Rapid and Accurate Quantification of Viable Bacteria in Reproducibly Generated Bioaerosols with a Certified Microbial Reference Material

Bioaerosols play a pivotal role in the transmission of respiratory diseases, rendering bioprotective products essential for mitigating bioaerosol-mediated disease spread. However, current limitations in reproducible bioaerosol generation and accurate assessment of filtration efficacy significantly impede the precision and traceability of quality evaluations for bioprotective products. In this study, we aimed to develop a microbial reference material and establish a rapid quantification method for viable bacteria in bioaerosols to enable consistent bioaerosol generation and reliable quality assessment, respectively. We demonstrated that strain type, culture conditions, and bacterial growth phase significantly affect the bioaerosol generation efficiency. A self-isolated <i>Staphylococcus aureus</i> strain was selected due to its high tolerance to the stress associated with bioaerosol generation. Using this strain, we developed a microbial reference material with certified and traceable values to ensure stable and reproducible production of the bioaerosol. Furthermore, the bacterial viability within bioaerosols was accurately assessed by evaluating esterase activity through a multiparameter flow cytometry (FCM) method. Finally, a rapid and accurate quantification method for viable bacteria in bioaerosols was developed, which demonstrated excellent linear correlation with conventional methods (<i>R</i>² = 0.982) and enabled the detection of mask filtration efficacy within 35 min. Collectively, we successfully developed a microbial reference material and a complementary FCM-based method for the rapid and accurate evaluation of bioprotective product quality. Our findings significantly enhance the bioaerosol-related detection efficiency and promote the standardization of bioaerosol generation and protection efficacy assessment.