Effects of incubation time and buffer concentration on in vitro activities of antifungal agents against Candida albicans

Nine selected isolates of Candida albicans were tested for their susceptibilities to amphotericin B and fluconazole by using three methods to assess the effect of incubation time and buffer concentration. By using a microdilution method with 0.0165 M 3-(N-morpholino)propanesulfonic acid (MOPS) and a 24-h incubation time, all of the isolates were found to be susceptible to amphotericin B and fluconazole. After 48 h of incubation, all isolates were still susceptible to amphotericin B. Seven of the nine isolates were resistant to fluconazole, and for the remaining two isolates, MICs increased by fourfold or more but the isolates remained susceptible (MIC, less than or equal to 10 mu g/ml). The nine isolates, along with three control strains, were further tested against amphotericin B and fluconazole by a standard broth macrodilution method with both 0.165 and 0.0165 M MOPS. The susceptibility results for fluconazole by the broth macrodilution method with the lower MOPS concentration correlated with the results of the 24-h broth microdilution method for determination of susceptibility or resistance in eight of nine tests and with the results of the 48 h broth microdilution method in three of nine tests. The results of the broth macrodilution method with the standard MOPS concentration did not correlate with any of the results obtained by the 24-h broth microdilution but correlated with results of seven of nine tests by the 48-h broth microdilution method. All nine test strains appeared to be susceptible when they were examined by a flow cytometric method. For clinical yeast susceptibility testing in microdilution panels, the 0.0165 M MOPS concentration combined with 24 h of incubation appeared to be the method of choice. The lower MOPS concentration may also be a useful modification to the tentative broth macrodilution method of the National Committee for Clinical Laboratory Standards. Use of the higher buffer concentration or longer incubation time may lead to false in vitro resistance for agents like fluconazole.