QUANTITATIVE FLOW CYTOMETRIC DETECTION OF SPECIFIC MICROORGANISMS IN SOIL SAMPLES USING RIBOSOMAL-RNA-TARGETED FLUORESCENT-PROBES AND ETHIDIUM-BROMIDE

Specific detection and accurate enumeration of microorganisms in the e nvironment have been hampered by the lack of suitable techniques, A th ree-parameter flow cytometric method (FCM) was developed to detect qua ntitatively 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 fluor escence in situ hybridization against the 16S rRNA (i.e., detection sp ecificity), 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 allo w oligonucleotide probes to enter the bacterial cells and, second, to achieve clear discrimination of fixed cells from debris in soil sample s. This method allowed effective qualitative and quantitative analysis by fluorescence in situ hybridization. The results showed that the de tection threshold by FCM was 3 x 10(4) cells/g of dry soil. Cell count s deduced from FCM analysis mere similar to those obtained by the colo ny forming unit assay when soils contained fewer than 3 x 10(6) cells/ g dry soil, This method should be useful for either quantitative monit oring of microorganisms inoculated in contaminated soil samples during bioremediation or detecting known bacterial strains in environmental samples. (C) Wiley-Liss, Inc.