A microfluidic chemostat for experiments with bacterial and yeast cells

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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.

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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.

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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

Correspondence to Alex Groisman or Andre Levchenko.

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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)

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

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