A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Summary

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Abstract

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

Protocol

1. Fabrication of silicon masters using standard SU-8 photolithography¹(not shown in this video).

1. Use standard SU-8 photolithography methods to create a SU-8 "master" (SU-8 2025, MicroChem, Newton, MA) for fabricating the PDMS mold that contains the microfluidic network and chemotaxis chamber. Such master molds can be fabricated at any microfabrication facility (e.g., Stanford Microfluidics Foundry; http://thebigone.stanford.edu/foundry/).
2. Prior to PDMS replication, expose the SU-8 master to fluorosilane vapor in a desiccator attached to a vacuum source to facilitate eas....

Discussion

The chemotaxis partition coefficient (CPC) and the chemotaxis migration coefficient (CMC) can be calculated as described in Mao et al⁵. If a cell is detected on the high concentration side, it is given a value of +1, whereas a cell detected on the low concentration side is given a value of -1. The values are summed up and divided by the total number of cells to generate the CPC. The sign of the CPC (positive or negative) gives the direction of migration (towards or away from a signal). Although the C.......