DEVELOPMENT AND FIELD APPLICATION OF A QUANTITATIVE METHOD FOR EXAMINING NATURAL ASSEMBLAGES OF PROTISTS WITH OLIGONUCLEOTIDE PROBES

A fluorescent in situ hybridization method that uses rRNA-targeted oli gonucleotide probes for counting protists in cultures and environmenta l water samples is described. Filtration, hybridization, and enumerati on of fixed cells with biotinylated eukaryote-specific probes and fluo rescein isothiocyanate-conjugated avidin were performed directly on 0. 4-mu m-pore-size polycarbonate filters of Transwell cell culture inser ts (Costar Corp., Cambridge, Mass.). Counts of various species of cult ured protists by this probe hybridization method were not significantl y different from counts obtained by the 4',6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) staining methods. However, counts of t otal nanoplankton (TNAN) based on probe hybridizations in several fiel d samples and in samples collected from a mesocosm experiment were fre quently higher than TNAN counts obtained by staining with DAPI or AO. On the basis of these results, 25 to 70% of the TNAN determined with p robes were not detectable by DAPI or AO staining. The underestimation of TNAN abundances in samples stained with DAPI or AO was attributed t o the existence of small nanoplanktonic cells which could be detected with probes but not DAPI or AO and the difficulty associated with dist inguishing DAPI- or AO-stained protists attached to or embedded in agg regates. We conclude from samples examined in this study that enumerat ion of TNAN with oligonucleotide probes provides estimates of natural TNAN abundances that are at least as high as (and in some cases higher than) counts obtained with commonly employed fluorochrome stains. The quantitative in situ hybridization method we have described here enab les the direct enumeration of free-living protists in water samples wi th oligonucleotide probes. When combined with species-specific probes, this method will enable quantitative studies of the abundance and dis tribution of specific protistan taxa.