Stability of Mueller-Hinton Agar Supplemented with Glucose and Methylene Blue for Disk Diffusion Testing of Fluconazole and Voriconazole

Abstract

The shelf life of Mueller-Hinton agar supplemented with 2% glucose and methylene blue (0.5 μg/ml) (MH-GMB) prepared in the laboratory to test disk diffusion of voriconazole and fluconazole was assessed using quality control (QC) strains of Candida albicans ATCC 90028, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019. MH-GMB agar plates were prepared as described in National Committee for Clinical Laboratory Standards document M44-P, and isolates were tested using 25-μg fluconazole disks and 1-μg voriconazole disks over a 36-day period. Zone diameters for fluconazole and voriconazole varied by no more than 4 mm over the study period, and 95 to 100% of results were within the established QC limits for the strains tested. Prepared MH-GMB agar plates provide acceptable performance for disk diffusion testing for at least 30 days when stored at 5°C.

The agar disk diffusion test for fluconazole that has been proposed by the National Committee for Clinical Laboratory Standards (NCCLS) (4) was developed by Meis et al. (3) and refined by Barry and colleagues (2). This method has been expanded to include voriconazole (6, 7) and has been shown to correlate well with NCCLS document M27-A2 (5) broth microdilution results for both fluconazole and voriconazole when tested against Candida spp. (6, 8).

Barry et al. (2) have demonstrated the precision and accuracy of fluconazole disk diffusion testing and have established quality control (QC) zone diameter limits for both fluconazole and voriconazole disk diffusion tests (1, 4, 8) (Table 1). The proposed NCCLS method employs Mueller-Hinton agar (MHA) supplemented with 2% glucose and 0.5 μg of methylene blue (GMB) per ml (4). At the present time, there is no commercial source of MHA supplemented with GMB (MH-GMB); however, MHA may be prepared and supplemented with GMB in the laboratory, or the surfaces of commercially available MHA plates may be flooded with a GMB solution (1). Although prepared and flooded plates have been shown to perform equally well in testing both fluconazole (1) and voriconazole (8), it was noted by Barry et al. (1) that the shelf life of the prepared MH-GMB plates has not been established. Currently, it is recommended that both prepared and flooded plates be used within 24 h of preparation (1, 2, 4, 8).

TABLE 1.

Fluconazole and voriconazole zone diameters determined on prepared MH-GMB over a 36-day period^a

Antifungal agent	Test strain	NCCLS QC zone diameter (mm)b	Incubation time (h)	Zone diameter (mm) on the indicated day after preparation of agar medium:
				3	15	17	25	29	31	36
Fluconazole	C. albicans ATCC 90028	28-39	24	35	35	34	32	34	35	36
			48	35	35	34	32	34	34	35
	C. parapsilosis ATCC 22019	22-33	24	30	30	30	28	29	30	29
			48	29	27	28	28	27	27	28
Voriconazole	C. albicans ATCC 90028	31-42	24	37	35	38	37	36	37	38
			48	35	36	37	37	36	37	37
	C. parapsilosis ATCC 22019	28-37	24	35	33	36	35	32	35	32
			48	34	32	34	33	33	33	32
	C. krusei ATCC 6258	16-25	24	26c	24	25	26c	25	22	24
			48	22	21	21	20	20	20	21

^aAs determined in NCCLS document M44-P (4) and references 2 and 8.

^bQC zone diameter ranges determined after 24 h of incubation.

^cValue outside the NCCLS-recommended range of 16 to 25 mm.

In this study, we used the disk diffusion QC strains recommended by the NCCLS (4) to establish the shelf life of prepared MH-GMB plates for use in disk diffusion testing of fluconazole and voriconazole. The use of prepared MH-GMB plates may be more convenient than flooded plates in those laboratories testing several isolates per day or in laboratories involved in large-scale antifungal surveys (1). Furthermore, the demonstration of an extended shelf life for prepared MH-GMB plates may encourage the commercial development of this specialty medium.

MH-GMB agar plates (150-mm diameter) were prepared as described previously and stored at 5°C in a cold room (2, 6). Plates were removed from the cold room, warmed to ambient temperature, and inoculated with the QC isolates on days 3, 15, 17, 25, 29, 31, and 36 after preparation of the medium. The NCCLS-recommended QC isolates tested were Candida albicans ATCC 90028, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019 (4) (Table 1). On each testing day, individual plates were inoculated with the three QC strains (one per plate) according to NCCLS guidelines (4). Each test plate received a 25-μg fluconazole disk (Becton Dickinson, Sparks, Md.) and a 1-μg voriconazole disk (Becton Dickinson). The plates were incubated in air at 35°C, and zone diameters were measured after 24 and 48 h of incubation.

The NCCLS-recommended QC ranges for each isolate and antifungal agent are shown in Table 1. C. krusei ATCC 6258 is not a recommended QC strain for fluconazole (1). The zone diameters for fluconazole and voriconazole obtained with each QC strain read at 24 and 48 h on each day of testing are shown in Table 1. As described previously, the zones were clear and easy to read (2, 6). The fluconazole zone diameters for each isolate varied by no more than 4 mm over the 36-day period, and all were within the established QC limits at both 24- and 48-h readings. The NCCLS guidelines specify reading the zones after 24 h of incubation (4); however, we have also included the 48-h readings to demonstrate the stability of the zones on extended incubation. Likewise, the voriconazole zone diameters for each isolate varied by no more than 4 mm over the 36-day period, and 40 of 42 zones (95%) were within the QC limits. Among the 14 voriconazole zones obtained with C. krusei ATCC 6258, two determinations were 1 mm outside the recommended limits. The fact that the two outliers were on days 3 and 25 suggests that the cause was technical and not the age of the medium.

These findings establish a minimum shelf life for prepared MH-GMB agar plates of 36 days when used to perform fluconazole and voriconazole disk testing against Candida spp. This estimate may vary slightly according to the source of media; however, it appears that prepared MH-GMB plates provide acceptable performance for at least 30 days. Similar data have not yet been generated for flooded plates, and this approach is best reserved for next-day testing of sporadic isolates in laboratories lacking the facilities for preparation of media. Hopefully, these findings will encourage commercial development of this medium.