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Applied and Environmental Microbiology
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Microbial Ecology

New Strategies for Cultivation and Detection of Previously Uncultured Microbes

Bradley S. Stevenson, Stephanie A. Eichorst, John T. Wertz, Thomas M. Schmidt, John A. Breznak
Bradley S. Stevenson
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
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  • For correspondence: steven77@msu.edu
Stephanie A. Eichorst
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
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John T. Wertz
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
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Thomas M. Schmidt
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
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John A. Breznak
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan
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DOI: 10.1128/AEM.70.8.4748-4755.2004
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ABSTRACT

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.

  • Copyright © 2004 American Society for Microbiology
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New Strategies for Cultivation and Detection of Previously Uncultured Microbes
Bradley S. Stevenson, Stephanie A. Eichorst, John T. Wertz, Thomas M. Schmidt, John A. Breznak
Applied and Environmental Microbiology Aug 2004, 70 (8) 4748-4755; DOI: 10.1128/AEM.70.8.4748-4755.2004

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New Strategies for Cultivation and Detection of Previously Uncultured Microbes
Bradley S. Stevenson, Stephanie A. Eichorst, John T. Wertz, Thomas M. Schmidt, John A. Breznak
Applied and Environmental Microbiology Aug 2004, 70 (8) 4748-4755; DOI: 10.1128/AEM.70.8.4748-4755.2004
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KEYWORDS

bacteria
Digestive System
Isoptera
soil microbiology

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