Use of Fluorescein Diacetate (FDA) as a Single-Cell Probe of Metabolic Activity in Dinoflagellate Cultures

Abstract

Our goal was to determine what percentage of cells remain metabolically active under environmentally relevant culture conditions. The approach combines epifluorescence microscopy and/or a standard bench-top fluorometer, plus the probe fluorescein diacetate (FDA). This stain is colorless in solution when added at a final concentration of 10 μM. It readily penetrates the cell membrane, and once inside the cell esterases cleave acetates to form fluorescein. Within 10 minutes, metabolically active cells become brilliantly yellow-green (fluorescence measured at 515–530 nm) while inactive cells do not fluoresce or are only weakly fluorescent. The FDA assay does not degrade the chlorophyll a fluorescence signal (>675 nm) nor does it interfere with normal motility or carbon uptake in most instances. Thirty clones of dinoflagellates were examined in this study. We found this assay useful to (1) optimize culture conditions and transfer schedules; (2) evaluate trauma of transfer into new media; (3) test for survivability in harsh environments, such as survival in prolonged darkness; (4) identify and study temporary resting stages; (5) detect the presence and abundance of bacteria in dinoflagellate cultures, and (6) evaluate metabolic vigor of cells of a population collected in nature in attempts to establish clonal cultures.

Keywords:

• Fluorescein diacetate
• dinoflagellate
• culture
• epifluorescence microscopy stain
• bacteria
• metabolic activity