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Updated: Feb 6, 2026

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy
Published on: September 11, 2014
Directed freeze-fracturing of Bacillus subtilis biofilms for conventional scanning electron microscopy
Felix M Fuchs1, Gudrun Holland2, Ralf Moeller1
1German Aerospace Center (DLR e.V.), Institute of Aerospace Medicine, Radiation Biology Department, Space Microbiology Research Group, Linder Hoehe, Cologne (Köln), Germany.
Abstract:
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.
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