(728f) Bacterial Patterning at the Micron Scale for Quantitative Interactions

Authors: 
Timm, C. M. - Presenter, University of Wisconsin-Madison
Retterer, S., Oak Ridge National Laboratory
Pelletier, D. A., Oak Ridge National Laboratory
Hansen, R. R., Colorado School of Mines

Bacteria in nature exist in spatially heterogeneous environments where dynamic interspecies interactions shape community development and function.  In the rhizosphere, the volume of soil directly influenced by the root of a plant, the primary source of carbon is produced by the root and secreted into the soil. In the otherwise carbon-poor soil system, the competition for nutrients in the rhizosphere is fierce, leading to observations of distinct bacterial species that dominate this environment. The species found in the rhizosphere often grow quickly and/or produce antimicrobials to suppress their competitors. Because it is difficult to quantify rhizosphere interactions in nature, model systems that allow the direct observation of spatially controlled species interaction could enable new insights into the processes that shape bacterial community development.

We have developed a microfluidic platform and patterning technique that allows the controlled deposition of multiple bacterial species.  Interactions between species can then be observed in a defined laboratory environment. The general process consists of depositing a thin, peel-able film of parylene on silicon, etching a defined pattern in the parylene, overlaying with a microfluidic delivery device, and peeling the parylene to leave desired patterns of bacteria. With this approach we can generate micron-scale patterns of multiple species of bacteria. At high culture densities (OD600>0.1) we consistently create colonies with diameters as small as 10µm.  At lower culture densities, stochastic processes influence colony formation.  Spots with different bacterial species can be spaced as closely as 100µm. Cells are viable throughout the delivery process and we observe growth when the patterns are overlaid with liquid or solid media.  Real-time monitoring of bacterial cells and colonies allows quantitative analysis of how interspecies interactions influences processes such as cell growth.