The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms

doi:10.1073/pnas.0701693104

The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms

*Top Institute Food and Nutrition, 6703 CT, Wageningen, The Netherlands;
^§MESA+ Institute for Nanotechnology, University of Twente, 7500 AE, Enschede, The Netherlands;
^†Laboratory of Microbiology, Wageningen University, 6703 HB, Wageningen, The Netherlands; and
^¶NIZO Food Research, 6710 BA, Ede, The Netherlands

Edited by James M. Tiedje, Michigan State University, East Lansing, MI, and approved September 17, 2007 (received for review February 28, 2007)

Abstract

A miniaturized, disposable microbial culture chip has been fabricated by microengineering a highly porous ceramic sheet with up to one million growth compartments. This versatile culture format, with discrete compartments as small as 7 × 7 μm, allowed the growth of segregated microbial samples at an unprecedented density. The chip has been used for four complementary applications in microbiology. (i) As a fast viable counting system that showed a dynamic range of over 10,000, a low degree of bias, and a high culturing efficiency. (ii) In high-throughput screening, with the recovery of 1 fluorescent microcolony in 10,000. (iii) In screening for an enzyme-based, nondominant phenotype by the targeted recovery of Escherichia coli transformed with the plasmid pUC18, based on expression of the lacZ reporter gene without antibiotic-resistance selection. The ease of rapid, successive changes in the environment of the organisms on the chip, needed for detection of β-galactosidase activity, highlights an advantageous feature that was also used to screen a metagenomic library for the same activity. (iv) In high-throughput screening of >200,000 isolates from Rhine water based on metabolism of a fluorogenic organophosphate compound, resulting in the recovery of 22 microcolonies with the desired phenotype. These isolates were predicted, on the basis of rRNA sequence, to include six new species. These four applications suggest that the potential for such simple, readily manufactured chips to impact microbial culture is extensive and may facilitate the full automation and multiplexing of microbial culturing, screening, counting, and selection.

Footnotes

^‡To whom correspondence should be addressed. E-mail: colin.ingham{at}wur.nl
Author contributions: C.J.I., A.v.d.B., J.E.T.v.H.V., and W.M.d.V. designed research; C.J.I., A.S., and J.B. performed research; A.S. and J.B. contributed new reagents/analytic tools; C.J.I. and D.M. analyzed data; and C.J.I., A.S., J.B., D.M., A.v.d.B., J.E.T.v.H.V., and W.M.d.V. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0701693104/DC1.
Abbreviations:

FDG,

fluorescein di-β-d-galactopyranoside;

FDP,

fluorescein 3,6-diphosphate;

HTS,

high-throughput screening;

L-agar,

Luria agar;

PAO,

porous aluminum oxide;

RIE,

reactive ion etching.