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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Food Processing and Sensory Quality Research » Research » Publications at this Location » Publication #236464
Title: Viable cell sorting of dinoflagellates by multi-parametric flow cytometry.

Author
item SINIGALLIANO, CHRISTOPHER - FLORIDA INT. UNIV.
item WINSHELL, JAMIE - FLORIDA INT. UNIV.
item GUERRERO, MARIA - FLORIDA INT. UNIV
item SCORZETTI, GLORIA - UNIV. OF MIAMI
item FELL, JACK - UNIV. OF MIAMI
item Eaton, Richard
item BRAND, LARRY - UNIV. OF MIAMI
item REIN, KATHLEEN - FLORIDA INT. UNIV.

Submitted to: Phycologia Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/26/2009
Publication Date: 7/1/2009
Citation: Sinigalliano, C.D., Winshell, J., Guerrero, M.A., Scorzetti, G., Fell, J.W., Eaton, R.W., Brand, L., Rein, K.S. 2009. Viable cell sorting of dinoflagellates by multi-parametric flow cytometry. Phycologia. 48:249-257.

Interpretive Summary: Dinoflagellates are unicellular, flagellated, often photosynthetic marine and freshwater protists. Difficulties in establishing pure cultures in the laboratory have been an impediment to the use of these marine phytoplankton for biotechnological applications such as drug discovery and gene mining. Moreover, these obstacles slow studies of the ecology, physiology, and population genetics of many dinoflagellate species. Here, a method in which electronic cell sorting is used for improved isolation of dinoflagellate strains is reported.

Technical Abstract: Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking of cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor as fragile dinoflagellates, such as Karenia brevis (Dinophyceae) survived electronic cell sorting to yield viable cells. The rate of successful isolation of large scale (> 4 l) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5% respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required re-picking as the cultures were determined not to be unialgal after a single round of isolation, whereas no cultures obtained in this study from electronic single cell sorting required re-sorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs. 54% respectively) and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual post-sort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single cell sorting has the potential to accelerate the discovery of new algal strains.