A. terreus Accessory Conidia Figure 2. Cell wall architecture of phialidic and accessory conidia of A. terreus. Comparative scanning electron microscopy (A and B) and transmission electron microscopy (C and D) of A. terreus phialidic conidia (A and C) and accessory conidia (B and D). Conidia were harvested and fixed in 5% glutaraldehyde for microscopy. Dashed arrow indicates melanin like layer and solid arrow indicates the cell wall (C and D); 10–15 conidia were imaged, p = 0.0003. doi:10.1371/journal.pone.0007673.g002 15 conidia were imaged, p = 0.0003) and appeared to lack the dark pigmented layer characteristic of PC. Accessory conidia differ from Phialidic conidia in germination properties, metabolic activity and adherence We hypothesized that differences in cell wall architecture of AC (as observed by SEM and TEM studies) may manifest as other phenotypic differences such as rate of germination and adherence properties of the conidial forms. Results of this study show that AC germinated rapidly (germ tubes were observed within 2 hours of incubation) in vitro when compared to PC (no germ tubes observed at 2 hours) at both temperatures tested. We did not determine the ratio of germinated to ungerminated conidia of both cellular forms for this study. Germination experiments in this study were in vitro observations, performed with only two representative A. terreus isolates, and will need to be confirmed with more isolates and in other environments. If confirmed, the ability of AC to germinate rapidly into tissue invasive hyphal forms may be a virulence factor for A. terreus, given that exit from conidial dormancy and subsequent germination is essential for the establishment of infection [4,16]. In keeping with the altered cell surface phenotype of AC when compared to PC, we found that AC were significantly more adherent to microspheres than PC (26% of AC adhered versus 16% of PC; p value = 0.01) reiterating the cell surface associated differences between these conidial forms. Aspergillus nidulans rodletless mutant strains are more adherent to microspheres than wild-type strains, resulting in changes in physico-chemical properties of the conidia, including reduced hydrophobicity and decreased polarization [9]. In contrast, no differences in adherence were observed between the rodletless mutants and wild type isolates of A. fumigatus. However, studies with A. fumigatus rodlet A mutants and germ tubes that have lost the hydrophobin layer indicate other changes in cell surface receptor expression that affect conidial binding potential to certain surfaces [17]. Thus, the smooth surface of A. terreus AC described in this study and its altered adherence phenotype may reflect a lack of surface hydrophobins. Additionally, studies analyzing differences in adherence of both conidial forms to other surfaces and the cell wall composition of AC are warranted to substantiate this observation. Next, we compared the metabolic activity of both conidial forms using the viability dye XTT. This dye is reduced to a colorimetric formazan product in the presence of metabolically active cells and has been used to quantify metabolic activity and viability of Aspergillus isolates [18]. Results of the present study showed that the ACs were metabolically active as early as thirty minutes as evidenced by an increase in XTT-formazan production; OD of PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7673