Analysis of vegetative cells and spore germination in food using flow cytometry

The detection of spore-forming bacteria in food is crucial because they potentially cause spoilage. However, traditional microbial tests are time-consuming and inefficient. Here, we report the use of flow cytometry with 5-cyano-2,3-ditolyl-2H-tetrazolium chloride (CTC) staining for the rapid detection and monitoring of spore germination. In particular, we investigated sporulation and metabolic activity in Paenibacillus, Sporosarcina, and Clostridium spp. and validated our method using real food samples. Our approach enabled the rapid detection of spore-forming bacteria and spore germination followed by outgrowth in food. Flow cytometry is a valuable tool for distinguishing vegetative cells and spores in food samples. In this study, we used flow cytometry with CTC staining to determine spore germination and vegetative cell populations. Our findings revealed that 0.2 % peracetic acid treatment inhibited spore germination, as indicated by decreased metabolically active spore count and increased vegetative cell count detected via flow cytometry. Additionally, flow cytometry enabled rapid and accurate differentiation of spores from vegetative cells, providing quantitative insights into bacterial viability within minutes. These findings indicate that flow cytometry is a reliable and practical tool for real-time microbial monitoring in food safety, offering advantages over conventional culture-based methods.