Abstract
The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution of community members and their activities. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. We highlight the utility of the platform and describe its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of P. aeruginosa, part of a series of mutants developed to study type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor each community member abundance and location within each community. We present a detailed method for assembling mixtures of bacteria into the wells of the array and use time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. We describe the seeding and assembly of the microwell platform, discuss the imaging procedures necessary for quantitative analysis of microbial communities within the array, and describe methods that can be used to reveal interactions between microbial species.
