IADR Abstract Archives
Real-time Approach to Flow Cell Imaging of Fungal Biofilm Development
Objectives: Flow cells have been used to assess Candida albicans biofilm development at one time point, however these studies did not evaluate the dynamic events leading to biofilm formation. Therefore, our objective is to develop a system to identify dynamic events of C. albicans biofilm formation under flow and in real time, and use this system to assess the role of hyphae in non-static biofilm development.
Methods: C. albicans biofilms were grown for 18 h either at 37°C (hyphae-inducing) or 30°C (non-hyphal inducing/yeast form cells) under flow through a ibidi® µ-slide™ flow chamber, and images were captured every 15 min using darkfield or interference reflection (IR) microscopy. Cell-seeded media containing 106 cells/ml was used for 2 h for the attachment phase, before switching to cell-free media for 16 h for the development phase. C. albicans low adhesion/hypofilamentous (Δefg1) and low adhesion (Δbcr1) mutants were compared with wild-type cells. Time-lapse movies were quantitated for fungal biomass, rates of attachment and detachment, and strength of adhesion.
Results: Wild-type cells grown at 37°C had the greatest total biomass at 18 h, while cells grown at 30°C had 72% less total biomass. Mutants had ~99% reduction in biomass. All fungal cells including adhesion-defective mutants had similar rates of attachment for the first 2 h. However, rates of detachment during the development phase were double for Δbcr1 and triple for Δefg1 cells compared with wild-type at 37°C, accounting for the total biomass reduction after 18h. IR imaging showed hyphae were more adherent than yeast cells to the surface.
Conclusions: C. albicans biofilm defective mutants had no differences in early attachment, but had elevated detachment rates during maturation; showing that separate gene sets expressed during biofilm maturation regulate attachment/detachment events. Real-time flow imaging allows analyses of total cell population events during biofilm development.
Methods: C. albicans biofilms were grown for 18 h either at 37°C (hyphae-inducing) or 30°C (non-hyphal inducing/yeast form cells) under flow through a ibidi® µ-slide™ flow chamber, and images were captured every 15 min using darkfield or interference reflection (IR) microscopy. Cell-seeded media containing 106 cells/ml was used for 2 h for the attachment phase, before switching to cell-free media for 16 h for the development phase. C. albicans low adhesion/hypofilamentous (Δefg1) and low adhesion (Δbcr1) mutants were compared with wild-type cells. Time-lapse movies were quantitated for fungal biomass, rates of attachment and detachment, and strength of adhesion.
Results: Wild-type cells grown at 37°C had the greatest total biomass at 18 h, while cells grown at 30°C had 72% less total biomass. Mutants had ~99% reduction in biomass. All fungal cells including adhesion-defective mutants had similar rates of attachment for the first 2 h. However, rates of detachment during the development phase were double for Δbcr1 and triple for Δefg1 cells compared with wild-type at 37°C, accounting for the total biomass reduction after 18h. IR imaging showed hyphae were more adherent than yeast cells to the surface.
Conclusions: C. albicans biofilm defective mutants had no differences in early attachment, but had elevated detachment rates during maturation; showing that separate gene sets expressed during biofilm maturation regulate attachment/detachment events. Real-time flow imaging allows analyses of total cell population events during biofilm development.