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Cultivating previously uncultured soil bacteria using a soil substrate membrane system

Nature Protocols volume 3, pages 1261–1269 (2008)Cite this article

Abstract

Most bacteria are recalcitrant to traditional cultivation in the laboratory. The soil substrate membrane system provides a simulated environment for the cultivation of previously undescribed soil bacteria as microcolonies. The system uses a polycarbonate membrane as a solid support for growth and soil extract as the substrate. Diverse microcolonies can be visualized using total bacterial staining combined with fluorescence in situ hybridization (FISH) after 7–10-d incubation. Molecular typing shows that the majority of microcolony-forming bacteria recovered using this protocol were resistant to growth using standard methods. The protocol takes <4 h of bench time over the 10-d period.

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Figure 1: Flow chart for microcultivation using the soil substrate membrane system.
Figure 2: Major steps involved in set up of the soil substrate membrane system.
Figure 3: Epifluorescence microscopy image of microcolony-forming soil bacteria present on growth membranes.

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Acknowledgements

This work was supported by a Macquarie University Research Fellowship. We thank Debra Birch for her advice and support with confocal microscopy imaging of microcolonies.

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Authors and Affiliations

  1. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, 2052, New South Wales, Australia

    Belinda C Ferrari

  2. Department of Biological Sciences, Macquarie University Sydney, Sydney, 2109, New South Wales, Australia

    Tristrom Winsley & Michael Gillings

  3. Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, Frederiksborgvej 399, Roskilde, DK-4000, Denmark

    Svend Binnerup

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  1. Belinda C Ferrari
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  4. Svend Binnerup
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Correspondence to Belinda C Ferrari.

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Ferrari, B., Winsley, T., Gillings, M. et al. Cultivating previously uncultured soil bacteria using a soil substrate membrane system. Nat Protoc 3, 1261–1269 (2008). https://doi.org/10.1038/nprot.2008.102

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