AB 25:39-52 (2016)  -  DOI: https://doi.org/10.3354/ab00662

Dual staining with CFDA-AM and SYTOX Blue in flow cytometry analysis of UV-irradiated Tetraselmis suecica to evaluate vitality

Ranveig Ottoey Olsen1, Ole-Kristian Hess-Erga2, Aud Larsen3, Friederike Hoffmann4,5, Gunnar Thuestad1, Ingunn Alne Hoell1,*

1Stord/Haugesund University College, Bjoernsonsgt. 45, 5528 Haugesund, Norway
2Norwegian Institute for Water Research, Thormoehlensgt. 53 D, 5006 Bergen, Norway
3Uni Research Environment and Hjort Centre for Marine Ecosystem Dynamics, 5006 Bergen, Norway
4Uni Research Environment, Thormoehlensgt. 49 B, 5006 Bergen, Norway
5University of Bergen, PO Box 7800, 5020 Bergen, Norway
*Corresponding author:

ABSTRACT: 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.


KEY WORDS: Phytoplankton · Ballast water · Water treatment · Live/dead analysis · Viability · Water analysis


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Cite this article as: Olsen RO, Hess-Erga OK, Larsen A, Hoffmann F, Thuestad G, Hoell IA (2016) Dual staining with CFDA-AM and SYTOX Blue in flow cytometry analysis of UV-irradiated Tetraselmis suecica to evaluate vitality. Aquat Biol 25:39-52. https://doi.org/10.3354/ab00662

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