cDNA were synthesized from the isolated RNA using iScript cDNA Synthesis Kit

Analysis of Aspergillus values from susceptibility testing is therefore necessary for some clinical isolates, but when inhibition of growth is not necessarily complete, deriving exact MIC values can be problematic. An alternative, minimal effective concentration based around echinocandin-induced morphology changes, has been proposed but determination of this value is rather subjective. Culture on a porous aluminium oxide support offers advantages in studying the effects of drugs on microorganisms over direct growth on agar or liquid culture. This ceramic material permits effective imaging of large “9357531 numbers of microcolonies cultured on its upper DCC 2618 web surface with imaging by fluorescence and scanning electron microscopy. Strips of PAO are highly porous but only 60 mm thick. Therefore, it is possible to add or remove compounds rapidly but with minimal disturbance by moving the material between agars of different compositions. This method has been previously used to osmotically stress bacteria and to dose microcolonies of bacteria and Candida spp. during rapid drug “7644474 susceptibility testing. Microcolony-based methods combined with fluorogenic dyes and imaging may offer particular advantages with respect to studying filamentous fungi, a group of organisms for which dispersed growth in liquid culture is not necessarily the most relevant. In this study, PAO culture was used to explore the lethality of caspofungin and anidulafungin to A. fumigatus, and also the recovery from these drugs, by imaging individual microcolonies and by quantifying the effects. limitation, but this was not relevant to the microcolony studies presented in this work. Growth of A. fumigatus and A. terreus with anidulafungin and caspofungin Results Germination and growth of A. fumigatus and A. terreus on PAO Dispersal and germination of conidia on PAO. Conidia purified from clinical isolates and a reference strains of A. fumigatus and A. terreus were inoculated onto 3668 mm strips of PAO placed on Sabouraud agar to a density of from 5 to 50 cfu/mm2. After incubation for 4 to 10 h at 37uC the fungi on PAO strips were stained with Fun-1/calcofluor white and the percentage germination assessed. Both germinated and ungerminated conidia of both species could be imaged and distinguished upon PAO by this method. The distribution of conidia immediately after inoculation was predominantly of single spores. The median swelling and outgrowth times for all strains tested were similar on PAO placed on Sabouraud agar compared to growth directly on the same agar. This 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 gr