Determining the viability of marine protists using a combination of vital, fluorescent stains

Determining the viability of protists and small microzooplankton has long been a focus of studies in marine biology and ecology. It is especially relevant in the issue of shipborne invasive species, and impending international guidelines and various national regulations on the allowable concentrations of organisms in discharged ballast water have spurred the growth of an industry that develops and manufactures ballast water management systems. The success of management systems and ability of ships to meet ballast water discharge standards is determined by the number of viable organisms in treated water. Here, we propose combining two vital, fluorescent stains (fluorescein diacetate FDA] and 5-chloromethylfluorescein diacetate CMFDA]) with direct microscopic observation to enumerate viable organisms ≥10 and <50 μm in minimum dimension (nominally protists). This approach was validated in four locations in the United States to determine the efficacy of the stains. Although the accuracy of the stains varied by geographic location and the taxonomic composition of the planktonic assemblage, combining fluorescent stains is a robust, powerful tool that can be optimized for the species present at each location. While this method was developed for analyzing viable organisms in treated ballast water, it may also be used or adapted for any field of research that examines a broad taxonomic range of autotrophic and heterotrophic plankton.