Measuring viability of dinoflagellate cysts and diatoms with stains to test the efficiency of facsimile treatments possibly applicable to ships’ ballast water and sediment

Expansion of harmful algal bloom (HAB) species through ships’ ballast water and sediment has been an increasing concern. Determining whether a microalgal cell, particularly for the toxic and HAB-forming species, is “viable” or “dead” is fundamental to understanding the effectiveness of the many ballast-water treatments that have been considered. To this end, we screened a variety of stains to assess the viability of dinoflagellate (Gymnodinium catenatum, GC) cysts and diatom (Corethron hystrix) vegetative cells to test the efficiency of ballast water treatments. Results showed that the stains fluorescing red or green are not sound candidates for viability measurements due to the interference of chlorophyll-induced red fluorescence or cytosolic green autofluorescence, while the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is limited by its toxicity, pseudo-positive judgment and the consequent confusion between cysts and vegetative cells. We further demonstrated that the stain Neutral Red (NR) is a sound candidate as the “vital stain” and can be easily applied for functionally defining the viability of both dinoflagellate cysts and diatoms. Another stain, the Evans Blue (EB), could be used as a “mortal stain” for the vegetative diatom cells but not a sensitive indicator of viability for GC cysts. The NR staining for GC cysts generally needs a higher dosage (0.005%) and longer staining time (24 h) than that were used for staining zooplankton, diatoms, and vegetative cells of dinoflagellates. In all cases, EB staining defined a “percentage of viable cells” significantly higher than that defined by NR. We conclude that the viability of a population is highly dependent on the species of stains used thus must be referred as a method-defined indicator.