Flow cytometric distinction of mature spores of Bacillus subtilis 168 using Auramine O

Area of Science:

• Microbiology
• Analytical Chemistry
• Biotechnology

Background:

• Accurate enumeration of bacterial spores is crucial for food safety and probiotic manufacturing.
• Distinguishing mature spores from vegetative cells in Bacillus subtilis populations can be challenging.

Purpose of the Study:

• To develop a simple, rapid, and effective method for distinguishing mature Bacillus subtilis 168 spores from mixed cell populations.
• To optimize spore staining and identification using Auramine O (AuO) and flow cytometry (FCM).

Main Methods:

• Utilized Auramine O (AuO) fluorescent staining for Bacillus subtilis 168.
• Employed flow cytometry (FCM) analysis to measure fluorescence intensity.
• Optimized staining by testing heat treatment at various temperatures (25, 40, 55, 70 °C) for 30 minutes.

Main Results:

• Auramine O (AuO) preferentially stains mature spores, enabling fluorescence-based discrimination.
• Heating at 55 °C for 30 minutes provided the most distinct separation between spores and other cell types.
• The optimized method demonstrated efficient and accurate spore identification via FCM.

Conclusions:

• The combined AuO staining and heat treatment with FCM offers a high-throughput, scalable method for spore identification.
• This technique is valuable for applications requiring fast and reliable bacterial enumeration, such as in food safety and probiotic production.