Quantification of bacterial populations in complex ecosystems using fluorescent in situ hybridization, confocal laser scanning microscopy and image analysis

A procedure for quantification of distinct bacterial populations in aggre-gated ecosystems was developed. It is based on fluorescent in situ hybridization coupled with confocal-laser-scanning microscopy and surface measurement of hybridized bacteria by image analysis. The proportion of a targeted bacterial species was ob-tained by comparison between the surface hybridized by a specific probe and the surface hybridized with a general bacterial reference probe. The accuracy of the re-sults obtained was evaluated by direct visual counting on the same sets of images. The analytical uncertainty of the image analysis procedure was determined and de-pended on the threshold values selected by the operator for cutting between signal and background fluorescence. The number of fields to be analyzed for reliable quantification was also determined. This procedure allows a quantification of the proportions of bacterial species in aggregated bacterial ecosystems which gives more accurate re-sults than visual counting because of the large number of bacteria counted. Moreover, since floc structures are preserved, it also gives information about the distribution of bacterial populations in the floc material which might reveal bacterial interactions within the community.