Quality Control Limits for Fluconazole Disk Susceptibility Tests on Mueller-Hinton Agar with Glucose and Methylene Blue

Abstract

An international collaborative study was performed in order to propose quality control limits for fluconazole disk diffusion susceptibility tests on Mueller-Hinton agar with 2% glucose and 0.5 g of methylene blue per ml. The supplements may be added before autoclaving the agar, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates generated data to propose zone size limits for tests of Candida parapsilosis ATCC 22019 (22 to 33 mm), C. tropicalis ATCC 750 (26 to 37 mm), and C. albicans ATCC 90028 (28 to 39 mm). C. krusei ATCC 6258 was not useful for this purpose.Immunocompromised patients are often colonized by or infected with fungi, especially Candida spp. Consequently, they frequently receive antifungal agents such as fluconazole throughout their period of hospitalization. Prolonged exposure to an azole can select strains with diminished susceptibilities (4), and consequently, increasing doses may be necessary. For that reason, clinical laboratories may be asked to monitor the susceptibility of isolates from such patients in order to determine when strains with diminished susceptibility have been selected (3). For many laboratories, the disk diffusion procedure may be the most practical method for this purpose. A standardized method and well-defined quality control procedures are essential in order to determine when significant changes are being observed over an extended period of time.In 1996, we proposed a disk diffusion procedure that was performed on RPMI 1640 broth with 2% glucose and 1.5% agar (1). Subsequent studies were carried out to find an agar medium that should be readily available in most clinical laboratories. Mueller-Hinton (MH) agar could be used for this purpose, but it had to be supplemented with 2% glucose in order to increase the number of clinical isolates that would grow adequately. Unfortunately, the added glucose resulted in a significant amount of growth within zones of inhibition and zone edges that were not well defined. Addition of a low concentration of methylene blue (0.5 g/ml) made the zones of inhibition clearer and easier to measure precisely (2,8).Plates with MH-glucose-methylene blue (GMB) agar can be prepared in advance and stored until needed, but the shelf life of such plates has not been determined. Prepared agar plates are practical for surveys, when the number of tests can be predicted in advance and prolonged storage of the plates is not necessary. An alternative approach is preferred for clinical laboratories where susceptibility tests are only sporadically needed. Fresh MH agar plates are commonly available in clinical laboratories, and they can be supplemented by flooding the surface with a GMB solution.Flooding procedure. A stock solution of methylene blue (5 mg/ml) was prepared and refrigerated at 2 to 8°C. To 100 ml of a 40% glucose solution, 200 l of the stock methylene blue was added to give 10 g of methylene blue per ml of 40% glucose (GMB solution). The GMB solution was dispensed int...

Susceptibilities of Candida albicans Mouth Isolates to Antifungal Agents, Essentials Oils and Mouth Rinses

“…Agar plates (90-mm diameter) containing Mueller-Hinton (MH) agar (Difco Laboratories) supplemented with 2% glucose were inoculated with yeast cells, previously suspended in a saline solution (0.85%) with the turbidity 0.5 in a McFarland scale. The plates were incubated at 36 ± 1°C for 24 h. After 24 h cultivation, the inhibitory diameter zone (dz) was measured [23]. The interpretative criteria for fluconazole according to CLSI guidelines [21] were: susceptible (S) C19 mm; susceptible dose dependent (SDD) 18-15 mm and resistant (R) B14 mm.…”

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

“…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.…”

“…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 g of methylene blue (GMB) per ml (4).…”

“…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).…”

“…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).…”