Developing a novel and versatile approach to study populations of microbes on surfaces

Abstract

Spatial structure, for example regarding antibiotic gradients, is an important topic of investigation in microbial ecology and evolution. Experiments investigating pop- ulation dynamics in spatially-structured environments are often performed on agar plates. Whilst inexpensive and straightforward, these provide only rudimentary temporal and spatial control of environmental conditions. In chemostats and microfluidic devices, for well-mixed and micrometre-scale environments, respectively, regulating media inflow and outflow enables environ- mental control. We combine proven use of agar surfaces with such flow-enabled control in a novel, low-cost fluidic device; the device comprises an elastomer supporting base with a thin agar sheet on top on which microbes grow. Indented channels in the base allow flow of media/antibiotics below the agar surface. A Raspberry-Pi-operated camera allows for time-lapse imaging suitable for quantita- tive image analysis. As a proof of principle, we used our device for extended and robust growth of non-motile E. coli and motile P. aeruginosa maintaining the initial speed with which colonies propagate over three days, whilst a continual speed decrease occurred on agar plates. Guided by simulations of flow and diffusion, we then used the device to create stable antibiotic gradients within the agar. Along these gradients, we found P. aeruginosa exhibit unique microbial growth patterns with local adaptations. Because flow below the agar surface can be controlled spatially and temporally, the device promises a range of applications for studying microbial ecology and evolution in spatially continuous environments at a substrate-air interface.