Use of ichip for high-throughput in situ cultivation of "uncultivable" microbial species

Abstract

One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The "uncultivable" microbial majority arguably represents our planet's largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated in situ, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significant phylogenetic novelty. The new method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.

FIG. 1.

Isolation chip, or ichip, for high-throughput microbial cultivation in situ. (A) Dipping a plate with multiple through-holes into a suspension of mixed environmental cells leads to capturing (on average) a single cell (B). (C) Ichip assembly: membranes cover arrays of through-holes from each side; upper and bottom plates with matching holes press the membranes against the central (loaded) plate. Screws provide sufficient pressure to seal the content of individual through-holes, each becoming a miniature diffusion chamber containing (on average) a single cell. (Artwork by Stacie Bumgarner, Whitehead Institute for Biomedical Sciences, Cambridge, MA.)

FIG. 2.

Microbial recovery in ichip, diffusion chamber, and standard petri dish as percentage of inoculated cells forming colonies. (A) Seawater data. (B) Soil data.

FIG. 3.

Novelty of seawater and soil microbial strains grown in ichips and petri dish. The equation of sequence novelty, in percent divergence from the known species, and taxonomic rank of novelty (genus level, family level, etc.) is very approximate.

FIG. 4.

Examples of microcolonies grown in ichips as seen under a compound microscope equipped for differential interference contrast at ×100. The diameter of the through-holes is 1 mm.