MIC determination.

PAO1 was grown in LB broth at 200 rpm at 37°C. The cell suspension was first adjusted to an OD₆₀₀ of 1 and then diluted 100 times with LB broth as the cell sample. Mineral oil was used as the carrier oil. Pure LB and LB with colistin at 100 μl/ml were used as the other two aqueous phases. For more precise control of the concentration, we added a 15-s prerun step with flow rates of the cell suspension, colistin, and LB broth set at 1.5, 0, and 1.5 μl/min, respectively. After the prerun step, we paused all of the pumps for 20 s while keeping the dish drive running and then initiated the ramping program. The flow rate of colistin was linearly ramped from 0 to 1.5 μl/min in 8 min, and the flow rate of LB broth was linearly decreased from 1.5 to 0 μl/min simultaneously, while the flow rate of PAO1 remained 1.5 μl/min and the carrier oil flow rate was 20 μl/min. The device was fixed on the MSP system to generate a spiral array from an i.d. of 15 mm to an o.d. of 65 mm at a 5,000-μm/s constant linear velocity (CLV) (31) with a track spacing of 900 μm and a total writing time of 8 min. Control experiments (broth dilution method) were conducted with tubes containing colistin concentrations ranging from 0 to 80 μg/ml. Growth of PAO1 cells in tubes was observed for MIC evaluation.