FLOW SORTING OF MICROORGANISMS FOR MOLECULAR ANALYSIS

Not only classical cultivation-based methods but also the new molecula r approaches may result in incomplete and selective information on the natural diversity of microbial communities. Flow sorting of microorga nisms from environmental samples allows the deliberate selection of ce ll populations of interest from highly diverse systems for molecular a nalysis. Several cellular parameters that can be measured by flow cyto metry are useful as sori criteria. Here, we report sorting of bacteria from activated sludge, lake water, and lake sediment according to dif ferences iri light scattering, DNA content, and/or affiliation to cert ain phylogenetic groups as assessed by fluorescein-labeled, rRNA-targe ted oligonucleotide probes. Microscopy of the sorted cells showed that populations of originally low abundance could be strongly enriched by flow sorting (up to 280-fold), depending on the original abundance of the cells of interest and the type of sample sorted. The purity of th e cells of interest could be further increased by repeated sorting, bu t this increase was limited by cell aggregation in the case of activat ed-sludge samples. It was possible to amplify almost full-length 16S r ibosomal DNA (rDNA) fragments from sorted microbial cells by PCR, even after fixation with paraformaldehyde and in situ hybridization. Dot b lot hybridization and sequencing demonstrated that most of the amplifi ed rDNA originated from those cells that had been selected for by flow sorting. Comparative analysis of 16S rDNA sequences revealed previous ly unknown species of magnetotactic or activated-sludge bacteria.