Flow cytometric distinction of mature spores of <i>Bacillus subtilis</i> 168 using Auramine O

We developed a simple and effective method for distinguishing mature spores (MSs) of Bacillus subtilis 168 from mixed cell population using Auramine O (AuO) staining combined with flow cytometry (FCM) analysis. AuO preferentially stains MSs, enabling fluorescence-based discrimination. To optimize the method, B. subtilis 168 was stained with AuO and heated at different temperatures (25, 40, 55, or 70 °C) before FCM analysis. Among the tested conditions, heating for 30 min at 55 °C yielded the most distinct separation between MSs and other cell types based on fluorescence intensity. This approach combines the high-throughput capability of FCM with temperature-enhanced fluorescent staining to achieve efficient and accurate spore identification. This method is simple, rapid, and scalable, with potential applications in food safety testing and probiotic product manufacturing, where fast and reliable bacterial enumeration is essential.