Aquatic Sciences Meeting, Albuquerque 2001

PC01 Harmful Algal Blooms

Date: Tuesday, February 13, 2001

Location: Southwest Hall

Parrow, M, W, North Carolina State University, Raleigh, NC, USA, mwparrow@unity.ncsu.edu

Glasgow, H, J, North Carolina State University, Raleigh, NC, USA, howard_glasgow@ncsu.edu

Burkholder, J, M, North Carolina State University, Raleigh, NC, USA, joann_burkholder@ncsu.edu

FLOW CYTOMETRIC DETECTION OF PFIESTERIA SPP. AND A CRYPTOPERIDINIOPSOID SPECIES USING rRNA-TARGETED FLUORESCENT OLIGONUCLEOTIDE PROBES

We have developed fluorescent in situ hybridization en suspension (FISHES) techniques with flow cytomertry for species-level detection and enumeration of Pfiesteria piscicida, P. shumwayae, and an as-yet-unnamed cryptoperidiniopsoid species in laboratory cultures and field samples. Species-specific oligonucleotides probes directed against 18S rRNA sequences were developed and ligated to three different fluorophores. The probes first were used for visual taxonomic identification and enumeration of Pfiesteria spp. and the cryptoperidiniopsoid in culture and field samples under epifluorescence microscopy. The hybridization procedure was then modified to allow conjunctive flow cytometric analyses. Probed culture volumes were analyzed on a multi-laser research grade COUL-TER® EPICS® ALTRATM flow sorter. Simultaneous 3-color taxonomic identification and enumeration required 350 and 488 nm laser emissions with electronic-gated signal integration and fluorescence compensation, to optimize signal separation and minimize spectral overlap. Flow cytometric population measurements of probed cultures corre-lated well with microscopic cell counts. Probed cells are now routinely sorted for visual corroboration. Application of these techniques to field samples has yielded promising results, demonstrating the potential for flow cytometry taxonomic monitoring of phytoplankton from field samples.

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Aquatic Sciences Meeting, Albuquerque 2001

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