Microbial growth rates and local external mass transfer coefficients in a porous bed biofilm system measured by 19 F magnetic resonance imaging of structure, oxygen concentration, and flow velocity.

19 F nuclear magnetic resonance (NMR) oximetry and 1 H NMR velocimetry were used to noninvasively map oxygen concentrations and hydrodynamics in space and time in a model packed bed biofilm system in the presence and absence of flow. The development of a local oxygen sink associated with a single gel bead inoculated with respiring Escherichia coli was analyzed with a phenomenological model to determine the specific growth rate of the bacteria in situ, returning a value (0.66 hr-1 ) that was close to that measured independently in planktonic culture (0.62 hr-1 ). The decay of oxygen concentration in and around the microbiologically active bead was delayed and slower in experiments conducted under continuous flow in comparison to no-flow experiments. Concentration boundary layer thicknesses were determined and Sherwood numbers calculated to quantify external mass transfer resistance. Boundary layers were thicker in no-flow experiments compared to experiments with flow. Whereas the oxygen concentration profile across a reactive biofilm particle was symmetric in no-flow experiments, it was asymmetric with respect to flow direction in flow experiments with Sherwood numbers on the leading edge (Sh = 7) being larger than the trailing edge (Sh = 3.5). The magnitude of the experimental Sh was comparable to values predicted by a variety of correlations. These spatially resolved measurements of oxygen distribution in a geometrically complex model reveal in innovative detail the local coupling between microbial growth, oxygen consumption, and external mass transfer.

Non-invasive imaging of oxygen concentration in a complex in vitro biofilm infection model using 19 F MRI: Persistence of an oxygen sink despite prolonged antibiotic therapy.

PURPOSE: Oxygen availability is a critical determinant of microbial biofilm activity and antibiotic susceptibility. However, measuring oxygen gradients in these systems remains difficult, with the standard microelectrode approach being both invasive and limited to single-point measurement. The goal of the study was to develop a 19 F MRI approach for 2D oxygen mapping in biofilm systems and to visualize oxygen consumption behavior in real time during antibiotic therapy. METHODS: Oxygen-sensing beads were created by encapsulating an emulsion of oxygen-sensing fluorocarbon into alginate gel. Escherichia coli biofilms were grown in and on the alginate matrix, which was contained inside a packed bed column subjected to nutrient flow, mimicking the complex porous structure of human wound tissue, and subjected to antibiotic challenge. RESULTS: The linear relationship between 19 F spin-lattice relaxation rate R1 and local oxygen concentration permitted noninvasive spatial mapping of oxygen distribution in real time over the course of biofilm growth and subsequent antibiotic challenge. This technique was used to visualize persistence of microbial oxygen respiration during continuous gentamicin administration, providing a time series of complete spatial maps detailing the continued bacterial utilization of oxygen during prolonged chemotherapy in an in vitro biofilm model with complex spatial structure. CONCLUSIONS: Antibiotic exposure temporarily causes oxygen consumption to enter a pseudosteady state wherein oxygen distribution becomes fixed; oxygen sink expansion resumes quickly after antibiotic clearance. This technique may provide valuable information for future investigations of biofilms by permitting the study of complex geometries (typical of in vivo biofilms) and facilitating noninvasive oxygen measurement.

Spatiotemporal mapping of oxygen in a microbially-impacted packed bed using 19F Nuclear magnetic resonance oximetry.

19F magnetic resonance has been used in the medical field for quantifying oxygenation in blood, tissues, and tumors. The 19F NMR oximetry technique exploits the affinity of molecular oxygen for liquid fluorocarbon phases, and the resulting linear dependence of 19F spin-lattice relaxation rate R1 on local oxygen concentration. Bacterial biofilms, aggregates of bacteria encased in a self-secreted matrix of extracellular polymers, are important in environmental, industrial, and clinical settings and oxygen gradients represent a critical determinant of biofilm function. However, measurement of oxygen distribution in biofilms and biofouled porous media is difficult. Here the ability of 19F NMR oximetry to accurately track oxygen profile development in microbial impacted packed bed systems without impacting oxygen transport is demonstrated. Time-stable and inert fluorocarbon containing particles are designed which act as oxygen reporters in porous media systems. Particles are generated by emulsifying and entrapping perfluorooctylbromide (PFOB) into alginate gel, resulting in oxygen-sensing alginate beads that are then used as the solid matrix of the packed bed. 19F oxygenation maps, when combined with 1H velocity maps, allow for insight into the interplay between fluid dynamics and oxygen transport phenomena in these complex biofouled systems. Spatial maps of oxygen consumption rate constants are calculated. The growth characteristics of two bacteria, a non-biofilm forming Escherichia coli and Staphylococcus epidermidis, a strong biofilm-former, are used to demonstrate the novel data provided by the method.

Structured inquiry-based learning: Drosophila GAL4 enhancer trap characterization in an undergraduate laboratory course.

We have developed and tested two linked but separable structured inquiry exercises using a set of Drosophila melanogaster GAL4 enhancer trap strains for an upper-level undergraduate laboratory methods course at Bucknell University. In the first, students learn to perform inverse PCR to identify the genomic location of the GAL4 insertion, using FlyBase to identify flanking sequences and the primary literature to synthesize current knowledge regarding the nearest gene. In the second, we cross each GAL4 strain to a UAS-CD8-GFP reporter strain, and students perform whole mount CNS dissection, immunohistochemistry, confocal imaging, and analysis of developmental expression patterns. We have found these exercises to be very effective in teaching the uses and limitations of PCR and antibody-based techniques as well as critical reading of the primary literature and scientific writing. Students appreciate the opportunity to apply what they learn by generating novel data of use to the wider research community.