Dual staining with CFDA-AM and SYTOX Blue in flow cytometry analysis of UV-irradiated Tetraselmis suecica to evaluate vitality
2016
Olsen, Ranveig Ottøy | Hess-Erga, Ole-Kristian | Larsen, Aud | Hoffmann, Friederike | Thuestad, Gunnar | Hoell, Ingunn Alne
After disinfection of ballast water, it is crucial to detect organisms and determine their vitality to assess the performance of the chosen treatment technique. Ultraviolet (UV) irradiation is a treatment technology commonly used for water disinfection. In this study, the phytoplankter Tetraselmis suecica was UV irradiated and subsequently stained with both 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and SYTOX Blue, staining metabolically active and membrane-permeable cells, respectively. This dual staining protocol can be used to assess samples during type approval of UV-based treatment systems. Non-irradiated and UV-irradiated samples were incubated in darkness, to simulate a ballast water transport, after which the vitality and viability T. suecica were monitored regularly over a period of 15 d. Flow cytometry (FCM) analysis separated the cells into 4 FCM populations (=single cells grouped together based on their fluorescence signals) according to differences in esterase activity and membrane integrity. UV-irradiated samples followed a different staining pattern compared to non-irradiated samples, where 1 specific FCM population of cells expressed esterase activity, but at the same time gave signals for disrupted membranes. This is useful as a sign of future death and is interpreted as an ‘early warning’ FCM population. FCM results were also compared to corresponding plate count results, differentiating vital, viable cells from vital, non-viable cells. We argue that dual staining with SYTOX Blue and CFDA-AM facilitates and improves FCM analysis when evaluating the performance of UV-based water treatment systems.
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