## 1 Introduction
These restrictions highlight the need for 3D imaging techniques like X-ray uCT that allow the user to visualize the internal and external structure of objects [27] non-invasively and non-destructively. X-ray uCT is one of the well-established techniques to study different porous substrates and visualize 3D geometry [28; 29]. X-ray uCT data can be used as an input for pore-scale modelling [30] and flow simulations [31] to predict the flow and unwanted residual development inside the porous substrate. X-ray uCT is also being used to study the biofilm morphology and its permeability properties in porous substrates [32; 33]. The low density of the EPS components, such as polysaccharides and a variety of proteins, glycoproteins, and glycolipids, however, makes it difficult to visualize biofilms using X-ray \(\upmu\)CT. Although the application of chemical compounds for contrast enhancement between the biofilms and the substrate has been in place for some time, no ideal contrast agent has thus far been identified, taking into account the boundary conditions of minimal impact on the biofilm properties and growth, while providing sufficient and homogeneously distributed contrast. [PERSON] et al. [34] used the idea of applying two chemical compounds, barium sulphate and potassium iodide (BaSO\({}_{4}\) and KI) as contrast agents. A medical suspension of BaSO\({}_{4}\) was used to differentiate between the aqueous phase (growth medium) and the biofilm, while KI diffused inside the biofilm and aided in delineation between the biofilm and porous medium. However, since BaSO\({}_{4}\) is not water-soluble, it interacts with biofilms and displaces them. Moreover, the sedimentation of BaSO\({}_{4}\) results in a non-homogenous distribution inside the substrate. In the experiment by [PERSON] et al. [35], iron sulphate (FeSO\({}_{4}\)) was used as a contrast agent and was supplemented during the growth of bacteria. In their observation, they found that the biofilm grown in the flow cell exhibits brown patches due to the presence of iron oxide. Therefore, the colloidal iron formed due to oxidation of Fe (III) may lead to irreversible chromism effects on the porous substrate. Similarly, 1-chloronaphthalene was used as a contrast agent by [PERSON] et al. [36]. However, 1-chloronaphthalene is known to be highly toxic, which was also observed during our tests to reproduce these results, as it chemically reacts with the substrate resulting in its dissolution. Given the aforementioned limitations, an alternative chemical compound with different physical and chemical properties, namely potassium bromide (KBr), was evaluated here to stain the biofilm _P. fluorescens_ Pf01. Aqueous solutions of KBr are neutral (pH 7) and do not require any addition of buffer and hence can be applied onto the mature biofilms easily. [PERSON] [37] has compared various cations for their affinity and stability towards aggregative polysaccharides like alginic acid. His study showed that alginic acid has higher affinity towards K\({}^{+}\) cations than other ions in the same periodic group. As the _P. fluorescens_ bacteria has the potential to synthesize alginic acid [38], the K\({}^{+}\) cations attach to alginic acid while Br readily binds to the nucleic acid [39] present in the biofilms. The linear X-ray attenuation coefficient is dependent on the chemical composition and density of the sample. It is directly proportional to the atomic number of the chemical element under observation. As KBr has higher density and both the elements K and Br have higher atomic numbers compared to CHON (carbon, hydrogen, oxygen, nitrogen) elements present in the biofilm and the surrounding medium, they exhibit higher X-ray attenuation at given X-ray energy. Comparatively, Br with higher atomic may contribute more in enhancing the contrast.