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Single-bacterium tracking is a powerful microscopy method for quantitative studying bacterial motility. This method requires analysis tools that can be used for microscopy video processing and extracting trajectories from the videos. In this paper, we report a software with capability of handling the above two requirements. Moreover, we have applied the developed software to a single-bacterium tracking study for Escherichia coli moving near glass surfaces. By performing video processing and bacterial trajectory extraction in an automated and convenient way, our proposed approach can greatly help single-bacterium tracking studies and could be listed as one of the useful tools for related quantitative investigations.