Quantitative flow cytometric detection of specific microorganisms in soil samples using rRNA targeted fluorescent probes and ethidium bromide.

Abstract

Specific detection and accurate enumeration of microorganisms in the environment have been hampered by the lack of suitable techniques. A three-parameter flow cytometric method (FCM) was developed to detect quantitatively Sphingomonas sp. strain 107 inoculated into soil samples. By combining light scattering profiles (i.e., morphological properties), ethidium bromide (EtBr) influx (i.e., wall permeability), and fluorescence in situ hybridization against the 16S rRNA (i.e., detection specificity), we could accurately discriminate the bacterium of interest from the indigenous microflora and soil debris. EtBr was used, first, to determine the optimal cell wall permeabilization treatment to allow oligonucleotide probes to enter the bacterial cells and, second, to achieve clear discrimination of fixed cells from debris in soil samples. This method allowed effective qualitative and quantitative analysis by fluorescence in situ hybridization. The results showed that the detection threshold by FCM was 3 x 10(4) cells/g of dry soil. Cell counts deduced from FCM analysis were similar to those obtained by the colony forming unit assay when soils contained fewer than 3 x 106 cells/g dry soil. This method should be useful for either quantitative monitoring of microorganisms inoculated in contaminated soil samples during bioremediation or detecting known bacterial strains in environmental samples.