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Aquatic Microbial Ecology

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AME 34:263-277 (2004)  -  doi:10.3354/ame034263

Counting heterotrophic nanoplanktonic protists in cultures and aquatic communities by flow cytometry

Julie M. Rose1,*, David A. Caron1, Michael E. Sieracki2, Nicole Poulton2

1Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, AHF 301, Los Angeles, California 90089-0371, USA
2Bigelow Laboratory for Ocean Sciences, PO Box 475, 180 McKown Point Road, West Boothbay Harbor, Maine 04575, USA

ABSTRACT: The food vacuole stain LysoTracker Green® was used to enumerate heterotrophic protists on a standard model flow cytometer. Appropriate stain concentration and staining time were determined using cultures of protists. Stained heterotrophic protists consistently formed distinct populations within cytograms of green fluorescence versus forward scatter. Cytometric counts of cultured species were compared to direct counts using light microscopy at cell abundances ranging from 103 to 106 cells ml-1. A regression of these data was highly significant and yielded a slope of 0.95. Stained populations were accurately counted during lag, exponential and early stationary growth phases. Growth rates calculated from cytometric counts were not statistically different from those based on microscopy. The method was applied to 26 natural plankton samples, and general region definitions on the cytograms were established that identified heterotrophic protistan assemblages. A regression of cytometric counts versus direct counts yielded a slope of 1.16. LysoTracker Green® can only be used with live samples because preservation destroys membrane potential, resulting in loss of fluorescence. However, the flow cytometric method employing LysoTracker Green® is highly applicable for monitoring the growth of many heterotrophic protists in cultures and has the potential to be extremely useful for field samples, providing comparable counts to microscopical methods while allowing much faster sample processing.

KEY WORDS: Heterotrophic protists · Flow cytometry · Nanoplankton · Fluorescent staining

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