title: FLOW CYTOMETRIC APPLICABILITY OF FLUORESCENT VITALITY PROBES ON PHYTOPLANKTON1.
abstract: The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein-AM], 5-chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2',7'-dichlorofluorescein diacetate [H2DCFDA]; and two membrane probes: bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] and SYTOX-Green) as vitality stains was tested on live and killed cells of 40 phytoplankton strains in exponential and stationary growth phases, belonging to 12 classes and consisting of four cold-water, 26 temperate, and four warm-water species. The combined live/dead ratios of all six probes indicated significant differences between the 12 plankton classes (P < 0.01) and between individual species (P < 0.05). No specific differences were observed among strains of one species, among species or strains from different origin, nor between cells in exponential and stationary growth phase except for FDA. FDA showed a significant (P < 0.05) drop of <20% in fluorescence intensity in stationary cells. Of the four esterase probes, the live/dead ratios of FDA and CMFDA were not significantly different from each other, and both performed better than Calcein-AM and H2DCFDA (P < 0.001). Of the two membrane probes, DIBAC4(3) stained rhodophytes and euglenophytes much better than SYTOX-Green. The 13 algal strains best stainable (high live/dead ratios) among all six probes belonged to nine genera from six classes of phytoplankton. In conclusion, FDA, CMFDA, DIBAC4(3), and SYTOX-Green represent a wide choice of vitality probes in the study of phytoplankton ecology, applicable in many species from different algal classes, originating from different regions and at different stages of growth.
authors.affiliation: Department of Biological Oceanography, Royal Netherlands Institute for Sea Research/NIOZ, P.O. Box 59, NL-1790 AB Den Burg, the Netherlands.
authors.forename: Louis
authors.initials: L
authors.lastname: Peperzak
authors.affiliation: Department of Biological Oceanography, Royal Netherlands Institute for Sea Research/NIOZ, P.O. Box 59, NL-1790 AB Den Burg, the Netherlands.
authors.forename: Corina P D
authors.initials: CP
authors.lastname: Brussaard
keywords: CMFDA (CellTracker Green); Calcein-AM; DiBAC4(3); FDA; H2DCFDA; SYTOX-GREEN; flow cytometry; live/dead assay; phytoplankton; vitality