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Aquatic Microbial Ecology

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AME 26:157-166 (2001)  -  doi:10.3354/ame026157

Flow cytometric analysis of phytoplankton viability following viral infection

Corina P. D. Brussaard1,*, Dominique Marie2, Runar Thyrhaug1, Gunnar Bratbak1

1Department of Microbiology, University of Bergen, 5020 Bergen, Norway
2Station Biologique, CNRS, INSU et Université Pierre et Marie Curie, BP 74, 29682 Roscoff cedex, France
*Present address: Department of Biological Oceanography, Netherlands Institute for Sea Research, PO Box 59, 1790 AB Den Burg, The Netherlands. E-mail:

ABSTRACT: Two flow cytometric assays using physiological probes were used on the phytoplankton species Phaeocystis pouchetii and Micromonas pusilla to examine the assays¹ utility in detecting viral infections. Dead cells were detected using the membrane impermeant nucleic-acid dye SYTOX-Green, which stains algal cells that have lost their membrane integrity. Live cells were detected using the membrane permeant dye Calcein-AM, which is hydrolyzed by intracellular esterases into a green fluorescent charged form. We found that both assays are easy to use, are reproducible and can indeed be used as markers of the viability of individual phytoplankton cells following infection by viruses. Cell death rates up to 0.8 d-1 for P. pouchetii and 0.5 d-1 for M. pusilla were calculated. The first day postinfection, death rates determined by the Calcein-AM assay were typically twice as high as those determined by the SYTOX-Green assay. Both viability tests were found to assess the physiological status of noninfected P. pouchetii cells, independent of viral infection. The optimal choice of viability assay depended on the phytoplankton species studied. Compared with existing assays, the protocols described permit examination of infected phytoplankton in more detail, yielding insight into the heterogeneity of the algal population.

KEY WORDS: Flow cytometry · Live/dead assays · Phytoplankton · Viability · Viral infection

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