Peracetic acid mode-of-action on aquaculture microbes evaluated by dual-staining flow cytometry

Flow cytometry with dual staining allows an exact quantification of live and dead cells. It is a potential tool for gaining new information on the mode-of-action of disinfection chemicals and supporting efficient microbial control in recirculating aquaculture systems (RAS). Here, we used a dual-staining flow cytometry to examine the disinfection efficiency of peracetic acid (PAA) against i) the fish pathogenic bacterium Aeromonas salmonicida and ii) on microbes present in RAS. The method allowed us to accurately quantify the A. salmonicida cell response to different PAA concentrations (1, 2, and 10 mg/L) in time and to calculate the PAA concentration needed to achieve 95% cell mortality (LC95 estimates). With the high PAA dose of 10 mg/L, LC95 was reached in <15 min, while with the intermediate dose of 2 mg/L, LC95 was reached only in the end of the 60 min trial and with the low 1 mg/L dose, the disinfection efficiency remained low. The addition of soluble organic matter increased the PAA decay and decreased the disinfection efficiency. In RAS water, disinfection efficiency increased with increasing PAA concentration (from 1 to 10 mg/L) but was significantly reduced with increasing organic matter concentration (TCOD from 7.6 to 30.3 mg O₂/L). The increase in the proportion of dead cells was found to be a reliable proxy for disinfection efficiency in RAS water. In the pure cell culture, both the increase in the proportion of dead cells and the fraction of reduced live cells reflected the disinfection efficiency of PAA. Altogether, the study demonstrated the PAA mode-of-action on aquaculture microbes and showed that flow cytometry with dual staining allows fast and precise examination of microbial viability under different conditions.