COMBINATION OF RIBOSOMAL-RNA-TARGETED HYBRIDIZATION PROBES AND IMMUNOPROBES FOR THE IDENTIFICATION OF BACTERIA BY FLOW-CYTOMETRY

Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were for the first time combined for the flow cytometric identificatio n of bacteria. Artificial mixtures of fixed cells were hybridized with fluorescein-labeled, rRNA-targeted oligonucleotide probes and stained indirectly with biotinylated antibodies and R-phycoerythrin (PE) conj ugated streptavidin. Finally, they were counterstained with 4',6-diami dino-2-phenylindole (DAPI) in order to discriminate cells from backgro und. Forward scatter, fluorescein- as well as PE- and DAPI-fluorescenc e were measured simultaneously for the differentiation of bacterial sp ecies by multiparameter flow cytometric analysis. The influence of det ergents in the hybridization buffer on nonspecific antibody binding wa s evaluated. Sodium dodecyl sulfate (SDS) induced strong nonspecific s taining and was, therefore, replaced by Tween 20. We found that the im munostaining steps can be performed before or after hybridization. Thi s combination of rRNA-targeted hybridization probes and immune-probes for flow cytometry makes possible the highly specific and automated id entification of micro-organisms at any desired taxonomic level.