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 g/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-g fluconazole disks and 1-g 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) Table 1). The proposed NCCLS method employs Mueller-Hinton agar (MHA) supplemented with 2% glucose and 0.5 g 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).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) accordin...

Species distribution and in vitro antifungal susceptibility patterns of Candida

“…In the studies of Pfaller M A et al, 14 and Lee S C et al, 15 it was seen that prepared MH-GMB agar plates provide acceptable performance for disk diffusion testing for at least 30 days when stored at 5°C.…”

Risk Factor Determination and Antifungal Susceptibility Patterns of Candida in Neonatal Oral Candidiasis

“…Among the different methods disc diffusion method is one of the simple, cost effective method that can give sensitivity result within 42 hrs. In two other studies, it was seen that prepared MH-GMB agar plates provide acceptable performance for disk diffusion testing for at least 30 days when stored at 5°C 25,26 . Distinction between species facilitates the understanding regarding the reservoir and mode of transmission Candida infection.…”

An efficient approach for the isolation, identification and evaluation of antimicrobial plant components on an analytical scale, demonstrated by the example of Radix imperatoriae

“…In this work, agar disk diffusion was the method of choice for antimicrobial screening [22][23][24]. The bacterial strains were inoculated from a glycerol freezing culture in 100 mL LB (lysogeny broth) medium and incubated overnight at 37°C in a shaker.…”