Recent advances in experimental design and data analysis to characterize prokaryotic motility

Bacterial motility plays a key role in important cell processes such as chemotaxis and biofilm formation, but is challenging to quantify due to the small size of the individual microorganisms and the complex interplay of biological and physical factors that influence motility phenotypes. Swimming, the first type of motility described in bacteria, still remains largely unquantified. Light microscopy has enabled qualitative characterization of swimming patterns seen in different strains, such as run and tumble, run-reverse-flick, run and slow, stop and coil, and push and pull, which has allowed for elucidation of the underlying physics. However, quantifying these behaviors (e.g., identifying run distances and speeds, turn angles and behavior by surfaces or cell-cell interactions) remains a challenging task. A qualitative and quantitative understanding of bacterial motility is needed to bridge the gap between experimentation, omics analysis, and bacterial motility theory. In this review, we discuss the strengths and limitations of how phase contrast microscopy, fluorescence microscopy, and digital holographic microscopy have been used to quantify bacterial motility. Approaches to automated software analysis, including cell recognition, tracking, and track analysis, are also discussed with a view to providing a guide for experimenters to setting up the appropriate imaging and analysis system for their needs.