Ferrari BC, Gillings M, Binnerup S, Winsley T (2008) Cultivating previously uncultured soil bacteria using a soil substrate membrane system. Nat Protoc 3(8): 1261-1269. https://doi.org/10.1038/nprot.2008.102
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 o4 h of bench time over the 10-d period.
The study used prepGEM, a highly sensitive enzyme-based DNA extraction protocol proven to be efficient for isolating DNA from parasites and successfully adapted here for DNA extraction from PCMs. This method was superior as it enabled direct extraction from growth membranes without the requirement of a centrifugation step to separate the membrane from the cells of interest.
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Suitable for extracting DNA from bacteria (Gram-positive and Gram-negative), protozoa, archaea, colonies and liquid cultures, biofilm and mucosal samples, swabs, and metagenomic DNA including soil, stool and water.
Produces single-stranded DNA suitable for PCR, qPCR, targeted sequencing (amplicon sequencing) from metagenomic samples, 16S rDNA sequencing.
Each kit contains prepGEM, lysozyme, GREEN+ buffer, enhancer and wash.