Protocol | Published:

In situ cultivation of previously uncultivable microorganisms using the ichip

Nature Protocols volume 12, pages 22322242 (2017) | Download Citation

Abstract

Most microbial species remain uncultivated, and modifying artificial nutrient media brings only an incremental increase in cultivability. We reasoned that an alternative way to cultivate species with unknown requirements is to use naturally occurring combinations of growth factors. To achieve this, we moved cultivation into the microbes' natural habitat by placing cells taken from varying environmental samples into diffusion chambers, which are then returned to nature for incubation. By miniaturizing the chambers and placing only one to several cells into each chamber, we can grow and isolate microorganisms in axenic culture in one step. We call this cultivation platform the 'isolation chip', or 'ichip'. This platform has been shown to increase microbial recovery from 5- to 300-fold, depending on the study. Furthermore, it provides access to a unique set of microbes that are inaccessible by standard cultivation. Here we provide a simple protocol for building and applying ichips for environmental cultivation of soil bacteria as an example; the protocol consists of (i) preparing the ichip; (ii) collecting an environmental sample; (iii) serially diluting cells and loading them into the ichip; (iv) returning the ichip to the environment for incubation; (v) retrieving the ichip and harvesting grown material; and (vi) domestication of the ichip-derived colonies for growth in the laboratory. The ichip's full assembly and deployment is a relatively simple procedure that, with experience, takes 2–3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take 1–4 h, depending on which specific procedures are used.

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Acknowledgements

Development and application of the ichip was supported by, in chronological order, NSF Grant OCE-0221267 to S.E.; DOE Grant DE-FG02-04ER63782 to K. Lewis (Northeastern University) and S.E.; NIH Grant R21 DE018026-01A1, DOE Grants DE-FG02-07ER64507 and DE-FG02-04ER63782, NIH Grant 1RC1DE020707-01 (all to S.E.); NIH Grant 1R01HG005816-01 to D. Fredricks (Fred Hutchinson Cancer Research Center) and S.E.; NIH Grant AI085612 to A.L.S.; and NSF Grant ARC-1203857 to S.E. We are grateful to J. Case for video production and editing, and C. Williams and A. Eilers for assistance with lab work for protocol troubleshooting and useful discussions.

Author information

Author notes

    • Brittany Berdy
    •  & Amy L Spoering

    These authors contributed equally to this work.

Affiliations

  1. Department of Biology, Northeastern University, Boston, Massachusetts, USA.

    • Brittany Berdy
    •  & Slava S Epstein
  2. NovoBiotic Pharmaceuticals, Cambridge, Massachusetts, USA.

    • Amy L Spoering
    • , Losee L Ling
    •  & Slava S Epstein
  3. LiDakSum Marine Biopharmaceutical Research Center, Ningbo University, Ningbo, China.

    • Slava S Epstein

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Contributions

S.E. conceived the ichip idea and developed its prototypes. A.L.S., L.L.L., and S.E. simplified and improved the original designs. B.B., A.L.S., and L.L.L. designed and executed growth experiments. All authors wrote the paper.

Competing interests

The ichip technology has been patented by Northeastern University and licensed exclusively to NovoBiotic Pharmaceuticals, LLC.

Corresponding author

Correspondence to Slava S Epstein.

Supplementary information

Videos

  1. 1.

    Example of an ichip experimental setup.

    This video walks through the basic setup of an ichip experiment with soil, beginning with membrane application and continuing through colony visualization. Video production: Joe Case.

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DOI

https://doi.org/10.1038/nprot.2017.074

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