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Applied and Environmental Microbiology, August 2004, p. 4748-4755, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4748-4755.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

New Strategies for Cultivation and Detection of Previously Uncultured Microbes

Bradley S. Stevenson,* Stephanie A. Eichorst, John T. Wertz, Thomas M. Schmidt, and John A. Breznak

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan

Received 16 February 2004/ Accepted 14 April 2004

An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O2 [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO2 (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO2. A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Michigan State University, 2209 Biomedical Physical Sciences, East Lansing, MI 48824-4320. Phone: (517) 355-6463, ext. 1608. Fax: (517) 353-8957. E-mail: steven77{at}msu.edu.

Applied and Environmental Microbiology, August 2004, p. 4748-4755, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4748-4755.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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