Monitoring by laser-flow-cytometry of the polycyclic aromatic hydrocarbon-degrading Sphingomonas sp strain 107 during biotreatment of a contaminated soil

A flow cytometric method (FCM) was used to detect and accurately enumerate a polycyclic aromatic hydro-carbon-degrading bacterial strain, Sphingomonas sp. 107, inoculated into a soil sample artificially contaminated with pyre ne. To compare the FCM method with colony forming unit (CFU) assays, a rifa mpicin-resistant Sphingomonas sp. 107 was obtained which could be distingui shed from the indigenous microflora, since there was no organism resistant to rifampicin in the soil that could transform indole to indigo (naphthalen e dioxygenase activity). By combining light-scattering profiles (i.e., morp hological properties), ethidium bromide influx (i.e., cell wall permeabilit y), and fluorescence in situ hybridization against the 16S rRNA (i.e., dete ction specificity), we could enumerate the bacterial population of interest from the indigenous microflora and soil debris during the biotreatment. Th e FCM technique revealed that the number of inoculated Sphingomonas cells d ecreased gradually for 15 days of incubation before reaching a steady level of 7 to 12 x 10(5) cells.g(-1) of soil. Similar values were obtained wit t he CFU assay. During this period, pyrene concentration decreased from 632 t o 26 mg.kg(-1) of dry soil. The FCM detection was improved by adding blocki ng reagent to the hybridization buffer to minimize the non-specific attachm ent of the fluorescent probe to soil particles. combined wit the improvemen ts in probe technology. FCM detection was shown to be a good alternative to the conventional culture methods for the analysis of bacterial populations in environmental samples. This technique could be potentially useful for t he detection of microorganisms that grow poorly in culture.