APPLICATION OF RIBOSOMAL-RNA-BASED PROBES FOR OBSERVING MARINE NANOPLANKTONIC PROTISTS

The use of small-subunit rRNA-based oligonucleotides as probes for det ecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hy bridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhance d by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and s tationary growth phases. Mixed assemblages of heterotrophic protists f rom enrichment cultures were also intensely labeled by rRNA-targeted o ligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists th at were otherwise undetectable when hybridized with fluorescein-labele d probes were easily visualized by this approach. In the latter sample s, hybridization with multiple, biotin-labeled probes was necessary fo r detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin s ystem should increase the utility of rRNA-targeted probes for identify ing protists and facilitate characterization of the population structu re and distribution of protists in aquatic environments.