Quantification of whole-cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells

Abstract

The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis.