Optical analysis of bacterial metabolic activity and spore germination via formazan formation

Despite improved sanitation protocols, foodborne illnesses caused by spore-forming bacteria remain a persistent concern. In this study, we developed a method to visualize bacterial metabolic activity via dark-field microscopy by exploiting the optical properties of formazan particles generated from the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by intracellular cofactors. Escherichia coli was used as a model organism to track formazan formation, which became pronounced following cell division, showing a strong correlation with metabolic activity. The method was further applied to various bacterial species with diverse respiratory and fermentation characteristics to evaluate the influence of metabolic profiles on formazan formation. Moreover, Bacillus subtilis spores were employed to investigate metabolic changes during germination and outgrowth. Time-lapse imaging revealed stage-specific formazan production, demonstrating the potential of this technique to monitor the metabolic reactivation process non-destructively. These findings suggest that MTT-based optical monitoring can serve as a valuable tool for assessing bacterial viability and spore germination dynamics, with potential applications in food safety and microbial risk control.