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.
Keywords: B. subtilis; Biofilm; Cross-section; Filter; SEM.
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