surface exposure of AC. We demonstrate that A. terreus accessory conidia are phenotypically distinct, structurally, in cell wall composition, and in in vitro antifungal susceptibility, and as such may contribute to the virulence of this organism. Methods Production of AC by A. terreus isolates from diverse sources One hundred A. terreus isolates recovered from the CDC culture collection were tested for AC production. Of these, 45 isolates were recovered from clinical samples (both colonizing; i.e. clinical isolates that were recovered from sputum, tracheal aspirates, or skin samples, and invasive sites) and 55 isolates were recovered from environmental sampling (soil, air sampling, leaf litter, etc.). All isolates were cultured on Sabouraud dextrose agar plates (SDA) and incubated at 37uC for three days after which phialidic conidia were harvested in phosphate-buffered saline (PBS), and 1,500 ml of the conidial suspension (56105 cells/ml) was transferred to 40 ml Sabouraud dextrose broth (SDB) with 0.1% Tween 20 (SDB Tween) [8]. After 5 days of incubation at 28uC in SDB Tween, the production of AC by each isolate was determined microscopically. For all assays described below, A. terreus isolates CLF 29 and CLF 52, recovered from an environmental and a clinical sample respectively, were employed. Conidial phenotypes by scanning electron microscopy (SEM) & transmission electron microscopy (TEM) For SEM and TEM studies, A. terreus isolates CLF 29 and CLF 52 were sub-cultured on SDA and in SDB Tween (as described above) for production of PC and AC respectively. To prepare PC inocula, Aspergillus colonies were gently probed with a loop and the resultant conidia were suspended in SDB. Accessory conidia were harvested by vortexing the fungal growth and filtering through Whatman 4 filter papers for removal of contaminating hyphal fragments. For SEM, PC and AC were fixed in 5% glutaraldehyde in cacodylate buffer (0.067 mol, pH 6.2) overnight at room temperature. Conidial samples were filtered through a 0.1 mm nucleopore polycarbonate filter, placed on top of a 13 mm glass cover slip, and incubated in a chamber for final dehydration using acetone vapors. The glass cover slip with the filter attached was then mounted on an aluminum stub with silver paint and sputter coated with gold. Finished samples were observed with a FEI XL 30 ESEM (FEI Co., Hillsboro, OR). For TEM, PC and AC were fixed in a 2% gluteraldehyde solution for one hour, followed by incubation overnight in EM buffer (0.01 M phosphate) at room temperature. Conidia were transferred into 1% osmium tetroxide for an hour to provide image contrast, dehydrated in a graded series of ethanol, after which Araldite 502 resin (Ted Pella, Inc) plastic infiltration was performed in a series of Araldite 502 resin and propylene oxide until conidia were in 100% Araldite 502 resin. The conidia in resin were placed into molds and incubated in a 60uC–70uC oven for 18–24 hours. Sections of 60 nm or less were cut using a Diatome diamond knife on a Leica Ultracut UCT and placed onto a copper grid. Grids were washed in uranyl acetate for 2 minutes, followed by lead citrate for 6 minutes, and were then examined in a Philips Techai G2 TEM. Germination and adherence of accessory and phialidic conidia For evaluation of germinating potential of the conidial types, PC and AC were obtained from A. terreus isolates CLF 29 and CLF 52 A. terreus Accessory Conidia as described above. One milliliter of SDB containing 16106co- nidia was transferred to a 24 well plate and incubated at either 30uC or 37uC for 2, 4, 6, 8, 12, and 22 hours. Germination of both conidial forms at different timepoints was assessed by light microscopy. A microsphere adhesion assay was performed to assess relative adhesive properties of both conidial forms [9]. In brief, PC and AC were each resuspended in 0.1 mol/liter KNO3 solution (pH 6.5) and mixed with latex particles (0.8 mm; Sigma) at a ratio of 20:1. The number of microspheres adherent to each conidium was counted using light microscopy in at least 20 different fields. Assessment of metabolic activity of the conidial forms Metabolic activity of both conidial forms was assessed using the colorimetric indicator 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) with an electron-coupling agent, menadione [10]. A. terreus AC and PC from isolates CLF 29 and CLF 52 were harvested as described above. XTT (Sigma) was diluted to 1 mg/ml in warm sterile saline. Menadione (Sigma) was prepared in acetone at a concentration of 200 mM. XTT-menadione solution was pre- pared by mixing 1 ml of XTT (1 mg/ml) with 200 ml diluted menadione in 20 ml of PBS. Isolated conidia were diluted to 56106 cells/ml in XTT-menadione solution, 200 ml plated in 96- well flat bottom plate, and incubated at 37uC in the dark. Optical Density (OD) was read at 0, 0.5, 1, 2, 3, 4, 5, 6, and 7 hours at 490 nm in a spectrophotometer plate reader. Wells with XTT alone were used as background controls. The OD of the well with XTT alone (background) was subtracted from the OD of well with AC or PC with XTT at every time point. Experiments were performed in triplicate. b1-3-glucan exposure on conidial forms For assessing surface b-glucan exposure of both conidial forms, A. terreus AC and PC from isolates CLF 29 and CLF 52 were harvested as described above and resuspended in 1X PBS (plus 0.01% Tween) along with A. fumigatus (ATCC1022) conidia as control. Monoclonal mouse IgG antibody to b1-3-glucan (Biosup- plies Australia, Parkville, Australia) was added at 1:20 concentra- tion and conidia were incubated on ice for 45 minutes. After washing with 1X PBS (plus 0.01% Tween), conidia were resuspended in goat anti-mouse Alexa Fluor 594 conjugated antibody (Molecular Probes) diluted 1:250 in 1X PBS/plus 0.01% Tween, and incubated on ice for 45 minutes. Conidia were washed with 1X PBS/0.01% Tween, resuspended in Fluoromount G (Electron Microscopy Sciences), mounted onto slides, and examined under a fluorescent microscope (Zeiss Axiovert 25). Measurement of Cell Membrane Ergosterol Levels and susceptibility to amphotericin B Cell membrane ergosterol levels were analyzed by a previously published method with slight modifications [11]. Conidial suspensions of both AC and PC from isolates CLF 29 and CLF 52 were harvested as described above and suspended in 150 ml of 25% alcoholic potassium hydroxide solution and mixed for 2 minutes, followed by incubation at 85uC for 2 hours. Sterols were extracted from each sample by adding 60 ml of sterile water and 150 ml of n-heptane, mixing for 2 minutes, and allowing the layers to separate for 30 minutes at room temperature. The n-heptane layer was recovered for spectrophotometric analysis at 230 and 281.5 nm with a Beckman spectrophotometer, and the ergosterol content was calculated based on the absorbance and wet weight of the initial pellet as previously described [12]. PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7673