The vast majority of bacteria live in highly organized communities called biofilms that are usually associated with surfaces or with interfaces. Compared with their planktonic lifestyle, the physiological behavior of biofilm community members is profoundly altered, resulting in an increased resistance to a wide variety of environmental stresses, including antibiotics, desiccation, predation and host immune response (Stoodley et al., 2002). Because the biofilm responds as a community rather than as an assemblage of individual cells, cell-to-cell coordination and therefore communication and signaling are imperative. Quorum sensing, the secretion and detection of a diffusible molecule by the community members, can lead microbial species to suddenly change their behavior, for example, the production of virulence factors (Fuqua and Greenberg, 2002). Beveridge and colleagues for decades have advocated for membrane vesicles as an important but overlooked component of the matrices of biofilms (Schooling and Beveridge, 2006), and more recently have suggested that membrane vesicles may have an important role for intercellular DNA transfer (Schooling et al., 2009). Mashburn and Whiteley (2005) have invoked membrane vesicles as a mechanism for the direct cell-to-cell transmission of a certain quorum-sensing signal that has been implicated in the initiation and control of biofilm formation (Davies et al., 1998).

To study the organization of bacterial biofilms at macromolecular resolution, we resorted to Myxococcus xanthus, a poster child for complex social behavior and biofilm formation. The key for meaningful ultrastructural studies was the faithful preservation of the biofilm using high-pressure freezing and freeze-substitution, which overcomes artifacts typically associated with classical chemical fixation, and room-temperature sample preparation protocols (reviewed in McDonald and Auer, 2006).

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