MUELLER HINTON AGAR 2% GLUCOSE W/ METHYLENE BLUE

The Mueller Hinton formulation was originally developed as a simple, transparent agar medium for the cultivation of pathogenic species. Mueller Hinton Agar, modified (as per CLSI for antifungal) is recommended for the diffusion of antifungal agents impregnated on paper disc through an agar gel as described in CLSI Approved Standard.
When supplemented with glucose to a final concentration of 2%, it provides for suitable fungal growth. The addition of methylene blue to a final concentration of 5?g/ml enhances zone edge definition.
Kirby-Bauer et al recommended Mueller Hinton Agar for performing antibiotic susceptibility tests using a single disc of high concentration. WHO Committee on Standardization of Susceptibility Testing has accepted Mueller Hinton Agar for determining the susceptibility of microorganisms because of its reproducibility. Mueller Hinton Agar with 5% sheep blood and Mueller Hinton Agar with Haemoglobin have been recommended for antimicrobial susceptibility testing of Streptococcus pneumoniae and Haemophilus influenzae. Similarly, Mueller Hinton Agar, modified (as per CLSI for antifungal) is recommended for antifungal susceptibity testing of discs.
Technique:
Preparation of Inoculum:
1. Inoculum is prepared by picking five distinct colonies of approximately 1mm from 24 hours old culture grown on Sabouraud Dextrose Agar and incubated at 35 ? 2?C. Colonies are suspended in 5ml of sterile 0.85% Saline.
2. Vortex the resulting suspension and adjust the turbidity to yield 1 x 106 – 5 x 106 cells /ml (i.e. 0.5 McFarland standard).
Test Procedure:
1. Prepare plates with Mueller Hinton Agar, modified (as per CLSI for antifungal) for carrying out susceptibility of antifungal discs. The medium in the plates should be sterile and have a depth of about 4 mm.
2. Dip a sterile non-toxic cotton swab on a wooden applicator into the standardized inoculum (turbidity so adjusted, as to obtain semi confluent growth on the Petri plate) and rotate the soaked swab firmly against the upper inside wall of the tube to express excess fluid. Streak the entire agar surface of the plate with the swab three times, turning the plate at 60? angle between each streaking. Allow the inoculum to dry for 5 – 15 minutes with lid in place.
3. Apply the discs using aseptic technique. Deposit the discs with centers at least 24 mm apart. (Not more than 12 discs should be placed on a 150-mm plate or not more than 5 discs on a 100-mm plate
4. Invert the plates and place in an incubator set to 35 ? 2?C within 15 minutes after the discs are applied.
5. Examine each plate after 20-24 hours of incubation. If plate was satisfactorily streaked the resulting zones of inhibition will be uniformly circular and there will be a semi-confluent lawn of growth. Read at 48 hours only when insufficient growth is observed after 24 hours? incubation.

Mueller Hinton Agar, Modi?ed (as per CLSI for antifungal) is recommended for testing performing Antifungal Disk Di?usion Susceptibility of yeasts