High Throughput 3D Tracking of Bacterial Chemotaxis in Complex Environments

Bacterial chemotaxis is an important survival mechanism by which motile bacteria bias their random walk trajectory to navigate chemical gradients. While chemotaxis has been studied extensively in E. coli, much less attention has been devoted to the diversity of bacteria with different flagellar architectures, or to motility in viscous or porous environments that more closely mimic the complexity of natural habitats. Current approaches for characterizing and comparing chemotaxis strategies are hampered by being either limited to the population scale, qualitative, low throughput, 2D, and/or dependent on theoretical models.

Here we present a simple yet powerful chemotaxis assay combining a recent high-throughput 3D tracking method with microfluidically created chemical gradients. With a typical throughput of above 3,000 individual trajectories in 5 minutes, we demonstrate that we can directly determine chemotactic drift velocities in diverse environments while simultaneously resolving 3D motility behavior, enabling unprecedented access to a mechanistic understanding and quantitative comparison of chemotactic strategies. We highlight the power of our assay by revealing the role of lateral flagella in the opportunistic pathogen V. alginolyticus for motility and chemotaxis in hydrogels.