Candida albicans viability after exposure to amphotericin B: Assessment using metabolic assays and colony forming units

Abstract

Metabolic assays are a preferred method for evaluation of Candida albicans viability after exposure to antimicrobial agents in cases in which the culture is a complex mixture of yeast and filamentous forms. There is a lack of published data indicating the strength of the correlation between metabolic assays and viable cell numbers determined by a standard assay such as colony forming units (CFU). We developed a kinetic metabolic assay (KMA) for quantifying viable cells which was tested on yeast cells in both exponential and stationary phase using alamarBlue and XTT as metabolic indicators. The KMA enabled quantification of the viable population over a range of 101 to 107 cells that linearly correlated (R2>0.98) with estimates made by enumeration of CFU regardless of the indicator or growth phase of the cells. Linear relationships were used to calibrate the KMA in terms of equivalent CFU. Viable cell populations were then determined after exposure to AmB. These results were compared with those obtained by direct enumeration of CFU. There were significant correlations between KMA-derived equivalent CFU and direct CFU estimates of viable cell populations for exponential-phase cells. However, the proportions of viable cells based on the KMA were consistently lower than those obtained directly by CFU. This trend was substantially more pronounced for stationary phase cells. These results show that even in the relatively simple case in which only the yeast form is present, the relationship between assessment by metabolic assays and CFU is perturbed by exposure to an antimicrobial and that, furthermore, growth phase alters the nature of the perturbation.