We developed a method to grow Staphylococcus epidermidis bacterial biofilms and characterize their rheological properties in situ in a continuously fed bioreactor incorporated into a parallel plate rheometer. The temperature and shear rates of growth modeled bloodstream conditions, a common site of S. epidermidis infection. We measured the linear elastic (G′) and viscous moduli (G′′) of the material using small-amplitude oscillatory rheology and the yield stress using non-linear creep rheology. We found that the elastic and viscous moduli of the S. epidermidis biofilm were 11 ± 3 Pa and 1.9 ± 0.5 Pa at a frequency of 1 Hz (6.283 rad per s) and that the yield stress was approximately 20 Pa. We modeled the linear creep response of the biofilm using a Jeffreys model and found that S. epidermidis has a characteristic relaxation time of approximately 750 seconds and a linear creep viscosity of 3000 Pa s. The effects on the linear viscoelastic moduli of environmental stressors, such as NaCl concentration and extremes of temperature, were also studied. We found a non-monotonic relationship between moduli and NaCl concentrations, with the stiffest material properties found at human physiological concentrations (135 mM). Temperature dependent rheology showed hysteresis in the moduli when heated and cooled between 5 °C and 60 °C. Through these experiments, we demonstrated that biofilms are rheologically complex materials that can be characterized by a combination of low modulus (∼10 Pa), long relaxation time (∼103 seconds), and a finite yield stress (20 Pa). This suggests that biofilms should be viewed as soft viscoelastic solids whose properties are determined in part by local environmental conditions. The in situ growth method introduced here can be adapted to a wide range of biofilm systems and applied over a broad spectrum of rheological and environmental conditions because the technique minimizes the risk of irreversible, non-linear deformation of the microbial specimen before analysis.
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