TEM Analysis of Aspergillus Biofilms

The biofilm preparation for TEM analyses was performed as previously described (Melloul et al., 2016 (link)). Briefly, samples were fixed with 2.5% glutaraldehyde-cacodylate buffer (pH 6.5) and then post-fixed with osmium tetroxide and dehydrated with different dilutions of alcohol (50–100%). The samples were then embedded into EPON resin and left to polymerize. Ultra-fine sections were obtained using a Leica UC7-RT ultramicrotome, and contrasted with lead-citrate and uranyl-acetate solutions. Specimens were mounted on grids to be examined under the microscope (JEOL 100 CX II instrument, Japan). TEM was used to measure the cell wall thickness of A. fumigatus in single and mixed biofilms using ImageJ program. For both biofilms, between 10 and 20 measurements of cell wall thickness were performed on 15 different hyphae.