The between Cyclosporin A and immunosuppressive and calcineurin Microcolony Analysis of Aspergillus Discussion A. fumigatus was found to germinate, grow and produce conidia on a PAO support. Conidial germination and hyphal extension and microcolony growth 
appeared efficient on aluminium oxide, these parameters were similar to growth on agar. The addition of echinocandins allowed the effects of these drugs to be studied at the microscopic level, in microcolonies up to several hundred microns in diameter. As 21278085” with other culture methods on PAO, imaging by fluorescence microscopy after staining with fluorogenic dyes or processing for SEM was facilitated by the inert support. This has advantages over previous microscopic studies of the effect of caspofungin on cells, with the ready addition and removal of reagents without disturbing the microcolony. Additionally, effective imaging was possible on the planar surface. This permitted relatively high throughput image processing and quantification compared to previous studies, which used liquid culture to investigate caspofungin and micafungin lethality. As far as we are aware, this is the first report of anidulafungin lethality. Caspofungin is the only echinocandin to have been studied both in liquid culture and on PAO. Broadly, the two studies reveal similar trends, with one exception. Lysis was most apparent on PAO at intermediate concentrations of caspofungin, i.e. those close to MIC values. Higher concentrations of both echinocandins limited microcolony growth to a greater extent but resulted in reduced lysis. This may be due to more rapidly growing cells being more vulnerable to lysis. Paradoxical growth has previously been described for A. fumigatus treated with echinocandins, i.e. when very high doses have a lesser effect on growth compared to intermediate dosage. Explanations of paradoxical growth often invoke compensatory mechanisms for limiting cell wall damage, such as the stimulation of chitin synthesis, which reduce drug effects at high concentrations. However, both at the microcolony pathway inhibitor cyclosporine A is known to have fungicidal activity for A. fumigatus. Cyclosporine has been shown to combine positively as an antifungal agent with caspofungin against some clinical isolates of A. fumigatus. Therefore, this interaction was tested on PAO. After 14 h culture on PAO, cyclosporine A induced rounded cells staining predominantly with Syto 9 when present at concentrations.3 mg/ml. At 3 mg/ml cyclosporine had little effect on cell morphology alone but did enhance the action of echinocandins. Microcolony Analysis of Aspergillus level, and in E-tests, we did not observe classical paradoxical effects in these studies. In contrast, for liquid culture increasing concentrations of caspofungin above the MIC has been reported to lead to enhanced lysis and cell death. The KU55933 site ability to separate growth inhibition and tip lysis using microcolony imaging should allow paradoxical effects of drugs to be studied effectively in the future. Apart from the obvious damage, two supporting lines of evidence indicated that cell death was occurring. The first is an excellent correlation between propidium iodide staining and lysis of hyphal tips. So-called “live-dead”staining is commonly used within bacteriology as an indicator of membrane permeability and therefore as an indirect measure of cell death. Whilst this staining method is not commonly used for Aspergillus, this dye pairing is sold for yeast