Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

NOTE: C. perfringens is considered a biosafety hazard level 2 (BSL2) organism. Although not all food samples will contain C. perfringens, all cultured samples should be treated as such. All proper precautions and personnel protective equipment (PPE) should be worn at all times. Decontaminate all material prior to disposal.

1. Prepare modified RPM^4,15

1. Combine 30 g/L fluid thioglycolate medium, 60 g/L gelatin, 5 g/L peptone, 5 g/L glucose, 5 g/L potassium phosphate dibasic, 3 g/L yeast extract, 1.5 g/L sodium chloride, 0.5 g/L ferrous sulfate in deionized water until evenly dispersed. We typically make 1 L batches.
2. Autoclave at 121 °C for 15 min.
3. Allow to cool to approximately 40 °C.
4. Once RPM is cool, add D-cycloserine to a final concentration of 440 mg/L.
   NOTE: Stock concentrations of D-cycloserine dissolved in sterile water at 50 mg/mL may be stored at -20 °C for future use.
5. Aseptically transfer 10 mL of RPM to 15 mL conical tubes. RPM can be prepared in batches and stored at 4 °C for up to one month.
6. Warm RPM to 37 °C prior to use.

2. Sample collection and RPM incubation

1. Transport food to the laboratory under ambient temperatures within 1 h. Food may be transported in original packaging or sterile containers. If not testing immediately, food may be stored at -20 °C until use. Make note of the type of food and the country of origin according to the packaging label, if available, as well as any other relevant information.
2. Remove approximately 1.0–2.0 g of food and transfer to sterile Petri dish or equivalent. Finely mince with a sterile razor blade or scalpel.
3. Inoculate into 10 mL of phosphate buffered saline (PBS) in a 15 mL conical tube. Mix well.
   1. To select for vegetative cells, transfer 5 mL of the PBS-food mixture into a 15 mL conical tube containing 10 mL of RPM. Secure the lid tightly.
   2. To select for spores, heat the remaining 5 mL of PBS-food mixture at 85 °C for 15 min and then transfer to a 15 mL conical tube containing 10 mL of RPM. Secure the lid tightly.
4. Vortex and invert the food-RPM cultures to ensure complete mixing.
5. Tightly seal the conical tubes and wrap lids with paraffin film or plastic wrap to ensure an anaerobic environment.
6. Incubate overnight (ON) at 37 °C.
7. The following morning note any turbidity or fermentation in RPM tubes.

3. TSC "sandwich" plating

1. Inoculate ON RPM cultures into TSC agar using a “sandwich” technique (Figure 2) described below.
2. Prepare TSC agar as per the manufacturer's instructions.
   1. Prepare the base layer of the TSC plates by transferring 10 mL of molten TSC agar to sterile Petri dishes and allow to solidify. These may be prepared in advanced and stored at 4 °C. Ensure plates are warmed to room temperature prior to use.
   2. For the top layer of the TSC plate, maintain the remaining molten TSC agar at 40 °C.
      NOTE: Although agar's melting point is above 85 °C, molten agar solidifies around 32–45 °C.
3. Carefully retrieve the ON RPM cultures and transfer 100 µL to the TSC agar base. Because of fermentation, some cultures may be under pressure. Be sure to wear all appropriate PPE.
4. Spread the RPM inoculum using sterilized glass beads or cell spreader.
5. Allow RPM to be "absorbed" into TSC agar for 5–10 min at room temperature.
6. Carefully transfer 20 mL of molten, 40 °C TSC agar to plate using a serological pipette.
7. Replace the Petri dish lid and allow TSC agar to completely solidify at room temperature.
8. Invert the Petri dish and incubate at 37 °C ON.
9. If serial dilutions are desired, perform dilutions of the ON RPM cultures into fresh, pre-warmed RPM prior to inoculation into TSC agar.

4. Sub-culturing of sulfite-reducing colonies

1. The following morning, remove plates from incubator and examine for bacterial growth. Aerobic bacteria may be present on the surface of the agar. Anaerobic bacteria will be found growing embedded within the agar. Sulfite-reducing bacteria will turn surrounding agar black. Possible C. perfringens colonies will be black and embedded in the agar.
2. C. perfringens suspected colonies will be positive for sulfite reduction and will appear black, embedded within the agar. Using a sterile, single-use eyedropper, "pluck" the black colonies from the agar and transfer to 10 mL of fresh RPM in a 15 mL conical tube. It is important that the air from the eyedropper be expelled prior to piercing agar.
3. If there is a dense amount of aerobic bacterial growth on the surface of the plate, use a sterile cell scraper to remove colonies from selected areas. Multiple C. perfringens suspected colonies can be sampled from the same TSC plate into separate RPM cultures.
4. Tightly secure conical tube lids and wrap in paraffin film or plastic wrap. Incubate ON at 37 °C.

5. DNA Extraction

1. Remove ON RPM cultures and examine for signs of growth including turbidity and fermentation.
2. Gently invert RPM culture to disperse any settled bacteria and carefully open. Transfer 1 mL of the culture to a microcentrifuge tube.
3. Centrifuge at top speed (approximately 15,000 x g) for 10 min to pellet bacteria.
4. Wash pellet with 1 mL of sterile PBS.
   1. In some circumstances, undigested gelatin will settle on top of pelleted bacteria. To remove gelatin, "fluff" off gelatin by gently agitating with PBS using a micropipette. This will "fluff-up" the gelatin while leaving the bacterial pellet intact.
   2. Remove the PBS-gelatin mixture with gentle aspiration. Re-suspend the remaining bacterial pellet with 1 mL of fresh PBS.
5. Centrifuge the resuspended bacterial pellet at top speed for 10 min and carefully aspirate the supernatant.
6. Immediately perform DNA extraction using a DNA extraction kit and a protocol specifically created for extraction DNA from Gram-positive bacteria (see the Table of Materials).
7. Use the extracted DNA immediately or store at -20 °C until PCR analysis.

6. Detection of C. perfringens via PCR genotyping

1. To determine if cultures are positive for C. perfringens toxinotypes, examine extracted DNA for different toxin genes via PCR.
   NOTE: Because we are most interested in the epsilon toxin producing strains type B and D C. perfringens, we evaluate for the presence of the alpha, beta, and epsilon toxin genes. Primers are listed in Table 2. Primers for 16s ribosomal DNA are used as a DNA extraction control. Additional primers can be used to test for toxinotypes A through G and can be found in published literature^2,12,13.
2. Use previously extracted C. perfringens DNA as a positive control. This control ensures that all components of the PCR reactions are working. We use DNA extracted from C. perfringens type B (ATCC 3626) as our positive control. Grow ATCC 3636 ON in RPM at 37 °C and extract DNA using the same methods described in steps 5.1–5.7. Store extracted DNA at -20 °C until use.
3. Set PCR conditions as follows: 94.0 °C for 3 min; 94.0 °C for 30 s; 47.9 °C for 30 s, 72.0 °C for 1 min (Repeat steps 2–4 for 35 cycles); 72.0 °C for 10 min.
4. To confirm PCR products, run PCR reactions on a 1.8 g/100 mL agarose gel using standard techniques. Expected PCR product sizes are listed in Table 2. Typical PCR results are displayed in Figure 3.