Influence of incubation time, inoculum size, and glucose concentrations on spectrophotometric endpoint determinations for amphotericin B, fluconazole, and itraconazole

Abstract

We addressed the influence of the incubation time (24 h versus 48 h), starting inoculum size (standard inoculum size, approximately 10(3) CFU/ml, versus large inoculum size, approximately 10(4) CFU/ml), and supplementation with 2% glucose of RPMI 1640 medium on the spectrophotometric determination of the MICs of amphotericin B, fluconazole, and itraconazole. We compared the MICs determined spectrophotometrically with those determined by the standard broth macrodilution method (National Committee for Clinical Laboratory Standards approved guideline M27-A). The agreement between the results of the spectrophotometric and standard methods for amphotericin B testing was 100%; this agreement was independent of the inoculum size and incubation time. On the other hand, the agreement for the results for fluconazole testing and itraconazole testing was dependent on the inoculum size and incubation time. With large inoculum size, excellent agreement can be achieved at 24 h. With standard inoculum size, acceptable agreement can be achieved only at 48 h. In contrast to previous observations, the addition of 2% glucose did not have any significant impact on the growth density at 24 h, nor did it improve the agreement with the standard method. Furthermore, supplemental glucose might falsely elevate the MIC at 48 h.

FIG. 1

Influence of inoculum size and glucose concentration on the OD curve and on the fluconazole MIC determination for an isolate of C. albicans. 1, standard inoculum size (∼10³ CFU/mL), RPMI 1640 medium; 2, standard inoculum size (∼10³ CFU/mL), RPMI 1640 medium supplemented with 2% glucose; 3, large inoculum size (∼10⁴ CFU/mL), RPMI 1640 medium; and 4, large inoculum size (∼10⁴ CFU/mL), RPMI 1640 medium supplemented with 2% glucose. The MICs determined by testing with large inoculum size and medium supplemented with 2% glucose were 2 μg/ml, and the MICs determined by testing under other conditions were 4 μg/ml.