Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

We have developed an easy method to test food samples for the different C perfringens toxinotypes. We are particularly interested in the prevalence of toxinotypes B and D which produce the epsilon toxin. Our protocol allows easy enrichment and detection of the various C perfringens toxinotypes without the use of anaerobic chambers or incubators with limited sub-culturing.

This method could also be used to test soil, water, fecal samples, and more. Demonstrating the procedure will be Zuha Anwar and Samantha Regan, technicians from the lab. Begin by preparing modified rapid perfringens medium, or RPM.

Combine ingredients according to manuscript directions, and autoclave the mixture at 121 degrees Celsius for 15 minutes. Allow the medium to cool to approximately 40 degrees Celsius, then add D-cycloserine to a final concentration of 440 milligrams per liter. Aseptically transfer the RPM to 15 milliliter conical tubes, and store at four degrees Celsius for up to one month.

Prior to using, warm the medium to 37 degrees Celsius. When collecting samples to be tested, transfer the food to the laboratory according to manuscript directions. If not testing immediately, the food can be stored at minus 20 degrees Celsius.

Make note of the type of food and the country of origin, as well as any other relevant information. To test the food, transfer approximately one to two grams to a sterile Petri dish, and finely mince it with a sterile razor blade or scalpel. Inoculate it into 10 milliliters of PBS in a 15 milliliter conical tube, and mix well.

Select for vegetative cells by transferring five milliliters of the PBS food mixture to a 15 milliliter conical tube with 10 milliliters of RPM and closing the lid tightly. Select for spores by heating the remaining five milliliters of the mixture to 85 degrees Celsius for 15 minutes. And then transferring it to a tube with RPM and closing the lid tightly.

To ensure complete mixing, vortex and invert the food RPM cultures. Then, tightly seal the conical tubes and wrap the lids with paraffin film to create an anaerobic environment. Incubate the tubes overnight at 37 degrees Celsius and note any turbidity or fermentation on the following morning.

Prepare Tryptose Sulfite Cycloserine or TSC Agar according to manufacturer's direction, and then use the sandwich technique to inoculate it with overnight RPM cultures. Prepare the base layer of the plates by transferring 10 milliliters of the molten Agar to sterile Petrie dishes and allowing it to solidify. Maintain the remaining TSC Agar at 40 degrees Celsius for the top layer, and carefully transfer 100 microliters of the overnight RPM culture to the Agar base.

Some cultures may be under pressure due to fermentation, so be sure to wear all appropriate personal protective equipment. Spread the inoculine with sterile glass beads or a cell spreader, and allow RPM to be absorbed into the Agar for five to 10 minutes. Then, use a serological pipette to carefully transfer 20 milliliters of the molten TSC Agar to the plate.

Replace the Petrie dish lid and allow TSC Agar to completely solidify at room temperature, then invert the dish and incubate it at 37 degrees Celsius overnight. If required, perform serial dilutions of the RPM culture prior to inoculation into TSC Agar. After the incubation, examine plates for bacterial growth.

Aerobic bacteria may be present at the surface of the Agar, while anaerobic bacteria will be embedded in the Agar. Sulfite reducing bacteria will turn the surrounding Agar black, and possible C perfringens colonies will be black and embedded in the Agar. If there is a dense amount of aerobic bacterial growth on the surface of the plate, use a cell scraper to remove colonies from selected areas.

Use a sterile single-use eye dropper to pluck the suspected C perfringens colonies from the Agar, making sure to expel the air prior to piercing the Agar. Transfer the colonies to 10 milliliters of fresh RPM in a 15 milliliter conical tube. Multiple colonies could be sampled from the same plate into separate RPM cultures.

Tightly secure the conical tube lids, wrap them in paraffin film, and incubate them overnight at 37 degrees Celsius. C perfringens is a BSL two organism. It is important to use proper safety precautions at all times, especially when opening overnight RPM cultures and to carefully decontaminate and dispose of used media.

On the next day, remove the overnight cultures and examine them for turbidity and fermentation, then proceed with DNA extraction. Gently invert the RPM culture to disperse any settled bacteria and carefully open the tube. Transfer one milliliter to a microcentrifuge tube and pellet the bacteria by centrifugation.

Wash the pellet with one milliliter of sterile PBS. If undigested gelatin has settled on top of the bacteria, fluff it off by agitating with PBS. Gently aspirate the gelatin and PBS and re-suspend the remaining bacterial pellet with one milliliter of fresh PBS.

It is important to remove any pelleted gelatin with limited disturbance of the cell pellet. Failure to remove gelatin may inhibit DNA extraction and PCR. Centrifuge the bacteria for 10 minutes and carefully aspirate the supernatant.

Then perform DNA extraction using a kit specifically designed for gram positive bacteria. Use the DNA immediately or store it at minus 20 degrees Celsius. Perform PCR on the extracted DNA to determine if the cultures are positive for C perfringens toxinotypes.

Use C perfringens DNA as a positive control to ensure that all PCR reagents are working. Run the PCR according to manuscript directions and analyze the products with agarose gel electrophoresis. This protocol was used to test a total of 216 food samples purchased from New York retail stores.

Samples included various meat samples, poultry samples, and seafood samples. C perfringens Type B was used as a positive control. It was found that 15 to 20%of sampled foods test positive for C perfringens.

Of the 34 positive samples, 31 samples contained the alpha toxin, one sample contained the alpha, beta, and epsilon toxin. And two samples contained the alpha and epsilon toxin. We have developed an easy method to test food samples for the different C perfringens toxinotypes.

We are particularly interested in the prevalence of toxinotypes B and D which produce the epsilon toxin.