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

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 x 7 mum, 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 beta-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.

Fig. 1.

Culture chip use and manufacture. (A) Cross-section of set-up for RIE of a 36 × 8-mm strip of aluminum oxide (not to scale). (B) Cross-sectional diagram of a small part of a chip, illustrating microbial growth in the central compartment (7–150 μm wide) and supply of nutrients from beneath the 60-μm thickness of the aluminum oxide and 10-μm-high walls. (C) Photograph of a platinum-coated chip placed on sheep's blood agar in a standard Petri dish.

Fig. 2.

Images of materials, growth compartments, and microbial culture on chips. (A) SEM of aluminum oxide showing pores on average 200 nm diameter. (B) Transmission light microscopy of hundreds of 20 × 20-μm compartments viewed from above. (C) SEM of 7 × 7-μm compartments from above at a 30° angle. (D) Culture of L. plantarum in six compartments of the same dimensions as C stained with a fluorogenic dye (Syto 9) after growth and imaged from above. (E) Detection of β-galactosidase activity using the fluorogenic substrate FDG, from E. coli containing plasmid pUC18 grown in a 20 × 20-μm compartment. (F) As in E with one plasmid-containing microcolony, viewed at lower magnification. (G) View of 20 × 20-μm format chip with one area supporting a GFP-expressing strain of E. coli in a background of nonfluorescent cells. (H) Previously uncultivated oligotrophic bacterium related to Dechloromonas sp. labeled by FDP metabolism and grown in a 20 × 20-μm compartment supplied by Rhine water, before recovery and identification by PCR.

Fig. 3.

Example of bias and distribution of culture on chips. (A) Original image (inverted transmission light microscopy image; lighter areas show growth of L. plantarum) of a single field of view of 1,170 20 × 20-μm compartments. (B) Heat-density map of local colony density, computed by using a Gaussian smoothing kernel used, to construct statistical models of chip performance and assess bias.

Fig. 4.

Culture and viable counting of microorganisms on chips. Dilution series of microorganisms plated on a series of 20 × 20-μm format chips. The number of deposited cfu per chip was calculated from the fraction of compartments supporting growth, as scored by microscopy, with the numbers corrected for inoculation of compartments with multiple cfu. Open triangles, E. coli; filled squares, C. albicans; filled diamonds, L. plantarum. Error bars show SD with lower limits not shown at lowest dilution factor because of log₁₀ transformation of the data.