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Applied and Environmental Microbiology, September 2003, p. 5519-5529, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5519-5529.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Quantitative Assessment of Picoeukaryotes in the Natural Environment by Using Taxon-Specific Oligonucleotide Probes in Association with Tyramide Signal Amplification-Fluorescence In Situ Hybridization and Flow Cytometry

Isabelle C. Biegala, Fabrice Not, Daniel Vaulot,* and Nathalie Simon

Station Biologique de Roscoff, UMR 7127 et CNRS, Université Pierre et Marie Curie, F-29682 Roscoff cedex, France

Received 26 December 2002/ Accepted 1 May 2003

Picoeukaryotes (cells of <3 µm in diameter) contribute significantly to marine plankton biomass and productivity, and recently molecular studies have brought to light their wide diversity. Among the methods that have been used so far to quantify aquatic microorganisms, fluorescence in situ hybridization of oligonucleotide probes combined with flow cytometry offers the advantages of both high resolution for taxonomic identification and automated cell counting. However, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for routine application of this combination of techniques. We developed a new protocol associating tyramide signal amplification-fluorescence in situ hybridization and flow cytometry, which allows the detection of picoeukaryotes in cultures during both the exponential and stationary phases. The use of surfactant and sonication proved to be essential for the detection and quantification of picoeukaryotes from the natural environment, with as little as a few tenths of a milliliter of 3-µm-pore-size prefiltered sea water. The routine application of the technique was tested along a coastal transect off Brittany (France), where the different groups of picoeukaryotes were investigated using already published specific probes and a newly designed probe that targets the order Mamiellales (Prasinophyceae, Chlorophyta). Among the picoeukaryotes, Mamiellales outnumbered by 1 order of magnitude both the cyanobacteria and the non-Chlorophyta, which were represented mainly by the Pelagophyceae class. Picoeukaryote abundance increased from open toward more estuarine water, probably following changes in water temperature and stability.

* Corresponding author. Mailing address: Station Biologique de Roscoff, BP 74, F-29682 Roscoff cedex, France. Phone: 33 (2) 98 29 23 23. Fax: 33 (2) 98 29 23 24. E-mail: vaulot{at}sb-roscoff.fr.

Applied and Environmental Microbiology, September 2003, p. 5519-5529, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5519-5529.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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