Response of online flow cytometry to controlled physicochemical disturbances in desalinated potable water matrices

Online flow cytometry (FCM) has emerged as a promising technology for real-time monitoring of microbial water quality in potable and non-potable water distribution systems. However, systematic calibration and validation of online FCM measurements under different disturbances remain limited, particularly with respect to distinguishing true microbial responses from measurement artifacts. In this study, the response of an online FCM platform was evaluated under different potential disturbances in desalinated potable water and reclaimed non-potable water matrices. Online and offline FCM showed strong agreement in intact cell count (ICC) under baseline recirculation with no statistically significant differences observed (p > 0.05), providing a reference state for calibration. Controlled spiking of defined concentration NaCl and Fe2O3 resulted in physicochemical responses, but ICC remained within +/- 7% of baseline values. In contrast, humic acid and citric acid both reduced ICC and shifted fluorescence distributions, although via distinct mechanisms: humic acid primarily interfered with fluorescence detection, whereas citric acid induced cell lysis. This study demonstrates broad online and offline FCM comparability under baseline and typical water quality disturbance conditions. It also identified humic acid interference can bias ICC via fluorescence and background effects, underscoring the need for specific gating and orthogonal validation in complex water matrices.