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

Abstract

Bacterial infections of central venous catheters (CVCs) cause much morbidity and mortality, and are usually diagnosed by concordant culture of blood and catheter tip. However, studies suggest that culture often fails to detect biofilm bacteria. This study optimizes X-ray micro-focus computed tomography (X-ray µCT) for the quantification and determination of distribution and heterogeneity of biofilms in in vitro CVC model systems. Bacterial culture and scanning electron microscopy (SEM) were used to detect Staphylococcus epidermidis ATCC 35984 biofilms grown on catheters in vitro in both flow and static biofilm models. Alongside this, X-ray µCT techniques were developed in order to detect biofilms inside CVCs. Various contrast agent stains were evaluated using energy-dispersive X-ray spectroscopy (EDS) to further optimize these methods. Catheter material and biofilm were segmented using a semi-automated MATLAB script and quantified using the Avizo Fire software package. X-ray µCT was capable of distinguishing between the degree of biofilm formation across different segments of a CVC flow model. EDS screening of single-and dual-compound contrast stains identified 10 nm gold and silver nitrate as the optimum contrast agent for X-ray µCT. This †These authors contributed equally to this work.Supplementary material is available with the online version of this paper. ã 2016 The Authors Printed in Great Britain

Insights into the Development of Phototrophic Biofilms in a Bioreactor by a Combination of X-ray Microtomography and Optical Coherence Tomography

“…In contrast, X-ray microtomography (µCT) allows for measurement of biofilms and structures non-invasively, inside and outside, for example, complex flow channels [25], glass capillaries [17], central venous catheters [26], or porous media like 3-D-packed bead columns [27]. The measurement of µCT is based on the x-ray attenuation of matter.…”

Testing Contrast Agents to Improve Micro Computerized Tomography (μCT) for Spatial Location of Organic Matter and Biological Material in Soil

“…While I 2 had a good staining efficiency (the K-edge measurement values are very similar to those obtained by the chemical analysis of C), the Ag stained had low efficiency. In previous studies, using conventional µCT, the use of Ag improved contrast of organic material in soil (Van Loo et al, 2014) and bacterial material in catheter biofilms (Niehaus et al, 2016). Ag has high affinity to bind to SOM, polyssaccharidic debris of plants and microbial cell walls, forming chelates and complexes with organic material, for example, containing carboxyl, phenolic, alcoholic and enolic-OH, and carbonyl structures of various types (Chenu and Plante, 2006;Van Loo et al, 2014).…”

“…The soil was sieved to a diameter that fits in the small tubes (500 µm) and to exclude small fractions that could spill through the bottom of the containers (38 µm). These soil containers were then placed inside 1.5 ml centrifuge tubes and the soil underwent the following staining procedures: 1) Control (no treatment); 2) Lugol (aqueous solution of I 2 KI, Sigma 32922), 5 µl of the solution was pipetted in the upper part of the soil container (the soil was visually saturated); 3) Eosin (Merck Certistain R , Y 15935), 5 µl of a 30% solution (wt/vol in ultrapure water) was pipetted into the soil container; 4) AgNO 3 (Roth, 7908), 5 µl of a 0.3% solution (wt/vol in acetate buffer, Niehaus et al, 2016) was pipetted into the soil container; 5) Fixed-sample and Eosin [modified from Busse et al (2018)], first, 5 µl of fixative solution (950 µl of formaldehyde solution and 50 µl glacial acetic acid) was pipetted into the soil container and then incubated overnight 4 • C. The next day the tube was slowly centrifuged at 100 g for 2 min and washed twice with Dulbecco's phosphate-buffered saline solution (DPBS, Thermo Fischer, D8537). Then Eosin was applied as explained for treatment "3."…”

“…The principle is that organic matter has strong affinity for some heavy elements and that these elements (with Z > 30) increase the X-ray attenuation, thus improving imaging contrast (Van Loo et al, 2014). Among the most effective agents for soil, silver (Ag) stands out (Van Loo et al, 2014;Niehaus et al, 2016). Several other heavy metals bind with SOM, such as Cu, Co, Fe, Mn, Mo, and Zn, but since they naturally occur in soil, they should be avoided as contrast agents to reduce confounding effects.…”

Unraveling Structural Details in Ga-Pd SCALMS Systems Using Correlative Nano-CT, 360° Electron Tomography and Analytical TEM

“…These methods are usually historically realized at synchrotron facilities [25,26]. However, with the development and transfer of synchrotron optics to lab instruments in recent years, micro-CT and nano-CT can be performed in the lab with amazingly comparable performance, except for the X-ray source brilliance [27][28][29][30][31][32][33][34]. State-of-the-art lab nano-CT instruments like the ZEISS Xradia 810 Ultra typically offer several imaging modalities.…”