In situ identification of polyphosphate-accumulating bacteria in activated sludge by dual staining with rRNA-targeted oligonucleotide probes and 4′,6-diamidino-2-phenylindol (DAPI) at a polyphosphate-probing concentration

Polyphosphate-accumulating bacteria in activated sludge samples from an anaerobic/aerobic batch-type reactor for enhanced biological phosphate removal (EBPR) were identified by dual staining with ribosomal RNA-targeted oligonucleotide probes and 4′,6-diamidino-2-phenylindol dihydrochloride (DAPI) at a polyphosphate-probing concentration. The staining with the oligonucleotide probes showed that there were four major bacterial groups in the sludge: the beta subclass of Proteobacteria, Gram-positive bacteria with a high G+C content, the alpha subclass of Proteobacteria, and bacteria belonging to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum. By staining with DAPI, many of the Gram-positive bacteria with a high G+C content and the alpha subclass of Proteobacteria fluoresced bright yellow. These bacteria were considered to accumulate a large amount of polyphosphate.