 1952; Evanc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fungi-Tape sampling tape	Scientific Device Laboratory	745	http://www.scientificdevice.com/
Con-Tact-It sampling tape	Birko Corporation, Denver, CO		http://www.birkocorp.com/
Clear office tape, generic	Various		Should be optically clear, have low intrinsic fluorescence
Food surface	Local Grocery Store		Tomat–s (red tomat–s on the vine, not waxed or oiled) used here
Trypticase Soy Broth	Difco Laboratories	211768	For non-selective liquid surface miniculture enrichment
Xylose-lysine-Tergitol 4 agar base	Difco Laboratories	223420	For Salmonella-selective agar (XLT-4)
Xylose-lysine-Tergitol 4 agar supplement	Difco Laboratories	235310	For Salmonella-selective agar (XLT-4)
Formalin solution	Sigma-Aldrich	HT5011	10% solution, neutral, buffered (cell fixative)
Absolute ethanol	Sigma-Aldrich	E7023	Molecular biology grade (pre-hybridization dehydration)
1.5 ml microcentrifuge tubes	Various		RNase- and DNase-free
Microscope slides and cover slips	Thermo Fisher Scientific, Inc.
NaCl solution	Sigma-Aldrich	S5150	Molecular biology grade, 5M solution (hybridization buffer component)
Tris-EDTA buffer solution (100X concentrate)	Sigma-Aldrich	T9285	1M Tris [pH 8.0], 0.1M EDTA (hybridization buffer component)
Sodium dodecyl sulfate solution	Sigma-Aldrich	L4522	10% solution in 18 megohm water (hybridization buffer component)
Sal3 and Salm-63 oligonucleotide probes	Integrated DNA Technologies		5’-labeled with 6-carboxyfluorescein (FAM) or Texas Red (for microscopy) or Cy5 (for cytometry), HPLC-purified
Variable speed microcentrifuge	Various		Use rotor diameter to calculate RPM needed for RCF values described in protocol
CoverWell perfusion chamber	Grace Bio-Lab Inc.	PC1R-2.0	Non-sterile
Gel loading pipette tips (FS MultiFlex)	Thermo Fisher Scientific, Inc.	05-408-151	Long, thin tips for easy access to small sampling ports and maneuverability within chamber
Aluminum heat block or precision-controlled heating station	Various		Eppendorf Thermomixer R dry block heating and cooling shaker used here
Bambino mini hybridization oven	Boekel Scientific	Model 230300	Slides are placed in 50 ml polypropylene centrifuge tubes for hybridization, heat transfer not direct
Slide Moat slide hybridizer	Boekel Scientific	Model 240000	Provides rapid, direct transmission of heat through glass slide
Vectashield H-1200 mounting medium with 4’,6-diamidino-2-phenylindole (DAPI)	Vector Laboratories	H-1200	Minimizes quenching of fluorescence during microscopy, provides DAPI counterstain
Fluorescence microscope	Various		Leitz Laborlux S used here
Digital camera	Various		Canon PowerShot A640 camera used here
Image acquisition software	Various		Axiovision software v. 4.6 (Carl Zeiss) used
Adobe Photoshop	Adobe		For minimal processing of images (overlay of images taken in different channels)
Flow cytometer	Various		FACSCanto flow cytometer (BD Biosciences, San Jose, CA) with red (647 nm) excitation used
Flow cytometry analysis software	Various		FlowJo software v. 8.7.1 (Tree Star, Inc.) used