Rapid and universal quantification of viable bacteria with growth activity in raw milk using a fluorescent d-amino acid-flow cytometry (FDAA-FCM) method

The plate counting (PC) method is commonly used to determine the total bacterial count (TBC), which includes all viable bacterial cells displaying growth activity. However, this method is time-consuming and may not effectively identify all actively growing bacteria, and it remains largely unknown whether alternative methods can rapidly and universally quantify the TBC of growth-active bacteria. In this study, we investigated the use of fluorescent d-amino acid (FDAA)-based labeling, which universally targets viable bacteria, in conjunction with typical bacterial species found in milk. Furthermore, we established an antibiotic cocktail to impede cell division during the FDAA labeling process. We optimized the FDAA labeling and cell division inhibition conditions to integrate with flow cytometry (FCM), enabling the rapid quantification of viable bacteria with growth activity. Ultimately, we successfully demonstrated the universal quantification of viable bacteria in real milk samples with a TBC higher than 8.9 × 104 cells/mL within a timeframe of 2.5 h using the FDAA-FCM method. These findings suggest that the FDAA-FCM method holds great promise for quantifying TBC in complex biological samples, offering a rapid and universally applicable approach.