Figure 1.
Automated isolation of pure bacterial cultures from the environment.
(a) Schematic of one microfluidic device showing part of a single food chambers. The constriction geometry can be varied to isolate species with particular physical characteristics. (b) Scanning electron microscope image of one food chamber that is connected to the outside environment with a sub-micrometer constriction. (c) Magnified image of the constriction shown in (b). (b–c) White particles seen on the SEM images formed during the metal sputtering step of SEM sample preparation. Freshly made polymer chips do not have visible particles on the surface.
Figure 2.
Schematic depicting the device fabrication process.
1) PMMA is spun on a 3-inch silicon wafer. 2) The constriction design is written with electron beam lithography and developed. 3) Chromium is sputtered (the height of the constrictions is determined by this step). 4) Metal lift-off is done to complete the constriction. 5) Positive photoresist is spun on the wafer. 6) Large features are aligned with the constrictions and exposed using photo-lithography. 7) Large features are developed, completing the master wafer. 8) PDMS is poured over the master and heat cured. 9) PDMS is peeled off and access holes are drilled. 10) The PDMS is bonded to a glass cover slip using oxygen plasma to complete the device.
Figure 3.
Isolation of pure bacterial cultures from heterogeneous populations.
(a-d) Food chamber connected to an external environment containing CFP-labeled P. aeruginosa and RFP-labeled E. coli via a 700 nm tall, 1.5 µm wide, 40 µm long constriction. (a) GFP filter, 40x magnification. (b) RFP filter, 40x magnification. E. coli are only observed in the main entrance. (c) GFP filter, 100x magnification. A single column of P. aeruginosa cells is in the constriction. (d) RFP filter, 100x magnification. (e-f) Food chamber connected to an external environment containing GFP-labeled and RFP-labeled E. coli via a 950 nm tall, 1.5 µm wide, 40 µm long constriction. (e) GFP filter, 100x magnification. A single column of GFP-labeled cells is visible. (f) RFP filter, 100x magnification. No RFP-labeled cells are visible in the constriction or food chamber.
Table 1.
Separation efficiency data of devices: 49 constrictions with various widths were used to separate a mixture of CFP P. aeruginosa and m-cherry E. coli.
Figure 4.
Isolation of Roseobacter sp. from Psychroserpens sp. (a-b) Food chamber connected to main entrance containing heterogeneous bacterial sample via a 950 nm tall, 5 µm wide, and 30 µm long constriction.
(a) Brightfield, 40x magnification. (b) Brightfield, 100x magnification. Both species are present in the main entrance. Only Roseobacter sp. cells are present in the constriction and food chamber.