Abstract: Microscopy is mostly the method of choice to analyse biofilms. Due to the high local heterogeneity of biofilms, single and punctual analyses only give an incomplete insight into the local distribution of biofilms. In order to retrieve statistically significant results a quantitative method for biofilm thickness measurements was developed based on confocal laser scanning microscopy and the programming language R. The R‐script allows the analysis of large image volumes with little hands‐on work and outputs statistical information on homogeneity of surface coverage and overall biofilm thickness. The applicability of the script was shown in microbial fuel cell experiments. It was found that Geobacter sulfurreducens responds differently to poised anodes of different material so that the optimum potential for MFC on poised ITO anodes had to be identified with respect to maximum current density, biofilm thickness and MFC start‐up time. Thereby, a positive correlation between current density and biofilm thickness was found, but with no direct link to the applied potential. The optimum potential turned out to be +0.1 V versus SHE. The script proved to be a valuable stand‐alone tool to quantify biofilm thickness in a statistically valid manner, which is required in many studies.
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