Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A microfluidic chemostat for experiments with bacterial and yeast cells

Nature Methods volume 2, pages 685–689 (2005)Cite this article

Abstract

Bacteria and yeast frequently exist as populations capable of reaching extremely high cell densities. With conventional culturing techniques, however, cell proliferation and ultimate density are limited by depletion of nutrients and accumulation of metabolites in the medium. Here we describe design and operation of microfabricated elastomer chips, in which chemostatic conditions are maintained for bacterial and yeast colonies growing in an array of shallow microscopic chambers. Walls of the chambers are impassable for the cells, but allow diffusion of chemicals. Thus, the chemical contents of the chambers are maintained virtually identical to those of the nearby channels with continuous flowthrough of a dynamically defined medium. We demonstrate growth of cell cultures to densely packed ensembles that proceeds exponentially in a temperature-dependent fashion, and we use the devices to monitor colony growth from a single cell and to analyze the cell response to an exogenously added autoinducer.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Design and operation of the two-layer microfluidic device.
Figure 2: A representative experiment showing development of a JM109 E. coli colony inside a chamber at 35 °C from a single cell captured at the beginning of the experiment.
Figure 3: Response of cells to addition and removal of exogeneous signal, and growth curves of colonies under different conditions.

Similar content being viewed by others

References

  1. Stoodley, P., Sauer, K., Davies, D.G. & Costerton, J.W. Biofilms as complex differentiated communities. Annu. Rev. Microbiol. 56, 187–209 (2002).

    Article  CAS  Google Scholar 

  2. Reynolds, T.B. & Fink, G.R. Bakers' yeast, a model for fungal biofilm formation. Science 291, 878–881 (2001).

    Article  CAS  Google Scholar 

  3. Douglas, L.J. Candida biofilms and their role in infection. Trends Microbiol. 11, 30–36 (2003).

    Article  CAS  Google Scholar 

  4. Anderson, G.G. et al. Intracellular bacterial biofilm-like pods in urinary tract infections. Science 301, 105–107 (2003).

    Article  CAS  Google Scholar 

  5. Jelsbak, L. & Sogaard-Andersen, L. Pattern formation: fruiting body morphogenesis in Myxococcus xanthus. Curr. Opin. Microbiol. 3, 637–642 (2000).

    Article  CAS  Google Scholar 

  6. Udvardi, M.K. & Day, D.A. Metabolite transport across symbiotic membranes of legume nodules. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 493–523 (1997).

    Article  CAS  Google Scholar 

  7. Tebo, B.M., Linthicum, D.S. & Nealson, K.H. Luminous bacteria and light emitting fish: ultrastructure of the symbiosis. Biosystems 11, 269–280 (1979).

    Article  CAS  Google Scholar 

  8. Scherz, R., Shinder, V. & Engelberg, D. Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization. J. Bacteriol. 183, 5402–5413 (2001).

    Article  CAS  Google Scholar 

  9. Hastings, J.W. & Nealson, K.H. Bacterial bioluminescence. Annu. Rev. Microbiol. 31, 549–595 (1977).

    Article  CAS  Google Scholar 

  10. Sternberg, C. et al. Distribution of bacterial growth activity in flow-chamber biofilms. Appl. Environ. Microbiol. 65, 4108–4117 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Millar, M.R., Linton, C.J. & Sherriff, A. Use of a continuous culture system linked to a modified Robbins device or flow cell to study attachment of bacteria to surfaces. Methods Enzymol. 337, 43–62 (2001).

    Article  CAS  Google Scholar 

  12. Li, N., Tourovskaia, A. & Folch, A. Biology on a chip: microfabrication for studying the behavior of cultured cells. Critical Reviews in Biomedical Engineering 31, 423–488 (2003).

    Article  Google Scholar 

  13. Andersson, H. & van den Berg, A. Microfluidic devices for cellomics: a review. Sens. Actuators B Chem. 92, 315–325 (2003).

    Article  CAS  Google Scholar 

  14. Sia, S.K. & Whitesides, G.M. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis 24, 3563–3576 (2003).

    Article  CAS  Google Scholar 

  15. Mao, H.B., Cremer, P.S. & Manson, M.D. A sensitive, versatile microfluidic assay for bacterial chemotaxis. Proc. Natl. Acad. Sci. USA 100, 5449–5454 (2003).

    Article  CAS  Google Scholar 

  16. Balaban, N.Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Bacterial persistence as a phenotypic switch. Science 305, 1622–1625 (2004).

    Article  CAS  Google Scholar 

  17. Park, S. et al. Influence of topology on bacterial social interaction. Proc. Natl. Acad. Sci. USA 100, 13910–13915 (2003).

    Article  CAS  Google Scholar 

  18. Mao, H.B., Yang, T.L. & Cremer, P.S. A microfluidic device with a linear temperature gradient for parallel and combinatorial measurements. JACS 124 (2002).

  19. Unger, M.A., Chou, H.P., Thorsen, T., Scherer, A. & Quake, S.R. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288, 113–116 (2000).

    Article  CAS  Google Scholar 

  20. Miller, M.B. & Bassler, B.L. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55, 165–199 (2001).

    Article  CAS  Google Scholar 

  21. Fuqua, C., Parsek, M.R. & Greenberg, E.P. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu. Rev. Genet. 35, 439–468 (2001).

    Article  CAS  Google Scholar 

  22. Withers, H., Swift, S. & Williams, P. Quorum sensing as an integral component of gene regulatory networks in gram-negative bacteria. Curr. Opin. Microbiol. 4, 186–193 (2001).

    Article  CAS  Google Scholar 

  23. Andersen, J.B. et al. gfp-based N-acyl homoserine-lactone sensor systems for detection of bacterial communication. Appl. Environ. Microbiol. 67, 575–585 (2001).

    Article  CAS  Google Scholar 

  24. Elowitz, M.B., Levine, A.J., Siggia, E.D. & Swain, P.S. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002).

    Article  CAS  Google Scholar 

  25. Ozbudak, E.M., Thattai, M., Kurtser, I., Grossman, A.D. & van Oudenaarden, A. Regulation of noise in the expression of a single gene. Nat. Genet. 31, 69–73 (2002).

    Article  CAS  Google Scholar 

  26. Blake, W.J., Kaern, M., Cantor, C.R. & Collins, J.J. Noise in eukaryotic gene expression. Nature 422, 633–637 (2003).

    Article  CAS  Google Scholar 

  27. McMurray, M.A. & Gottschling, D.E. An age-induced switch to a hyper-recombinational state. Science 301, 1908–1911 (2003).

    Article  CAS  Google Scholar 

  28. Flardh, K. Growth polarity and cell division in Streptomyces. Curr. Opin. Microbiol. 6, 564–571 (2003).

    Article  CAS  Google Scholar 

  29. Xia, Y.N. & Whitesides, G.M. Soft lithography. Angew. Chem. Int. Ed. 37, 551–575 (1998).

    Article  Google Scholar 

  30. Groisman, A., Enzelberger, M. & Quake, S.R. Microfluidic memory and control devices. Science 300, 955–958 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by US National Institutes of Health grant GM066786 (A.L. and A.M.S.), National Science Foundation grant MCB-0331306 (A.G.) and National Science Foundation CAREER Award MCB-9875479 (A.M.S.).

Author information

Authors and Affiliations

  1. Department of Physics, University of California San Diego, 9500 Gilman Dr., MC 0374, La Jolla, 92093, California, USA

    Alex Groisman & J Kyle Campbell

  2. Department of Biomedical Engineering and Whitaker Institute of Biomedical Engineering, Johns Hopkins University, Baltimore, 21218, Maryland, USA

    Caroline Lobo, HoJung Cho & Andre Levchenko

  3. Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, 24061, Virginia, USA

    Yann S Dufour & Ann M Stevens

Authors
  1. Alex Groisman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Caroline Lobo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. HoJung Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J Kyle Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yann S Dufour
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ann M Stevens
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andre Levchenko
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Alex Groisman or Andre Levchenko.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Fabrication and structure of the microfluidic chemostat device. (PDF 560 kb)

Supplementary Fig. 2

Bacterial and yeast strains can be grown to high densities in chemostatic chambers. (PDF 145 kb)

Supplementary Fig. 3

Deformation of capillaries with applied gauge pressure. (PDF 82 kb)

Supplementary Fig. 4

Statistics of loading of particles into chambers of the chemostat device at different concentrations of the particle suspension. (PDF 91 kb)

Supplementary Fig. 5

Comparison of on-chip and batch culture fluorescence of cells exposed to various autoinducer concentrations. (PDF 99 kb)

Supplementary Fig. 6

Variability in colony growth between chambers of the same size on the same and different chips. (PDF 130 kb)

Supplementary Fig. 7

Growth of colonies of JM109 E. coli cells expressing Gfp(LVA) in the medium containing 10 nM of AI as a function of time at 24 °C and 35 °C in two representative experiments. (PDF 131 kb)

Supplementary Methods (PDF 260 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Groisman, A., Lobo, C., Cho, H. et al. A microfluidic chemostat for experiments with bacterial and yeast cells. Nat Methods 2, 685–689 (2005). https://doi.org/10.1038/nmeth784

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth784

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing