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Identification and quantification of toxic and nontoxic strains of the harmful dinoflagellate Alexandrium tamarense using fluorescence in situ hybridization and flow cytometry

Lisa K. Eckford-Soper, Keith Davidson, Eileen Bresnan

Limnol. Oceanogr. Methods 11:540-548 (2013) | DOI: 10.4319/lom.2013.11.540

ABSTRACT: The co-occurrence of morphologically identical toxic and nontoxic ribotypes of the biotoxin producing marine dinoflagellate Alexandrium tamarense presents a significant problem for its identification and enumeration, particularly in a regulatory monitoring context. To address this, we have developed a fluorescence in situ hybridization-flow cytometry (FISH-FC)-based method of cell identification and enumeration. This employed the taxa specific oligonucleotide probes TamToxC and TamA to fluorescently label (with the fluorochromes CY.3 and FITC) Group I (toxic) and Group III (nontoxic) A. tamarense ribotypes, respectively. Detection was by fluorescence activated flow cytometric analysis. The FISH-FC method allowed effective discrimination between laboratory cultures of Group I and Group III ribotypes, with toxic and nontoxic cells creating distinct, easily identifiable, clusters in a flow cytometer bi-plot of side scatter (SSC) versus the green (FL1) fluorescence detection channel. Comparison of estimates of cell abundance obtained by the FISH-FC technique with those obtained by microscopy (Sedgwick Rafter technique) showed no statistically significant difference across a range of concentrations. Subsequently, the methodology was successfully applied on natural seawater samples, spiked with known concentrations of toxic and nontoxic A. tamarense cells at environmentally relevant concentrations.