||Dual Staining of Natural Bacterioplankton with 4',6-Diamidino-2Phenylindole and Fluorescent Oligonucleotide Probes Targeting Kingdom-Level 16S rRNA Sequencest.
Hicks, R. E. ;
Amann, R. I. ;
Stahl, D. A. ;
||Minnesota Univ.-Duluth. Dept. of Biology. ;Illinois Univ. at Urbana-Champaign. ;Environmental Research Lab., Gulf Breeze, FL.;Office of Naval Research, Arlington, VA.;Minnesota Sea Grant Inst., St. Paul.
||ONR-N00014-88-K-0093, EPA-R-815285-01-02; EPA/600/J-92/386;
16S ribosomal RNA ;
Oligonucleotide probes ;
Fluorescent dyes ;
Nucleic acid hybridization ;
Stains and staining ;
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A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4', 6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.