Crystal violet staining to quantify 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 time-consuming. 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.

Candida tropicalis biofilms: Effect on urinary epithelial cells

“…The remaining yeasts attached to the monolayer were quantified using the CV staining method, according to Negri et al [11]. The mean absorbance of yeasts was expressed as the absorbance per area of each well and standardized by the number of adhered yeasts per area of each well using C. tropicalis standard curve [11]. All the procedures were repeated in triplicate in at least three separate assays.…”

“…After 2 h of contact at 37 C under 5% CO 2 , the coupons containing the biofilms and the suspension with planktonic cells were removed, then, each well washed once with PBS. The remaining yeasts attached to the monolayer were quantified using the CV staining method, according to Negri et al [11]. The mean absorbance of yeasts was expressed as the absorbance per area of each well and standardized by the number of adhered yeasts per area of each well using C. tropicalis standard curve [11].…”

Phenotypic switching ofCandida tropicalisis associated with cell damage in epithelial cells and virulence inGalleria mellonellamodel

“…22,23 According to the literature, C. tropicalis has ability to adhere and proliferate into different epithelial cell ) resulted in significantly higher mortality than inoculation of 2£10 6 yeast cells per larvae (P D 0.0159, log-rank test) and 1£10 6 yeast cells per larvae (P D 0.0001, log-rank test). Control included larvae injected with PBS.…”

Effect of miR-146 targeted HDMCP up-regulation in the pathogenesis of nonalcoholic steatohepatitis

“…Crystal violet staining (GenMed, Minnesota, the USA) in a co-culture system of transwell plates to test the inflammatory cell infiltration ability of HFFA-cultured L02 cells was applied, based on its ability of being absorbed by cells colonized in monolayer[18]. Transwell method was applied as previously described while HL-60 cell was chosen to represent inflammatory cell in NASH for its neutrophil like phenotype[19].…”