Combination of fluorescent in situ hybridization and microautoradiography - A new tool for structure-function analyses in microbial ecology

Natuscka Lee, Per Halkjær Nielsen, Kjær Holm Andreasen, Stefan Juretschko, Jeppe Lund Nielsen, Karl Heinz Schleifer, Michael Wagner

Research output: Contribution to journalArticlepeer-review

518 Scopus citations

Abstract

A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA- targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli and Herpetosiphon aurantiacus under aerobic incubation conditions with added [3H] glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33P(i)) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.

Original languageEnglish
Pages (from-to)1289-1297
Number of pages9
JournalApplied and Environmental Microbiology
Volume65
Issue number3
DOIs
StatePublished - Mar 1999

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