A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol

• Published in: Analytical biochemistry 2011-12, Vol.419 (1), p.26-32
• Main Authors: Hassan, Rabeay Y.A., Bilitewski, Ursula
• Format: Article
• Language: English
• Subjects: 2,6-Dichloroindophenol - metabolism; Candida albicans; Candida albicans - growth & development; Candida albicans - metabolism; Candida glabrata; Candida glabrata - growth & development; Candida glabrata - metabolism; carbon; Catalysis; Color; Complex I activity; complexing; Electrochemistry; electron transfer; Electron Transport - drug effects; electron transport chain; Enzyme Activation; enzyme activity; Fungal Proteins; galactose; Galactose - metabolism; glucose; Glucose - metabolism; Metabolic activation; Microbial Viability; Microbiological Techniques; NAD (coenzyme); NAD - metabolism; NADH Dehydrogenase - metabolism; NADH dehydrogenases; oxidation; Oxidation-Reduction; Oxygen - metabolism; pathogens; Respiratory chain inhibitors; rotenone; Rotenone - pharmacology; Saccharomyces cerevisiae; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Spectrophotometry; viability; Yeasts
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2011.07.025

Candida albicans is an opportunistic fungal pathogen with comparably high respiratory activity. Thus, we established a viability test based on 2,6-dichlorophenolindophenol (DCIP), a membrane-permeable electron transfer agent. NADH dehydrogenases catalyze the reduction of DCIP by NADH, and the enzymatic activity can be determined either electrochemically via oxidation reactions of DCIP or photometrically. Among the specific respiratory chain inhibitors, only the complex I inhibitor rotenone decreased the DCIP signal from C. albicans, leaving residual activity of approximately 30%. Thus, the DCIP-reducing activity of C. albicans was largely dependent on complex I activity. C. albicans is closely related to the complex I-negative yeast Saccharomyces cerevisiae, which had previously been used in DCIP viability assays. Via comparative studies, in which we included the pathogenic complex I-negative yeast Candida glabrata, we could define assay conditions that allow a distinction of complex I-negative and -positive organisms. Basal levels of DCIP turnover by S. cerevisiae and C. glabrata were only 30% of those obtained from C. albicans but could be increased to the C. albicans level by adding glucose. No significant increases were observed with galactose. DCIP reduction rates from C. albicans were not further increased by any carbon source.