Imaging biofilm in porous media using X-ray computed microtomography

Abstract

SummaryIn this study, a new technique for three-dimensional imaging of biofilm within porous media using X-ray computed microtomography is presented. Due to the similarity in X-ray absorption coefficients for the porous media (plastic), biofilm and aqueous phase, an X-ray contrast agent is required to image biofilm within the experimental matrix using X-ray computed tomography. The presented technique utilizes a medical suspension of barium sulphate to differentiate between the aqueous phase and the biofilm. Potassium iodide is added to the suspension to aid in delineation between the biofilm and the experimental porous medium. The iodide readily diffuses into the biofilm while the barium sulphate suspension remains in the aqueous phase. This allows for effective differentiation of the three phases within the experimental systems utilized in this study. The behaviour of the two contrast agents, in particular of the barium sulphate, is addressed by comparing two-dimensional images of biofilm within a pore network obtained by (1) optical visualization and (2) X-ray absorption radiography. We show that the contrast mixture provides contrast between the biofilm, the aqueousphase and the solid-phase (beads). The imaging method is then applied to two three-dimensional packed-bead columns within which biofilm was grown. Examples of reconstructed images are provided to illustrate the effectiveness of the method. Limitations and applications of the technique are discussed. A key benefit, associated with the presented method, is that it captures a substantial amount of information For example, the quantification of changes in porous media effective parameters, such as dispersion or permeability, induced by biofilm growth, is possible using specific upscaling techniques and numerical analysis. We emphasize that the results presented here serve as a first test of this novel approach; issues with accurate segmentation of the images, optimal concentrations of contrast agents and the potential need for use of synchrotron radiation sources need to be addressed before the method can be used for precise quantitative analysis of biofilm geometry in porous media.

Development of X-ray micro-focus computed tomography to image and quantify biofilms in central venous catheter models in vitro

“…Barium sulphate was previously used by Davit et al (2011), but due to the fact that samples were stacked on top of each other and scanned in duplicate for about an hour each, movement of barium sulphate was a high probability. Movement during scanning does not allow precise calculation of the centre of rotation, therefore inhibiting the 3D reconstruction of data.…”

“…Importantly, studies have demonstrated X-ray µCT identification of Gram-negative bacteria using silver microspheres as a contrast agent , and multispecies water biofilms using barium sulfate and propidium iodide (Davit et al, 2011). Other studies have also used gold and silver nanoparticles to identify yeast species by surface-enhanced Raman scattering (Fakhrullin et al, 2009), highlighting that these contrast stains, combined with X-ray µCT, are applicable across a wide range of potential pathogens.…”

“…In contrast, Davit et al (2011) did not analyse their data and instead used the CT scans for qualitative purposes. We were able to capture data in our study at a higher resolution than both of these previous studies.…”

“…This permitted non-destructive, whole-volume imaging for comparative, developmental and quantitative studies of morphology, where contrast agents were used to distinguish organs and various tissues in these animals (Metscher, 2009). This method has also been used to detect biofilms on model porous media in previous studies (Davit et al, 2011;Iltis et al, 2011). Biofilms were successfully imaged in both studies when X-ray µCT was combined with contrast agents.…”

“…Biofilms were successfully imaged in both studies when X-ray µCT was combined with contrast agents. Davit and colleagues used a lab-based µCT scanner (Skyscan1174) with a pixel resolution of 9 µm and a medical suspension of barium sulphate (Davit et al, 2011), whereas Iltis and colleagues performed experiments using a dedicated synchrotron beamline (8.3.2 beamline, Advanced Light Source) at a pixel resolution of 4.5 µm with 10 µm hollow silver-coated microspheres as a contrast agent . Stains at high concentrations (or in this case, contrast particles) may not be specific enough to allow clear demarcation between individual cells, while spatial resolution might actually be sufficient (Thurner et al, 2005).…”