Ultrafast and reproducible quantitative analysis of bacterial chemotaxis by microfluidic cell counting and imaging

Abstract The capillary assay is a classical method for quantifying bacterial chemotaxis, but it relies on overnight plating and colony counting or optical density measurements, which are time‑consuming and highly heterogeneous. Here, we developed an ultrafast chemotaxis quantification platform by coupling the capillary assay with automated microfluidic counting and imaging. This strategy preserves the simplicity and robustness of the conventional capillary assay while replacing overnight colony counting with rapid, real-time bacterial enumeration. We first characterized the microfluidic counting performance and validated its robustness across varying flow rates, sampling windows, and channel geometries. Then we successfully measured dose‑dependent chemotactic behaviors towards classic chemo-attractants and chemo-repellents and discovered novel chemotaxis behavior towards metal gradients. Lastly, to further explore the possibility of eliminating the manual sample-transfer step, we designed and validated a proof-of-concept integrated microfluidic platform that combines chemotaxis, counting, and microscopic observation within a single device.