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

In biotechnological processes, cell density and physiology are critical parameters for controlling the feed rate, harvest time, and process performance. We developed an automated flow cytometry approach that enables continuous, real-time (fully automated, hourly) monitoring of bacterial populations in continuous bioreactors. The method employed a double-staining protocol that combined DAPI to assess total DNA content and Alexa Fluor 488-EdU via Click-iT technology to identify the proportions of cells undergoing active DNA replication through EdU incorporation. The integrated workflow included fixation, permeabilization, staining, and measurement steps and was applied to three Gram-negative strains: Bradyrhizobium sp., Escherichia coli, and Stenotrophomonas rhizophila. Automated analysis captured growth dynamics and cell cycle progression, providing insights into population behavior under different dilution rates. In this study, automated on-line sampling enabled hourly flow cytometry measurements of cell concentration and physiological indicators during continuous cultivation, supporting real-time monitoring and control in industrial biotechnology.