A double-staining automated flow cytometry method for real-time monitoring of bacteria in continuous bioreactors

In industrial biotechnology, cell density is a critical parameter that influences key process control variables such as feed rate, harvest timing, and product recovery. Traditional biomass measurements are indirect, incapable of quantifying population heterogeneity, and often prone to inaccuracies due to manual handling and interference from culture media compounds. This study introduces an automated flow cytometry approach to enable continuous, real-time monitoring of bacterial cultures in a continuous bioreactor. Our method employs a double-staining protocol that combines DAPI for assessing total DNA content with Alexa Fluor 488 via Click-iT technology to detect the percentage of cells with active DNA replication through EdU incorporation. This streamlined protocol, which integrates cell fixation, permeabilization, staining, and measurement, was applied to three Gram-negative strains: Bradyrhizobium sp. , Escherichia coli , and Stenotrophomonas rhizophila . The approach successfully captured both growth dynamics and cell cycle progression, providing rapid and quantitative insights into culture composition. The enhanced temporal resolution achieved by this method facilitates timely adjustments of process parameters, ultimately improving bioprocess efficiency and product quality. These results underscore the potential of automated flow cytometry as a powerful tool for real-time monitoring and control in industrial biotechnology.