Crystal violet staining to quantity Candida adhesion to epithelial cells

Abstract

In vitro studies of adhesion capability are essential to characterise the virulence of Candida species. However, the assessment of adhesion by traditional methods is timeconsuming. The aim of the present study is the development of a simple methodology using crystal violet staining to quantify in vitro adhesion of different Candida species to epithelial cells. The experiments are performed using Candida albicans (ATCC 90028), C. glabrata (ATCC 2001), C. parapsilosis (ATCC 22019) and C. tropicalis (ATCC 750). A human urinary bladder epithelial cell line (TCC-SUP) is used. Yeast and epithelial cells were stained with crystal violet, epithelial cells were then destained using intermediate washing, and the dye in the yeast cells was extracted with acetic acid. The method was validated for the different Candida reference species by comparison with traditional microscope observation and enumeration. The method was then used to assess Candida adhesion to epithelial cells and also to silicone. For all Candida spp. high correlation values (r2= 0.9724–0.9997) between the number of adherent yeasts (microscope enumeration) and absorbance values were obtained for an inoculum concentration >10 6 cells/mL. The proposed technique was easy to perform and reproducible, enabling the determination of adhesion ability of Candida species to an epithelial cell line.