## 4 Discussion and Conclusions  
This work focused on in-situ visualisation of biofilms inside a porous substrate using X-ray \(\upmu\)CT. The toxic effect of different chemical compounds and the diffusion and drainage of contrast agents in biofilms were assessed. From the study, 2% \(w/v\) KBr proved to be relatively less toxic as there was no reduction in biofilms population size unlike in other chemical compounds (Figure 5) and based on the contrasting power of KBr vs. its toxicity on biofilms, 5% \(w/v\) KBr was used for this application. Figure 11: Normalised image of the porous substrate after PBS wash. The projections seen on the grid plate represent contrasted biofilms. Figure 10: 2D cross-sectional images of the grid plate. (**a**) after TSB removal (**b**) After application of contrast agent followed by PBS wash (Section 2.10) (**c**) contrasted biofilm obtained by subtracting Figure 10a from Figure 10b. Previously, commercially available cellulose sponges were used for studying biofilm formation in sponge pores by X-ray \(\upnu\)CT, because of their use as a cleaning appliance both in household and industrial settings. The non-sterile conditions and humid environment at these cleaning tasks, favour the growth of bacteria in these sponges. However, the random distribution of pores with large size variability in these sponges lead to diffusion of biofilms and makes it difficult to characterize the features using X-ray \(\upnu\)CT. The hydrophilic nature of the sponge [53] leads to absorption of contrast agents within the substrate and the homogeneous application could not be realised. The presence of dense particles in the solid matrix of the sponge [54] interferes with stained biofilms and this overlap in grey values make it difficult to segment the image. Furthermore, the partial volume effect [55] has significant contribution in substrates with large size variability. Because of these limitations, in the current study an experimental porous substrate was constructed in polymethylmethacrylate (PMMA), which acted as a model porous substrate constituting pores with a similar diameter. The toxicity tests by plate counting for bacteria incubated in 5% \(w/v\) KBr solution showed a \(1\,log_{10}\) reduction in biofilms population size. However, the simulated data for 5% \(w/v\) KBr application showed a significant distinction between the contrasted grid holes and the surrounding aqueous medium. Therefore, KBr at 5% \(w/v\) was applied as a contrast agent for this study to visualize biofilms in-situ using X-ray \(\upnu\)CT based on its uptake volume in biofilms and its contrasting power. If we consider an ideal case, i.e., if all the applied contrast agent is taken up by biofilms, according to simulation, for 5% \(w/v\) KBr application, the contrasted biofilms in the grid hole should have an attenuation coefficient value around 2.4 cm\({}^{-1}\). However, in the real experiment, we have a maximum of 1.78 cm\({}^{-1}\) as the attenuation coefficient value of the contrasted biofilm (Figure 8b) in the grid hole. Further, if we consider the attenuation coefficient value to increase linearly with the volume of the contrast agent, the percentage of contrast agent in biofilms can be determined after subtracting the water contribution of 0.45 cm\({}^{-1}\). Therefore, for the attenuation coefficient value of 1.33 cm\({}^{-1}\), we can infer that the concentration of KBr is about 3.4% in the biofilms present in the grid hole after contrast agent application (Figure 8b). Similarly, for attenuation coefficient value of 0.39 cm\({}^{-1}\), the concentration of KBr is about 1% in the biofilms present in the grid hole after PBS wash (Figure 9a). These calculations give an estimate on the amount of contrast agent that is present in the biofilms. To record the morphological changes, a confocal laser scanning microscope was used to visualize the three batches of biofilms (control, 5% \(w/v\) KBr solution and acute application).