As a negative control, a linear curve was observed for the CCK1R and Lrp5 BRET signal

suggests conidia can be inoculated to single cfu on PAO and germination occurs with the same efficiency to that seen on agar. Growth of mycelia on PAO. Hyphal extension rates were calculated from transmission light microscopy, and were found to be similar on PAO compared to culture directly on the same agar medium. Scanning electron microscopy suggested that the tips of vegetative mycelia of A. fumigatus were lifted off the surface of the PAO during growth; observation by light microscopy supported this conclusion. Visible colonies of all strains tested were smaller on PAO compared to agar, when observed after 24 and 36 h. The formation of conidiophores on PAO was delayed by 12 to 24 h. Taken together, these data suggest that PAO is suitable for the culture of fungi to microcolonies with no nutrient limitation caused by the interposition of a 40% porous filter between agar and the fungus, the changes in surface properties of the growth surface having a major impact. ANOVA with Tukey post hoc test was to determine first time point with a significant increase in area comparing unswollen conidia at T = 0 with subsequent time points to determine swelling time. Outgrowth times were determined in a similar way, comparing the microcolony area of swollen conidia with subsequent time points. b Mean of 20 determinations /2 S.D. The concentrations of anidulafungin and caspofungin that triggered the greatest degree of cellular lysis for strain JBZ11 of A. fumigatus were used in Sabouraud agar plates in viable counts. Viability was scored by counting the number of microcolonies that germinated after 14 h. Anidulafungin reduced viability to 86% and caspofungin to 88% relative to the untreated control. Therefore, the ability of these drugs to prevent microcolony formation was limited. Using higher concentrations viable counts were 81% and 90% of the control. Therefore, again, at the microcolony level the fungicidal activity of these drugs was marginal. These trends were similar for other echinocandin sensitive isolates, i.e. strains ATCC204305, JBZ17 and JBZ32 of A. fumigatus and strain CWZ59 of A. terreus. In contrast, amphotericin B reduced viability,86% when used at the MICs of JBZ11, JBZ13 and JBZ32. Quantification of cell lysis by anidulafungin and caspofungin. Cell lysis was scored for a range of concentrations of caspofungin and anidulafungin after 14 h. Lysis was particularly common at intermediate concentrations around the MIC. In contrast, whilst higher concentrations of echinocandins were more effective at limiting growth lysis was seen less often. Anidulafungin appeared somewhat more effective than caspofungin, with.50% cell lysis at the most effective concentrations. Fungal microcolonies growing on echinocandins appeared heterogeneous in their response. Subpopulations of lysed cells and intact ones coexisted within the same microcolony, ones that were derived, in most cases, from a single conidium. In both cases, a total count of microcolonies after 14 h suggested that neither drug reduced the number of microcolonies, despite having a significant impact on the number of intact hyphal tips within a microcolony. Caspofungin DCC 2618 site resistance increased the concentration of this drug required for optimal tip lysis commensurate with the increased MIC compared to sensitive strains. A series of control experiments were performed, using different staining or fixing and imaging methods, to check that the observed tip lysis was not affected by sample prep