PHYLOGENETIC IDENTIFICATION AND IN-SITU DETECTION OF INDIVIDUAL MICROBIAL-CELLS WITHOUT CULTIVATION

The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of seve ral indications that we currently know only a minor part of the divers ity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to d etect specific rRNA sequences of uncultured bacteria in natural sample s and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging f rom simple two-component bacterial endosymbiotic associations to multi species enrichments containing magnetotactic bacteria to highly comple x marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals ifs closest cultu rable relatives and may, together with information on the physicochemi cal conditions of its natural habitat, facilitate more directed cultiv ation attempts. For the analysis of complex communities such as multis pecies biofilms and activated-sludge flocs, a different approach has p roven advantageous. Sets of probes specific to different taxonomic lev els are applied consecutively beginning with the more general and endi ng with the more specific (a hierarchical top-to-bottom approach), the reby generating increasingly precise information on the structure of t he community. Not only do rRNA-targeted whole-cell hybridizations yiel d data on cell morphology, specific cell counts, and in situ distribut ions of defined phylogenetic groups, but also the strength of the hybr idization signal reflects the cellular rRNA content of individual cell s. From the signal strength conferred by a specific probe, in situ gro wth rates and activities of individual cells might be estimated for kn own species. In many ecosystems, low cellular rRNA content and/or limi ted cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to cir cumvent these problems are discussed in detail.