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).
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.
1. Surface Sampling with Sterile Adhesive Tape
2. Solid-phase Enrichment and Liquid Surface Miniculture
3. Fixation and Dehydration
4. Hybridization
5. Detection
6. Representative Results
Figure 1. Use of adhesive tape for sampling of Salmonella spp. from the surface of an artificially contaminated tomato.
Figure 2. Typical results for direct-to-tape sampling and FISH detection of Salmonella Typhimurium ATCC 14028 from the surface of a tomato (100 X oil objective). A two-probe cocktail of Texas Red-labeled probes (Sal3/Salm-63) was used to label these cells.
Figure 3. Microcolonies of Salmonella Typhimurium ATCC 14028 formed on the surface of Xylose Lysine Tergitol-4 (XLT-4) agar after an 8 h on-tape enrichment at 37°C. The initial inoculum was sampled from the surface of an artificially contaminated tomato. Solid-phase enrichment increases the numbers of cells available for detection and also enhances cellular rRNA content.
Figure 4. Use of adhesive tape for sampling of S. enterica serovar Typhimurium from the surface of an artificially contaminated tomato, followed by direct analysis via FISH and flow cytometry (panel A) or after 5 h non-selective liquid surface miniculture enrichment in a perfusion chamber filled with 500 μL Trypticase Soy Broth (panel B).
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; Evancho et al., 2001; Fung et al., 1980; Lakshmanan & Schaffner, 2005; Langvad, 1980). A recent modification described the combination of tape-based sampling with fluorescence in situ hybridization (FISH) for culture-independent analysis of microorganisms colonizing the surfaces of stone monuments (La Cono & C. Urzì, 2003). We have applied a similar approach for rapid sampling and detection of Salmonella present on fresh produce, including tomatoes, spinach and Jalapeño peppers (Bisha and Brehm-Stecher, 2009a). Adhesive tape can be used to remove cells from these foods and samples can then be processed for FISH and viewed via fluorescence microscopy. In this way, as few as 103 CFU cm-2 Salmonella (the limit of detection for microscopy-based methods) can be detected on fresh produce within ~1.5 – 2 h. Alternatively, short (8 h) solid phase enrichments can be performed by placing the cell-charged tape face down on selective agar, and the resulting microcolonies detected by FISH. By overlaying the tape with ≤ 500 μL liquid media, tape-sampled Salmonella cells can be detached and detected directly after FISH using flow cytometry (Figure 4, panel A). Brief incubation (~5 h) of these non-selective liquid minicultures allows substantial enrichment of bacteria, with Salmonella cells easily detected in complex mixtures of target and non-target cells after FISH (Figure 4, panel B). Collectively, our results demonstrate that this approach provides a novel method for extraction, presentation and identification of specific bacteria present on tomatoes and other fresh produce. Although we have described the use of DNA-based probes here, it is likely that this approach may also be expanded to include alternative probe chemistries, such as peptide nucleic acids (PNAs), for which Salmonella-specific probes have also been described (Almeida et al., 2010).
The authors have nothing to disclose.
Funding for this work was provided by a Grow Iowa Values Fund award to BFBS.
Material Name | タイプ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Fungi-Tape sampling tape | Scientific Device Laboratory, Des Plaines, IL | 745 | http://www.scientificdevice.com/ | |
Con-Tact-It sampling tape | Birko Corporation, Denver, CO | http://www.birkocorp.com/ | ||
Clear office tape, generic | Various suppliers | Should be optically clear, have low intrinsic fluorescence | ||
Food surface | Local grocery | Tomatoes (red tomatoes on the vine, not waxed or oiled) used here | ||
Trypticase Soy Broth | Difco, Sparks, MD | 211768 | For non-selective liquid surface miniculture enrichment | |
Xylose-lysine-Tergitol 4 agar base | Difco, Sparks, MD | 223420 | For Salmonella-selective agar (XLT-4) | |
Xylose-lysine-Tergitol 4 agar supplement | Difco, Sparks, MD | 235310 | For Salmonella-selective agar (XLT-4) | |
Formalin solution | Sigma-Aldrich, St. Louis, MO | HT5011 | 10% solution, neutral, buffered (cell fixative) | |
Absolute ethanol | Sigma-Aldrich, St. Louis, MO | E7023 | Molecular biology grade (pre-hybridization dehydration) | |
1.5 ml microcentrifuge tubes | Various suppliers | RNase- and DNase-free | ||
Microscope slides and cover slips | Thermo Fisher Scientific, Waltham, MA | |||
NaCl solution | Sigma-Aldrich, St. Louis, MO | S5150 | Molecular biology grade, 5M solution (hybridization buffer component) | |
Tris-EDTA buffer solution (100X concentrate) | Sigma-Aldrich, St. Louis, MO | T9285 | 1M Tris [pH 8.0], 0.1M EDTA (hybridization buffer component) | |
Sodium dodecyl sulfate solution | Sigma-Aldrich, St. Louis, MO | L4522 | 10% solution in 18 megohm water (hybridization buffer component) | |
Sal3 and Salm-63 oligonucleotide probes | Integrated DNA Technologies, Coralville, IA | 5’-labeled with 6-carboxyfluorescein (FAM) or Texas Red (for microscopy) or Cy5 (for cytometry), HPLC-purified | ||
Variable speed microcentrifuge | Various suppliers | Use rotor diameter to calculate RPM needed for RCF values described in protocol | ||
CoverWell perfusion chamber | Grace Bio-Labs Inc., Bend, OR | PC1R-2.0 | Non-sterile | |
Gel loading pipette tips (FS MultiFlex) | Thermo Fisher Scientific, Waltham, MA | 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 suppliers | Eppendorf Thermomixer R dry block heating and cooling shaker used here | ||
Bambino mini hybridization oven | Boekel Scientific, Feasterville, PA | Model 230300 | Slides are placed in 50 ml polypropylene centrifuge tubes for hybridization, heat transfer not direct | |
Slide Moat slide hybridizer | Boekel Scientific, Feasterville, PA | 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, Inc., Burlingame, CA | H-1200 | Minimizes quenching of fluorescence during microscopy, provides DAPI counterstain | |
Fluorescence microscope | Various suppliers | Leitz Laborlux S used here | ||
Digital camera | Various suppliers | Canon PowerShot A640 camera used here | ||
Image acquisition software | Various suppliers | Axiovision software v. 4.6 (Carl Zeiss) used | ||
Adobe Photoshop | Adobe Inc. | For minimal processing of images (overlay of images taken in different channels) | ||
Flow cytometer | Various suppliers | FACSCanto flow cytometer (BD Biosciences, San Jose, CA) with red (647 nm) excitation used | ||
Flow cytometry analysis software | Various suppliers | FlowJo software v. 8.7.1 (Tree Star, Inc.) used |