In situ detection and identification of bacteria prior to their cultivation

Karl-Heinz Schleifer, Ph.D.
Technical University Munich
Freising, Germany

Monday, August 9, 1999
12:00 Noon—Pacific Forum

Only a fraction of the bacteria that are observed in the microscope can currently be cultivated. The vast majority of microbial diversity remains to be discovered. A possible solution to this problem may be the direct analysis of microbial communities with rRNA-targeted oligonucleotide probes. We are using two approaches for the characterization of environmental samples with nucleic acid probes. For less complex communities, DNA is extracted and rRNA sequences of different microorganisms are obtained by in vitro amplification of rRNA genes and cloning of the amplificates in E. coli. Following comparative sequence analysis, sequence-specific oligonucleotide probes are designed, labeled with fluorescent markers, and used for the in situ detection and identification of whole cells in the original sample, the so-called FISH method. Using this approach we could identify unusual ectosymbionts of the ciliate Euplotidium as bacteria belonging to a recently described new phylum. These ectosymbionts contain tubulin-structures and a unique extrusion apparatus.

However, this PCR-assisted approach is too laborious for studying the microbial diversity of complex communities. Therefore, we introduced the so-called top-to-bottom approach. Nested sets of fluorescently labeled rRNA-targeted oligonucleotide probes with increasing narrow specificities are used for in situ detection and identification of microorganisms at different taxonomic levels. This approach was successfully used for studying the bacterial diversity of activated sludge. However, the phylogenetic identification does not generally provide much information about the function of the organisms. To learn more about the function of uncultured bacteria, a combination of FISH and microradioautography was applied to determine simultaneously the identity of the organisms and their specific substrate uptake profiles. This can be carried out under aerobic, anoxic, and anaerobic conditions. The technique should be helpful to identify active bacteria and to design appropriate media and conditions for isolation of hitherto uncultured microorganisms.

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