Flow cytometric analysis of activated sludge with rRNA-targeted probes

Samples from a wastewater treatment plant were hybridized with fluorescein-labeled oligonucleotide probes specific for members of the domains Bacteria and Eucarya; the alpha, beta, and gamma subclasses of the class Proteobacteria; or the genus Acinetobacter. Subsequently, they were counterstained with the DNA-specific dye Hoechst 33342 and analyzed by flow cytometry. By quantifying forward angle light scatter and Hoechst- and probe-conferred fluorescence as measures for cell size, DNA content, and rRNA content, respectively, not only relative abundances but also assessments of general metabolic activity for each of these groups were obtained. Hybridizations with a positive control probe binding to all bacteria showed that in the activated-sludge samples examined, 70 to 80% of the Hoechst-stained cells could unambiguously be identified by this method. The majority of the detected cells (approximately 40%) were beta-subclass Proteobacteria. Flow cytometric and microscopic counts were in general agreement. Discrepancies were found in particular for those populations that occurred predominantly in flocs (alpha subclass of the Proteobacteria) or chains (Acinetobacter spp.). Although the dispersal of aggregates needs to be improved, flow cytometry combined with rRNA-based in situ probing appears to be a powerful tool for the rapid and highly automated analysis of the microbial communities in activated sludge.