The use of flow cytometry and oligonucleotide probes for bacterial community structure analysis in biological wastewater treatment systems

Abstract

Investigation of microbial community structure in activated sludge is often complicated. When not only total bacterial counts, but also counts of bacterial subpopulations are to be determined, existing methods are not successful. In the present article, a method for analysis of activated sludge with two, differently labelled oligonucleotide probes and affiliated flow cytometric detectors is described. Oligonucleotide probes EUB338-Cy3 and fD1-FITC, specific for all bacteria were used to set the experimental model for flow cytometry analysis. This model was than used to analyze activated sludge sample with 16S rRNA targeted probe NSO190-FITC, specific for amonia oxidizing bacteria from b - subclass of Proteobacteria. In the examined sample 16.3% of bacteria hibridized with this probe. Finally, effects of tripan blue on cell autofluorescence and probe-specific fluorecence were investigated. Tripan blue did not alter the FITC-probe-specific fluorescence, while the level of autofluorescence and nonspecific fluorescence dropped.