Bacterial chemotaxis in response to continuous chemical gradients has been extensively studied at the individual cell and population levels using a variety of well-established in vitro methods (Englert et al., Microfluidic techniques for the analysis of bacterial chemotaxis. Methods Mol Biol 571:1–23, 2009). In nature, bacteria are surrounded by heterogeneous chemical gradients; hence, it is essential to understand chemotaxis behavior under such conditions. Here, we describe a setup that allows visualization of the chemotaxis response of motile cells to the complex microenvironment of a biofilm maintained under static conditions. The biofilm is separated from the motile cells by a semi-permeable membrane. Cells swimming toward the biofilm are captured on the membrane and imaged using confocal laser scanning microscopy (CLSM). Chemotaxis toward specific molecules produced by the biofilm, such as autoinducer-2 (AI-2), can be studied using this setup. This system can be adapted to study chemotaxis toward poly-species biofilms, or even mammalian cells.
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