Developing reliable anti-biofilm strategies or efficient biofilm-based bioprocesses strongly depends on having a clear understanding of the mechanisms underlying biofilm development, and knowledge of the relevant mechanical parameters describing microbial biofilm behavior. Many varied mechanical testing methods are available to assess these parameters. The mechanical properties thus identified can then be used to compare protocols such as antibiotic screening. However, the lack of standardization in both mechanical testing and the associated identification methods for a given microbiological goal remains a blind spot in the biofilm community. The pursuit of standardization is problematic, as biofilms are living structures, i.e., both complex and dynamic. Here, we review the main available methods for characterizing the mechanical properties of biofilms through the lens of the relationship linking experimental testing to the identification of mechanical parameters. We propose guidelines for characterizing biofilms according to microbiological objectives that will help the reader choose an appropriate test and a relevant identification method for measuring any given mechanical parameter. The use of a common methodology for the mechanical characterization of biofilms will enable reliable analysis and comparison of microbiological protocols needed for improvement of engineering process and screening.

In this study, we utilize the free motion of beads incorporated in bacterial suspension to investigate the behavior of the medium surrounding the beads during biofilm formation. The use of imaging techniques such as digital image correlation enables tracking of the movement of beads, which serve as markers in the processed images. This method is applied to detect and characterize biofilm formation. The main originality of this study lies in characterizing the evolution of the typology of bead movements during biofilm formation. The aim is to identify bead behaviors that represent the start of biofilm formation. By observing inert bead movements introduced into the bacterial environment, changes in trajectory typologies are detected and appear to be related to sessile bacterial activity, bacterial hindrance, and adhesion or formation of extracellular material. We use our approach to discriminate between the presence or absence of antibiotics mixed with bacteria and to assess their effectiveness. The results highlight the potential of our approach as nondestructive tracking of biofilm dynamics over time based on optical microscope images.