Real-time confocal imaging Analysis of mitochondrial inner membrane potential

that this approach may be more widely usable, for example to investigate fungi difficult to study in other ways because of slow growth and/ or limitations of liquid culture methods such as aggregation. It is possible to change the properties of PAO, which may allow a systematic investigation as to how filamentous fungi are affected by surfaces. Imaging by fluorescence microscopy can detect marginal growth and recovery from echinocandins. As gradients of drugs can be created beneath PAO supports, more complex drug effects can be investigated for example issues of synergy or antagonism between multiple antibiotics or growth under extreme stress. 7 Microcolony Analysis of Aspergillus or by using an E-strip aligned with the long axis of a 36 mm68 mm PAO strip. E-tests were performed directly on Sabouraud agar as recommended by the manufacturer, with 36 h incubation at 37uC. Cyclosporine A was obtained from Sigma Aldrich and was diluted from a stock solution of 25 mg/ml prepared in methanol. All experiments on growth on PAO, drug effects on PAO and conventional MIC testing were performed in triplicate. Recovery of A. fumigatus from echinocandins PAO strips were moved from plates containing echinocandins to those lacking the drugs in order to look at the potential for recovery from echinocandins. Because of the low volume of PAO strips relative to the agar beneath carry-over of drugs to plates lacking echinocandins was K-858 site minimal. Scanning electron microscopy Fixation of microcolonies for imaging by SEM was by one of two methods: 1) gel fixation was achieved by transferring the strip of 10455325 PAO to an agar plate containing Sabouraud medium with 1% glutaraldehyde for 30 min; 2) vapour fixation was performed by inverting an agar plate above the glutaraldehyde/paraformaldehyde fixative for 2 h. In both cases, treatment with osmium tetroxide, ethanol dehydration, critical point drying, sputtering with tungsten and imaging by an FEI Magellan electron microscope were as previously described. Staining and fluorescence microscopy Staining of A. fumigatus and A. terreus with pairs of dyes was performed by transferring the PAO strip to a microscope slide covered in a thin layer of low melting point agar containing the dye. Dye concentrations and staining times were as previously described. The low melting point agar was formulated with water to deliver an osmotic shock or with Sabouraud medium to stain under the same nutrient conditions as growth. Quantification and data analysis Image processing. TIFF format images were processing using ImageJ software. Images were inspected individually and only images with well separated microcolonies processed. Multiple images were then assembled into stacks for batch processing using an ImageJ macro that performed the operations of: application of a median filter, thresholding to black microcolonies on a white background and using the “analyze particle”function to determine the dimensions of each microcolony within the field of view. Objects only partially within a field of view were excluded from this analysis. Datasets were exported into Microsoft Excel for further calculations. Calculations of tip lysis and Syto9 vs propidium iodide staining. The frequency “8560673 of lysed and unlysed tips was calculated from at least 50 microcolonies per condition. Hyphal tips,4 mm Methods Culture of A. fumigatus and A. terreus and exposure to drugs on porous aluminium oxide All strains of Aspergillus species used in this study were clinical