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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
Author(s) -
EckfordSoper Lisa K.,
Davidson Keith,
Bresnan Eileen
Publication year - 2013
Publication title -
limnology and oceanography: methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.898
H-Index - 72
ISSN - 1541-5856
DOI - 10.4319/lom.2013.11.540
Subject(s) - alexandrium tamarense , biology , flow cytometry , dinoflagellate , fluorescence in situ hybridization , context (archaeology) , enumeration , microbiology and biotechnology , chromatography , chemistry , algal bloom , botany , biochemistry , gene , ecology , phytoplankton , chromosome , paleontology , mathematics , combinatorics , nutrient
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.