Fluorescence assay for the detection of adherent Candida yeasts to target cells in microtest plates.
We describe an assay based on photometric analysis for the measurement of adherence of Candida species to epithelial target cells (Vero cell line). Adherent Candida cells were detected by staining the cells with the fluorescent dye Calcofluor white (CFW), which binds to chitin and glucan in the yeasts. The tests were performed on microtest plates, which were analysed automatically by fluorescence plate readers. The assay is based on the following steps: (i) coating of the microtest plates with target cells (e.g. Vero cells); (ii) infection with Candida: (iii) staining of Candida with CFW; (iv) rinsing to remove non-adherent Candida cells and unbound dye; (v) detection of adherent fluorescent Candida cells. The test was able to detect 4 x 10(4) cells ml-1. The standard deviation was +/- 8%. Day-to-day variation was +/- 10% at most. The adherence of strains of different Candida species was assayed by a standard procedure. The results confirmed the order of adherence, with C. albicans ranking first, followed by C. tropicalis, C. parapsilosis and C. glabrata.
Borg-von Zepelin
M
M
Department of Medical Microbiology, University of Göttingen, Germany.
Wagner
T
T
D000818:Animals; D001557:Benzenesulfonates / Q000494:pharmacology; D002175:Candida / Q000502:physiology*; D002176:Candida albicans / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D013045:Species Specificity; D014709:Vero Cells
D001557:Benzenesulfonates; D005456:Fluorescent Dyes; C007061:C.I. Fluorescent Brightening Agent 28