Combination of rRNA-Targeted Hybridization Probes and Immuno-Probes 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 identification 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) conjugated streptavidin. Finally, they were counterstained with 4',6-diamidino-2-phenylindole (DAPI) in order to discriminate cells from background. Forward scatter, fluorescein- as well as PE- and DAPI-fluorescence were measured simultaneously for the differentiation of bacterial species by multiparameter flow cytometric analysis. The influence of detergents in the hybridization buffer on nonspecific antibody binding was evaluated. Sodium dodecyl sulfate (SDS) induced strong nonspecific staining and was, therefore, replaced by Tween 20. We found that the immunostaining steps can be performed before or after hybridization. This combination of rRNA-targeted hybridization probes and immune-probes for flow cytometry makes possible the highly specific and automated identification of micro-organisms at any desired taxonomic level.