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Appl. Environ. Microbiol., Mar 1997, 938-944, Vol 63, No. 3
Copyright © 1997, American Society for Microbiology

Flow Cytometric Analysis of Characteristics of Hybridization of Species-Specific Fluorescent Oligonucleotide Probes to rRNA of Marine Nanoflagellates

J Rice, MA Sleigh, PH Burkill, GA Tarran, CD O'Connor and MV Zubkov
Departments of Biology and Biochemistry, School of Biological Sciences, University of Southampton, Southampton SO16 7PX, and Plymouth Marine Laboratory, Plymouth PL1 3DH, United Kingdom

Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples. Flow cytometry also permits measurement of intensity of probe binding by cells. Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate. Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase; cell size changed in a comparable manner. Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved.

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