Isolation of Microorganisms Using Sub-Micrometer Constrictions

We present an automated method for isolating pure bacterial cultures from samples containing multiple species that exploits the cell's own physiology to perform the separation. Cells compete to reach a chamber containing nutrients via a constriction whose cross-sectional area only permits a single cell to enter, thereby blocking the opening and preventing other cells from entering. The winning cell divides across the constriction and its progeny populate the chamber. The devices are passive and require no user interaction to perform their function. Device fabrication begins with the creation of a master mold that contains the desired constriction and chamber features. Replica molding is used to create patterned polymer chips from the master, which are bonded to glass microscope cover slips to create the constrictions. We tested constriction geometries ranging from 500 nanometers to 5 micrometers in width, 600 to 950 nanometers in height, and 10 to 40 micrometers in length. The devices were used to successfully isolate a pure Pseudomonas aeruginosa culture from a mixture that also contained Escherichia coli. We demonstrated that individual strains of the same species can be separated out from mixtures using red and green fluorescently-labeled E. coli. We also used the devices to isolate individual environmental species. Roseobacter sp. was separated from another marine species, Psychroserpens sp.