Contrast-Enhanced micro-computed tomography for 3D imaging of biofilms in opaque materials: Insights from water treatment plant sand filters

Microorganisms form biofilms in various environments, including porous media, where they alter substrate properties and fluid flow. Visualizing biofilms in 3D within the pore system is essential to understand their effect. Micro-computed tomography (µCT) is a powerful technique to visualize the pore system, but biofilms are usually invisible due to low contrast with water. Contrast-enhancing staining agents (CESAs) can improve their visibility by changing the attenuation of the biofilm or water phase. Traditional CESAs such as BaSO4, silver-coated microspheres or 1-chloronapthalene proved to be successful, but often stain the water phase and have drawbacks such as sedimentation or toxicity. After the successful use of isotonic Lugol and Hf-WD 1:2 POM on cyanobacteria at the surface, this study investigated the use of those CESAs to stain biofilms within opaque materials and, in particular, on biofilms colonizing field-derived sand filters of water treatment plants. Both CESAs increased the biofilms' visibility, including channels within the Hf-WD 1:2 POM stained biofilms and improved segmentation. Their visualization was consistent with Scanning Electron Microscopy (SEM). The CESAs quickly stained the biofilms, and their effect was stable. Isotonic Lugol enhanced the X-ray attenuation the most but might have caused shrinkage. The use of BaSO4 solution, added for comparison and positive control, was complicated due to its fast precipitation. BaSO4 was unable to visualize the isotonic Lugol-stained biofilms but corresponded with the Hf-WD 1:2 POM-stained biofilms. However, the stability and detail shown by isotonic Lugol and Hf-WD 1:2 POM make the latter two preferred. The study showed the potential of µCT and these CESAs for biofilm visualization and water research. Future research should apply these CESAs and focus on the CESA-biofilm interaction to understand the binding mechanisms and their effect on the structure.