Dead or alive? Viability assessment of micro- and mesoplankton

The rapid and efficient analysis of plankton samples (e.g. enumeration, identification, biomass determination) has been an important driver for recent technological developments in (semi-) automated analysis and imaging instruments. Most focus has been on identification and abundance estimates, while less attention has been given to viability, i.e. the assessment of whether the organisms are dead or alive. However, a wide spectrum of scientific applications requires accurate viability determinations, e.g. the monitoring of invasive species in ship ballast water. The transfer of species through ballast water forms a major threat to marine ecosystems, resulting in significant environmental and economic losses. A variety of viability stains and viability assessment methods are available, but there has been no systematic investigation how these methods perform for larger organisms (epsilon 50 m). We review the current procedures for viability determination for large plankton and present a cross-comparison of three methods: cell digestion assay (CDA), SYTOX Green nucleic acid staining and Neutral Red vital staining. The CDA and SYTOX Green methods did not perform well and gave various problems linked to the multicellular nature of zooplankton, autofluorescence and/or constraints set by the definition of cell death. Although some issues remain and there is no universal method, the Neutral Red vital stain proved the most robust viability method in this study and is broadly applicable to both phytoplankton and zooplankton larger than 50 m.