My Clostridium tyrobutyricum seems not producing spores. I incubated the Clostridium tyrobutyricum in 10 ml of RCM at 28 degrees Celcius for 5 days anaerobically, then heat the whole tube in 75 degrees for 10 minutes, ten-fold serial diluted to -5, and plate -1 to -5 dilution on RCA. The plate count for not heated culture is around 5 log CFU/ml, but the heated sample has no growth. I am wondering what troubleshooting procedure I can make.
Can you check if your Clostridium tyrobutricum isolate is producing spores by microscopy? Phase contrast microscopy is often used to asses and observe endospores. The Malachite Green staining method is a simple procedure that is able to differentiate spores from vegetative cells. It is also important to ensure you're actually obtaining anaerobic conditions during growth in RCM by using suitable indicators either in anaerobic boxes or cabinets as a quality check.
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