Photomicrographic images and quantification of A. fumigatus and P. aeruginosa biofilms. A. Monomicrobial biofilm of AF53470 grown on plastic cover slips for 48 h at 35°C. B. Monomicrobial biofilm of PA56402 grown on plastic cover slips for 48 h at 35°C. C. Polymicrobial biofilm formed in coculture by AF53470 sporelings and PA56402 grown on plastic cover slips for 48 h at 35°C. The biofilms were photographed using a Nikon Microscope Camera System equipped with SPOT image processing computer software . With the SPOT program, each Objective (10× to 100×) of the microscope was calibrated using a stage micrometer as previously described in the SPOT Software User Guide (Chapter 4, pages 76 and 77). The photomicrographs shown in Figure 1 were captured using the 60× Objective providing a total magnification of 600×. D. Quantification of 24-h and 48-h monomicrobial and polymicrobial biofilms of AF53470 and PA56402. The biofilm quantification experiment by crystal violet binding assay was performed two times with eight replications for each group. The data were analyzed by two-way ANOVA and paired Student’s t-test using GraphPad Prism 5.0. The vertical bar on each histogram represents the standard error of the mean for two independent experiments. The laboratory isolates AF36607 and PA27853 also produced similar monomicrobial and polymicrobial biofilms on plastic cover slips and Costar 6-well cell culture plates.