## 4 Discussion and Conclusions
Although cell elongation was observed in the 5% \(w/v\) KBr incubated bacteria for 48 h compared to control, for 4h application of 5% \(w/v\) KBr no significant changes in morphology were observed. Based on the diffusion experiments, it can be assumed that 30 min application of contrast agent is sufficient for diffusion into biofilms. Considering biofilms as a porous membrane the imbibition of contrast agent solution can be explained by diffusion where the movement of fluid takes place from the higher concentration region to the lower concentration region. As biofilms are made up of around 90% of water [48] the surrounding contrast agent solution which is at higher concentration tends to diffuse into the biofilms. This diffusion is accelerated by the chemical affinity of EPS matrix towards K\({}^{+}\) ions [56; 57] and Br\({}^{-}\) ions [39]. [PERSON] et al. [58] has elaborated on the ability of bacteria to accumulate intracellular potassium several times more than the surrounding medium. Hence the osmotic pressure along with chemical affinity towards K\({}^{+}\) ions facilitated easy passage of KBr into biofilms. However, the reason for non-uniformity in attenuation coefficient value along the depth of the grid hole could be due to the low volume of biofilms. _Pseudomonas fluorescens_ are aerobic bacteria and biofilms were therefore predominantly observed at the liquid-air interface in the flow cell [59]. These biofilms were allowed to settle on the grid plate by draining out the growth medium, leading to a higher density of biofilm at the entrance of the holes than along the hole depth [60]. Therefore, on the addition of contrast agent solution, a more rapid diffusion was observed at the entrance than along hole depth. Similarly, if we consider household sponges, due to their porous open-cell structure the food soils and liquid may get impregnated randomly throughout the sponge. With sufficient aeration in the sponge, biofilms may tend to grow at the liquid-air interface in the pores of the sponge. However, since these spongesundergo dynamic actions like squeezing and rinsing, there are considerable chances of biofilms being embedded in the pore volume. Therefore, along with the porous structure of the substrate, the forces induced by the external stimuli presumably determine the location of biofilms inside the substrate. The diffusion experiment aimed at evaluating the diffusion of the contrast agent and PBS along with the thickness of biofilms over time. There was a sudden decrease in the attenuation coefficient value of the contrasted biofilms to almost its half when submerged with PBS solution. For segmentation, the entire grid hole was assumed to be filled with biofilms, but in reality, this is often not the case. Although enough care was taken during the drainage and application process of the fluids, some amount of biofilms present in the grid holes might have been removed. This void in the meantime gets filled with the PBS solution and hence while segmentation by thresholding based on histogram shape-based method, there is a fair chance of including the PBS for attenuation coefficient calculation. The solubility of KBr in water is beneficial to its application on the developed biofilms and simplifies the experimental procedure. There was no precipitation of salts and a homogeneous distribution of contrast agent solution was achieved. Although both KI and KBr could be considered as contrast agents for its K\({}^{+}\) ions, the plate counting results showed that, unlike 2% \(w/v\) KI, for 2% \(w/v\) KBr solution there was no reduction in the bacterial population size. In addition, with the increase in the extracellular concentration of iodine its intake into the biofilms decreases [61] hence, 5% \(w/v\) KI may not yield sufficient contrast enhancement. On the other hand, for 5% \(w/v\) KBr there is sufficient contrast enhancement (Figure 4) with minimal effect on biofilm structure (Figure 6).