Fluorometric observation of viable and dead adhering diatoms using TO-PRO-1 iodide and its application to the estimation of electrochemical treatment

Abstract

A rapid method for the direct measurement of viable and dead adhering diatoms was developed using a fluorescent dye, TO-PRO-1 iodide. By staining the marine diatom, Nitzchia closterium, with TO-PRO-1 iodide, viable and dead cells were identified as red and yellow cells respectively, under an epifluorescence microscope employing blue excitation. Only dead cells were stained with TO-PRO-1 iodide. Viable cells were observed as red because of autofluorescence arising from intracellular chlorophyll, whereas dead cells were observed as yellow because of the fluorescence of TO-PRO-1 iodide. The percentage of TO-PRO-1-iodide-stained was correlated with the percentage of dead cells in N. closterium cells exposed to heat (60 °C, 15 min). Other microalgae containing intracellular chlorophyll could be also distinguished as viable or dead cells by this fluorometric staining method. This method was applied for the assessment of N. closterium cells killed by the electrochemical treatment and used to monitor biofouling populations and their viability directly on the electrode surface. When 1.0 V was applied against a saturated calomel electrode, 99% of the cells attached to graphite electrode were killed in 1 h.