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Applied and Environmental Microbiology, March 1999, p. 1289-1297, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Combination of Fluorescent In Situ Hybridization and Microautoradiography---a New Tool for Structure-Function Analyses in Microbial Ecology

Natuscka Lee,1 Per Halkjær Nielsen,2 Kjær Holm Andreasen,3 Stefan Juretschko,1 Jeppe Lund Nielsen,2 Karl-Heinz Schleifer,1 and Michael Wagner1,*

Lehrstuhl für Mikrobiologie, Technische Universität München, D-80290 Munich, Germany,1 and Environmental Engineering Laboratory, Aalborg University, DK-9000 Aalborg,2 and Krüger A/S, DK-2860 Søborg,3 Denmark

Received 19 October 1998/Accepted 8 December 1998

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 33Pi) 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.

* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie, Technische Universität München, D-80290 Munich, Germany. Phone: 49 89 2892 2373. Fax: 49 89 2892 2360. E-mail: wagner{at}mikro.biologie.tu-muenchen.de.

Applied and Environmental Microbiology, March 1999, p. 1289-1297, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.