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Biofilms are aggregates of cells that form on surfaces or at the air–water interface. Cells in a biofilm are encased in a self-secreted extracellular matrix (ECM) that provides them with mechanical stability and protects them from antibiotic treatment. From a soft matter perspective, biofilms are regarded as colloidal hydrogels, with the cells playing the role of colloids and the ECM compared with a cross-linked hydrogel. Here, we examined whole biofilms of the soil bacterium Bacillus subtilis utilizing methods that are commonly used to characterize hydrogels in order to evaluate the uptake of water and the water properties in the biofilms. Specifically, we studied wild-type as well ECM mutants, lacking the protein TasA and the exopolysaccharide (EPS). We characterized the morphology and mesh size of biofilms using electron microscopy, studied the state of water in the biofilms using differential scanning calorimetry, and …