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In situ cultivation of previously uncultivable microorganisms using the ichip

Nature Protocols volume 12, pages 2232–2242 (2017)Cite this article

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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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Figure 1: Microcolonies obtained from a diffusion chamber.
Figure 2: Conceptual design of the ichip.
Figure 3: An alternative, miniaturized version of the ichip used to grow oral microorganisms.
Figure 4: Example of a pipette tip rack that is commonly used to build ichips.
Figure 5: Novelty of seawater and soil microbial strains grown in ichips and Petri dishes.
Figure 6: Domestication of isolates through repeated cycles of in situ incubation.
Figure 7: Microcolonies grown in an ichip.

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References

  1. Staley, J.T. & Konopka, A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu. Rev. Microbiol. 39, 321–346 (1985).

    Article  CAS  Google Scholar 

  2. Rappe, M.S. & Giovannoni, S.J. The uncultured microbial majority. Annu. Rev. Microbiol. 57, 369–394 (2003).

    Article  CAS  Google Scholar 

  3. Epstein, S.S. The phenomenon of microbial uncultivability. Curr. Opin. Microbiol. 16, 636–642 (2013).

    Article  CAS  Google Scholar 

  4. Brown, H.P. et al. Culturing of 'unculturable' human microbiota reveals novel taxa and extensive sporulation. Nature 533, 543–546 (2016).

    Article  Google Scholar 

  5. Lagier, J.-C. et al. Culture of previously uncultured members of the human gut microbiota by culturomics. Nat. Microbiol. 1, 16203 (2016).

    Article  CAS  Google Scholar 

  6. Hurst, C.J. Divining the future of microbiology. ASM News 71, 262–263 (2005).

    Google Scholar 

  7. Lewis, K., Epstein, S., D'Onofrio, A. & Ling, L.L. Uncultured microorganisms as a source of secondary metabolites. J. Antibiot. 63, 468–476 (2010).

    Article  CAS  Google Scholar 

  8. Kaeberlein, T., Lewis, K. & Epstein, S. Isolating 'uncultivable' microorganisms in pure culture in a simulated natural environment. Science 296, 1127–1129 (2002).

    Article  CAS  Google Scholar 

  9. Bollmann, A., Lewis, K. & Epstein, S.S. Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates. Appl. Environ. Microbiol. 73, 6386–6390 (2007).

    Article  CAS  Google Scholar 

  10. Bollmann, A., Palumbo, A.V., Lewis, K. & Epstein, S.S. Isolation and physiology of bacteria from contaminated subsurface sediments. Appl. Eviron. Microbiol. 76, 7413–7419 (2010).

    Article  CAS  Google Scholar 

  11. Ben-Dov, E., Kramarsky-Winter, E. & Kushmaro, A. An in situ method for cultivating microorganisms using a double encapsulation technique. FEMS Microbiol. Ecol. 68, 363–371 (2009).

    Article  CAS  Google Scholar 

  12. Aoi, C. et al. Hollow-fiber membrane chamber as a device for in situ environmental cultivation. Appl. Environ. Microbiol. 75, 3826–3833 (2009).

    Article  CAS  Google Scholar 

  13. Ling, L.L. et al. A new antibiotic kills pathogens without detectable resistance. Nature 517, 455–459 (2015).

    Article  CAS  Google Scholar 

  14. Nichols, D. et al. Use of Ichip for high-throughput in situ cultivation of 'uncultivable' microbial species. Appl. Environ. Microbiol. 76, 2445–2450 (2010).

    Article  CAS  Google Scholar 

  15. Sizova, M.V. et al. New approaches for isolation of previously uncultivated oral bacteria. Appl. Environ. Microbiol. 78, 194–203 (2012).

    Article  CAS  Google Scholar 

  16. Epstein, S.S. & Rossel, J. Enumeration of sandy sediment bacteria: search for optimal protocol. Mar. Ecol. Prog. Ser. 117, 289–298 (1995).

    Article  Google Scholar 

  17. Epstein, S.S. et al. Enumeration of sandy sediment bacteria: are the counts quantitative or relative? Mar. Ecol. Prog. Ser. 151, 11–16 (1997).

    Article  Google Scholar 

  18. Nichols, D. et al. Short peptide induces an 'uncultivable' microorganism to grow in vitro. Appl. Environ. Microbiol. 74, 4889–4897 (2008).

    Article  CAS  Google Scholar 

  19. Epstein, S.S. General model of microbial uncultivability. in: Uncultivated Microorganisms. (ed. Epstein, S.S.), Series: Microbiology Monographs Vol. 10 (Series ed. Steinbuchel, A.) 131–150 (Springer, 2009).

    Chapter  Google Scholar 

  20. Porter, K.G. & Feig, Y.C. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25, 943–948 (1980).

    Article  Google Scholar 

  21. Epstein, S.S., Lewis, K., Nichols, D. & Gavrish, E. New approaches to microbial isolation. in: Manual of Industrial Microbiology and Biotechnology, (eds. Baltz, R.H., Demain, A.L., and Davies, J.E.) 3–12 (ASM, 2010).

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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.

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Author notes

  1. Brittany Berdy and Amy L Spoering: These authors contributed equally to this work.

Authors and 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

Authors
  1. Brittany Berdy
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  2. Amy L Spoering
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  4. 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.

Corresponding author

Correspondence to Slava S Epstein.

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Competing interests

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

Supplementary information

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. (MP4 28557 kb)

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Berdy, B., Spoering, A., Ling, L. et al. In situ cultivation of previously uncultivable microorganisms using the ichip. Nat Protoc 12, 2232–2242 (2017). https://doi.org/10.1038/nprot.2017.074

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