Confocal Laser Scanning Microscopy of Candida Infecting Epithelial Tissue

Objectives: The objectives of this study were to characterize the invasion of selected Candida albicans strains within an in vitro tissue model of oral candidosis using confocal laser scanning microscopy (CLSM). Initial experiments examined C. albicans strains that had been isolated from a range of oral conditions including oral candidosis, oral cancer and healthy oral mucosa. Methods: Isolates were cultured in yeast nitrogen base medium for 24 h at 37°C and the yeast cells harvested by centrifugation. Cells were washed (×3) in phosphate buffered saline and adjusted to a density of 4×10⁷ cell/ml. One-ml of this cell preparation was used to overlay reconstituted human oral epithelium (0.5 cm² area; SkinEthic, Nice, France). Inoculated tissue was incubated at 37°C in a CO₂ enriched environment for 24 h. One half of this tissue was then stained using concanavalin A-Alexa 594 conjugated lectin and the remaining portion retained for RNA analysis. Results: Confocal laser scanning microscopy (CLSM) revealed a strain dependent tissue invasion with differences evident both in terms of depth and pattern of tissue penetration by candidal hyphae. Conclusions: CLSM provides an ideal tool for investigating the process of tissue invasion by C. albicans and preliminary results have shown strain variability in the invasion process which could relate directly to the pathogenic potential of these yeast in vivo.