Monodisperse Emulsion Drop Microenvironments for Bacterial Biofilm Growth

In this work, microfluidic technology is used to rapidly create hundreds of thousands of monodisperse double and triple emulsion drops that serve as 3D microenvironments for the containment and growth of bacterial biofilms. The size of these drops, with diameters from tens to hundreds of micrometers, makes them amenable to rapid manipulation and analysis. This is demonstrated by using microscopy to visualize cellular differentiation of Bacillus subtilis biofilm communities within each drop and the bacterial biofilm microstructure. Biofilm growth is explored upon specific interfaces in double and triple emulsions and upon negative and positive radii of curvature. Biofilm attachment of matrix and flagella mutants is studied as well as biofilms of Pseudomonas aeruginosa. This is the first demonstration of biofilms grown in microscale emulsion drops, which serve as both templates and containers for biofilm growth and attachment. These microenvironments have the potential to transform existing high-throughput screening methods for bacterial biofilms. Bacterial biofilms are grown at oil-water interfaces in microscale, monodisperse double and triple emulsions created using drop-based microfluidics. Cellular differentiation of a model biofilm-forming bacterium, Bacillus subtilis, is observed in these emulsion microenvironments. Surfactant and oil chemistry direct the preferential localization of biofilm growth at liquid interfaces. These emulsions can be applied towards high-throughput screening of bacterial biofilms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.