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bioRxiv · 10.1101/2022.02.13.480190

R-based method for quantitative analysis of biofilm thickness by using Confocal Laser Scanning Microscopy

Fruehauf, H. M.·Stoeckl, M.·Holtmann, D.
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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 G. 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 vs 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. Practical applicationBiofilm communities are ubiquitous. They can be found in every habitat in which water, nutrients and a colonisable surface are present. Depending on the surface, biofilms can cause economic losses due to bio-corrosion (pipelines and ship walls are prominent examples) or are a severe threat to human health when important medical devices or body tissues are colonised [1]. Desirable biofilms are catalytic biofilms how they are used in bioelectrochemical production processes, for example. In all cases, quantitative and qualitative biofilm analysis is necessary in order to prevent or promote biofilm formation. In bioelectrochemistry quantitative biofilm analysis is essential to link productivity (current or chemicals) with biomass deposited on the electrode. Microscopic analysis (e.g. with CLSM) of stained biofilms allows the recording of high volumes of image data but often image analysis then remains at a qualitative stage. In terms of biofilm thickness determination this limits analysis to an estimated thickness of a small amount of images, mostly. The presented R-script allows the calculation of biofilm thickness based on a larger amount of image sets and allows conclusions on the homogeneity of biofilm coverage on the electrode surface. The script is a stand-alone tool if only biofilm thickness should be determined and does not require any image segmentation or processing.

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Fruehauf, H. M., Stoeckl, M., Holtmann, D.. 2022-02-15. R-based method for quantitative analysis of biofilm thickness by using Confocal Laser Scanning Microscopy. https://doi.org/10.1101/2022.02.13.480190

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