Both conidial forms were assessed for their susceptibilities to AMB using the FUN 1 based assay as described previously and the CLSI microbroth dilution method [13]. For the flow cytometry based assay (FCM), PC and AC were obtained from A. terreus isolates CLF 29 and CLF 52, and 200 ml of the conidial suspension (56106 cells/ml SDB) was dispensed into polystyrene round- bottom FACS tubes. Two hundred microliters of AMB (Sigma) (diluted in SDB) were added to yield final drug concentrations of 2 mg/ml to 64 mg/ml [14] and the tubes were incubated at 37uC for 4 hours. FUN-1 was added to a final concentration of 0.5 mmol, incubated in the dark for 40 minutes at 37uC with shaking, and acquired in FACScan cytometer with data analyzed using CellQuest software. The CLSI microbroth method was performed as previously described with a slight modification wherein both the conidial forms were counted using a hemocytometer before the conidial inocula (56105) were dispensed into microtiter wells containing serial twofold dilutions of AMB, and plates were incubated for 48 h at 35uC. MIC was defined as the lowest concentration of drug that produced 100% reduction in the optical density at 550 nm (OD550) relative to the growth control. In these studies, the Student T-test was used to determine statistical significance. Results and Discussion Both clinical and environmental A. terreus isolates produce accessory conidia Although it has been previously documented that A. terreus can produce AC, it was not known if all strains within this species can produce these structures or if there is variability in quality and quantity of AC production between A. terreus strains recovered from clinical and environmental origin. In this study, we examined 100 A. terreus isolates from both clinical and environmental samples for their ability to produce AC in vitro. We found that although there were differences in the number of AC that each isolate produced, all A. terreus isolates, regardless of their origin, produced these structures (data not shown). We further speculated that the ability to produce AC may better facilitate A. terreus to establish infection, and thus isolates recovered from clinical samples may have the potential to produce more AC than isolates recovered from environmental samples. Results of our study demonstrate that isolates from clinical samples produced on average 2.36105 conidia/ml while isolates from environmental samples produced on average 1.96105 conidia/ml - thus there was no correlation A. terreus Accessory Conidia between the origin of the isolate and AC production. Interestingly, our study shows a wide variability in the size and shape of AC ranging from globose (Figure 1A), to ellipsoidal, to club-shaped (Figure 1B). Some isolates produced AC in clusters, some in pairs (Figure 1C), and in other cases AC arose singly from hyphae (Figure 1A–B). The variation in AC morphology was strain dependent and was fairly consistent for a given strain. Thus, this study demonstrates that all the tested A. terreus isolates regardless of origin (clinical versus environmental) have the ability to produce AC, albeit in different quantities and shapes. Accessory conidia have distinct cell wall architecture Little or nothing is known thus far about the cell wall architecture of the PC and AC of A. terreus and to address this, we performed SEM and TEM imaging studies on both these conidial forms with the PC of A. fumigatus as a control. Previous studies have revealed that the A. fumigatus conidial cell wall has an outermost melanized layer consisting of interwoven rodlet fascicles and other hydrophobins, giving the conidia their green, echinulate morphology and rendering them hydrophobic [15]. Homologous structures have also been demonstrated in A. oryzae, A. niger, and A. nidulans [9]. In keeping with these studies, our study revealed that A. fumigatus PC were 2–4 mm in diameter, and had an ornamented surface with distinct protuberances giving it an echinulate morphology (data not shown), whereas the surface of A. terreus PC had a striated outer surface with rod shaped structures that were arranged closely on the surface; PC were 2–4 mm in diameter (Figure 2A). In contrast to PC of both these aspergilli, A. terreus AC were bigger, about 4–7 mm in diameter exhibiting a smooth conidial surface with no rod shaped structures or protuberances evident (Figure 2B). Beneath the melanized conidial surface of A. fumigatus lies the cell wall that is composed of b1-3 glucan, b1-3 glucan synthase, chitin, chitin synthases, and galactomannan, among other cell associated molecules [15]. In this study, TEM analysis of both A. terreus and A. fumigatus PC revealed that the outermost cell surface layer of conidia from both species was composed of a thick, dark melanin-like layer (Figure 2C, indicated with a dashed arrow) under which a thin cell wall layer was evident (Figure 2C, indicated with a solid arrow). In marked contrast, the outermost dark, melanin-like outer layer was absent in A. terreus AC and a smooth, thicker non-pigmented cell wall was observed (Figure 2D, indicated with a solid arrow). Collectively, the SEM and TEM studies demonstrate that the outer surface of AC was smoother and thicker than PC (,12567 nm AC versus 5063 nm PC; 10– Figure 1. Aspergillus terreus Accessory conidia production. Light microscopic images of accessory conidia recovered from A. terreus isolates IBT 12713 (A), P12 (B), E5 (C) demonstrating globose (A) and club-shaped (B) and in pair (C) phenotypes. Scale bars represent 20 mm. doi:10.1371/journal.pone.0007673.g001 PLoS ONE | www.plosone.org 3 October 2009 | Volume 4 | Issue 10 | e7673