The overall goal of this procedure is to couple adhesive tape-based sampling of fresh produce with whole cell molecular detection of salmonella species using rapid fluorescence in C two hybridization, also known as fish. This is accomplished by first applying sticky tape to the desired material and extracting surface bound cells. Cell charge tapes may be analyzed directly or subjected to solid or liquid phase enrichments.
The second step of the procedure is on tape, self fixation and dehydration in an ethanol series for cells alluded into or enriched in broth. The second step involves cell fixation in suspension, followed by Resus suspension of cells in storage buffer. The third step of the procedure is to perform a rapid on tape hybridization step using a salmonella specific oligonucleotide probe cocktail, followed by removal of excess probe with a wash buffer rinse for samples analyzed via fax, the fish step is carried out in suspension, and excess probe may be removed using a simple dilution step prior to analysis.
The final step of the procedure is to counterstain the hybridized tape bound cells with DPI and examine them using fluorescence microscopy. For facts-based analyses, a counter stain is not needed. Ultimately, this method provides a means for visual or cytometric detection of salmonella species that may be present on sampled fresh produce.
I first had the idea for this method when reading about an Italian group that was using a similar approach to examine bacterial communities on Roman aero stone relics. But r and I led a discussion of this work in a class that I was teaching on rapid methods in food microbiology. A few weeks later, a salmonella outbreak traced to fresh produce began ultimately sickening nearly 1500 people in 43 different states.
While our experimental approach offers a timely and practical avenue for the detection of the specific bacterial types on fresh produce, it is also portable enough to enable testing in the field or in a food production environment. Start by selecting a tape to use for sampling. Some options include the commercially available fungi tape or contacted which are sterile and also packaged for ease of use.
With a permanent marker, draw one centimeter squares on the non-sick side of a 10 centimeter piece of adhesive tape using a sterile aluminum template. This will serve as a visual guide for noting which portion of the tape has been used to sample the food or environmental surface form a CS shaped loop of tape with the sticky side facing the surface to be sampled. To do this, hold the sticky ends with the thumb and middle finger and position the index finger against the drawn square on the back of the tape.
Place the tape on the surface to be sampled and gently press the marked area against the surface without releasing the edges of the tape. Use the index finger to ensure that the sticky side of the tape comes in full contact with the sample surface. Avoiding bubbles using an even motion.
Slowly pull the tape away from the sample. Physically extracting surface bound microbes fasten the cell charge. Tape sticky side up onto a glass microscope slide Using generic transparent office tape, ensure proper tension and stretching of the tape so that a flat non wrinkly surface is created.
This will help minimize problems with deformation or curling of the tape. During subsequent heating. During hybridization, place the tape face down on xylose slicing tertile four or XLT four agri plates so that the sampled cells are in direct contact with the agri surface to facilitate easy removal of the tape from the agri surface.
Following enrichment, the templated sample contact portion of the tape is placed flush with the agri surface, and one end of the tape is loosely adhered to. The sidewall of the Petri dish plates are inverted to avoid condensation and incubated at 35 degrees Celsius. To allow sufficient growth of salmonella species, the length of the incubation period needed to enrich the cells to a detectable level will depend on initial salmonella contamination levels.
After the desired enrichment period, open the agar plate, the tape will retain its tackiness during incubation. Gently press the tape against the agar using the index finger to ensure maximum recovery of micro colonies formed At the tape agar interface, grasp the tape from the edge attached to the wall of the Petri dish and remove it with a slow even motion. Using generic transparent office tape mount the cell charge tape sticky side up onto a microscope slide so that a flat non wrinkly surface is created.
Perform on tape cell fixation for 30 minutes at 25 degrees Celsius by covering the sample contact area with 500 microliters of 10%neutral buffered formin. Discard the fixative into a sealable container under a chemical hood to minimize exposure to irritating or toxic vapor, wash the tape once in one x phosphate buffered saline or PBS by gently flooding the tape surface with PBS dehydrate the sample. Using a progressive ethanol series, prepare pre hybridization hybridization and washing buffers using the commercial molecular biology grade solutions filter using a point 22 micrometer syringe and preheat hybridization and washing buffers to 55 degrees Celsius.
Add fluorescently labeled oligonucleotide probes to preheated hybridization buffer for a final probe concentration of five nanograms per microliter overlay the templated sample contact area of the tape with 300 microliters of hybridization buffer containing the probe cocktail and hybridized tape bound cells in a moist sealed incubation chamber set to 55 degrees Celsius for a direct contact incubator, such as the slide mode instrument hybridized for 15 minutes. Following hybridization, the slides are removed and the probe containing hybridization buffer overlay is discarded. Briefly and gently rinse with preheated washing buffer by pipetting.
A small volume of buffer over a tilted slide allow slides to air dry for onap detection of salmonella species via fluorescence microscopy. Overlay the slide with about 10 microliters of vector shield H 1200, mounting medium containing dpi, the nuclear counter stain incubate in the dark for 10 minutes. Place immersion oil on the cover, slip and examine samples using a high magnification oil objective.
The DPI filter is used to bring the sample into focus. The microscope is then switched to the appropriate filter, and salmonella cells are scored visually according to their green or red fluorescence, depending on which dye is used to label the probes. Fore cytric detection transfer PBS suspended samples to five milliliter round bottom sampling tubes and examine using a flow cytometer with excitation at 647 nanometers.
Analyze data using FlowJo software. Adhesive tape sampling of terica serovar, Ty Murium from the surface of an artificially contaminated tomato, facilitates direct post fish analysis via microscopy or flow cytometry cell charge. Tapes were processed for rapid fluorescence in C two hybridization using a combination of salmonella specific DNA probes and labeled with Texas Red, depending on initial local concentration of salmonella cells on the tape.
Solid phase enrichment yielded results ranging from isolated micro colonies to confluent growth. Tape sampled cells could be analyzed via combined fish and flow cytometry, either immediately after sampling or following a five hour liquid surface. Mini culture enrichment at 35 degrees Celsius.
Although we have described the use of the tape fish technique for food safety, the method also has good potential for use in other disciplines, including environmental microbiology, plant pathology, or clinical microbiology. In parallel with This procedure, additional experiments like biochemical testing or PCR may be performed on solid or liquid phase enrichments from tape extracted samples. Tests can be used to answer questions about a salmonella that may be present, questions that are beyond the scope of the fish procedure.
