Effects of cell density and growth medium on biofilm formation. A. Effect of conidial density on A. fumigatus-P. aeruginosa polymicrobial biofilm formation. One ml aliquots of AF53470 conidial suspension containing 1 × 102 - 1 × 107 conidia/ml were incubated in 24-well cell culture plates in duplicates at 35°C in SD broth for 18 h, washed and then inoculated with 1 × 106 PA56402 cells in 1 ml SD broth and further incubated for 24 h for the development of A. fumigatus-P. aeruginosa polymicrobial biofilm. The biofilm was washed and the embedded cells were resuspended in 1 ml sterile water and assayed for A. fumigatus and P. aeruginosa by CFU counts. The experiment was performed at two different times using independently prepared conidial suspensions and bacterial cultures and the vertical bar on each data point on the graph represents the standard error of the mean. B. P. aeruginosa monomicrobial biofilm formation in various growth media with and without bovine serum. One ml aliquots of growth media containing 1 × 106
P. aeruginosa cells were incubated in quadruplicates in 24-well cell culture plates with and without 10% bovine serum for 24 h at 35°C for biofilm formation. The adherent monomicrobial biofilm was washed (3 times), resuspended in 1 ml sterile distilled water and the biofilm growth was assessed by CFU assay. The experiment was performed two different times with PA56402 using independently prepared bacterial cultures, and one time with PA27853. Both sets of isolates provided similar results. The data were analyzed by paired Student’s t test using GraphPad prism 5.0. The vertical bar on each histogram denotes standard error of the mean for two independent experiments using PA56402. Legends: SD, Sabouraud’s dextrose broth; SD-BS, Sabouraud’s dextrose broth with 10% bovine serum; BHI, Brain Heart Infusion broth; BHI-BS Brain Heart Infusion broth with 10% bovine serum; RPMI, RPMI640; RPMI-BS, RPMI1640 with 10% bovine serum.