Directed freeze-fracturing of Bacillus subtilis biofilms for conventional scanning electron microscopy

Biofilms have been intensively investigated over the past decades. Bacillus subtilis is able to form highly structured colony biofilms, and as one of the most studied Gram-positive model organisms, has helped to decipher the complex genetic regulation of biofilms. Several methods have been developed to analyze the architecture of biofilms. In this paper, we describe a method which allows the analysis of the internal structures of biofilms by scanning electron microscopy (SEM). The method uses a modified freeze-fracturing of chemically fixed biofilm to generate defined, well-preserved fractures at millimeter-scale which allows to analyze systematically the internal biofilm structure from macro- to nano-scale.