In vitro test of virulence using the macrophage killing assay. Macrophages were seeded onto a glass slide and subsequently co-incubated with C. albicans strains at a multiplicity of infection of 2. (A) Live macrophage cells from four fields per strain tested were counted and the averages were compared using ANOVA. Results from co-incubations at T = 24 hours are shown. An asterisk (*) indicates statistical significance, p < 0.0001. (B) Live (green) and dead (red) macrophage cells were co-stained with calcein AM and ethidium bromide homodimer-1 (LIVE/DEAD Viability/Cytotoxicity kit, Invitrogen), respectively, and visualized by fluorescence microscopy. Representative images from the 24 hour timepoint for each strain are shown.