Variability of agar dilution-replicator method of yeast susceptibility testing

The agar dilution method of in vitro susceptibility testing of fungi was analyzed with a Steers-type inoculum replicator, ten strains, and three drugs. The replicator reproducibly delivered the same inoculum to each series of plates. The minimal inhibitory concentrations of ketoconazole (an imidazole) and 5-fluorocytosine, but not that of the polyene nystatin, were dependent on the initial inoculum size. With the former two drugs, but not with the latter, minimal inhibitory concentrations were highly variable depending on the time of reading. Results with agar and broth dilution methods were divergent, and the differences in minimal inhibitory concentrations were variable in serial comparisons by the two methods. If the agar dilution minimal inhibitory concentrations were determined at first appearance of control growth, a commonly used end point, small variations in the time of reading (as could occur by variation in observer perception of when initial growth appears) induced large variations in the minimal inhibitory concentrations of 5-fluorocytosine and ketoconazole, particularly with rapidly growing strains. Results at 35 and 30 degrees C were similar. The differences in results with the three drugs suggest different mechanisms of action. The variability quantitated with the agar dilution method could result in variability in results between laboratories and even observers in the same laboratory.