Bacterial phenotypic heterogeneity is important to increase survival probability when the environment turns into a severe condition for bacteria. Conventional statistical analysis of bulk populations typically masks physiologically important signals from minorities owing to the averaging process of the mixture of minorities and majorities in the bacterial population. To study minor bacteria with phenotypic heterogeneity, single-cell analysis of a bacterial population is required. Although standard single-cell analysis investigates the condition of every bacterium at the moment of processing, a technique to investigate the temporal reaction of a single bacterium against local chemical exposure has not been developed. Here, we developed a microfluidic device with a narrow gap and size that was slightly smaller than the typical size of bacteria (approximately 1 μm). This device can trap a single bacterium at the narrow gap by applying pressure to attract bacteria, and expose the trapped single bacterium to chemical solution by adjusting the pressure balance between the narrow and wide ends of the gap. We achieved single-bacterium trapping of Escherichia coli and Bacillus subtilis, local chemical delivery to the single bacterium trapped at the narrow gap for 30 min, which is comparable to the bacterial generation time.

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