Method Optimization for High-throughput antifungal screening

A successful antifungal susceptibility test in vitro, which should be adapted for robotic system for high-throughput procedures, is wholly depending upon standardization on every part of the assay. A number of factors may influence on the evaluation of MIC (minimal inhibitory concentration) values. The sensitivity of the antifungal susceptibility test in vitro is dependent on inoculums preparation, the solubility and stability of the drugs, pH and composition of the medium, the duration and the temperature of incubation and criteria for determining the endpoints. In this study, we compare effect of MIC evaluation on three different fungal strains (C. albicans, C. kruzei and S. cervisiae) for three standard antifungal drugs (amphotericin B, flucanazole, myconazole) in different pH of YNG medium (pH 6 and pH 7) and in RPMI. The optical density of cultures was measured at 620nm on spectrophotometer TECAN, Ultra after 24 and 48 hours of incubation. The MIC endpoint for amphotericin B was defined as the minimum concentration in which no growth could be observed. Readings for the other two antifungal drugs was the lowest drug concentration that met the criterion: %T>%K+0.5(100-%K) where T is transmission and K is the drug-free well. Parallel, the turbidity measurements of the same plates were read in Nepheloskan Ascent (Labsystems). Comparison of MIC values showed that measurements of turbidity and optical density have no difference for myconazole and amphotericin B but for flucanazole the MIC values were more precisely determinate with turbidity measurements. Influence of pH on MIC values didn’t showed significant difference compared with MIC values in RPMI media. All tests were performed in 96-well plate format and automatized for TECAN Genesis 150 robotic system. According to present advantages of turbidity measurements in antifungal high-throughput screening, the monitoring of continuously growth in 10 different time points of flucanazole resistant C. albicans incubated with three standard drugs will be present.