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Mueller Hinton Agar is recommended for antimicrobial disc diffusion susceptibility testing of common, rapidly growing bacteria by the Bauer-Kirby method,^2-4 as standardized by the Clinical and Laboratory Standards Institute (CLSI).¹

NOTE: The recommended medium for disc diffusion susceptibility testing of Streptococcus pneumoniae is Mueller Hinton Agar with 5% Sheep Blood. The recommended medium for Haemophilus influenzae is Haemophilus Test Medium (HTM) Agar. The recommended medium for Neisseria gonorrhoeae is GC II Agar with IsoVitaleX™ Enrichment. Interpretive criteria are provided in the CLSI Document M100-S16 (M2),⁵ which is included with CLSI Document M2-A9, Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard, Ninth Edition.¹

Mueller Hinton Agar was originally developed for the cultivation of pathogenic Neisseria.⁶ However, these organisms are now commonly isolated on selective media.

Because clinical microbiology laboratories in the early 1960s were using a wide variety of procedures for determining the susceptibility of bacteria to antibiotic and chemotherapeutic agents, Bauer, Kirby and others developed a standardized procedure in which Mueller Hinton Agar was selected as the test medium.^2,3 A subsequent international collaborative study confirmed the value of Mueller Hinton Agar for this purpose because of the relatively good reproducibility of the medium, the simplicity of its formula, and the wealth of experimental data that had been accumulated using this medium.⁷

The CLSI has written a performance standard for the Bauer-Kirby procedure and this document should be consulted for additional details.¹ The procedure is recommended for testing rapidly growing aerobic or facultatively anaerobic bacterial pathogens, such as staphylococci, members of the Enterobacteriaceae, aerobic gram-negative rods; e.g., Pseudomonas spp. and Acinetobacter spp., enterococci and Vibrio cholerae. The procedure is modified for testing fastidious species; i.e., Haemophilus influenzae, Neisseria gonorrhoeae and S. pneumoniae and other streptococci.

Mueller Hinton II Agar is manufactured to contain low levels of thymine and thymidine,^8,9 and controlled levels of calcium and magnesium.^10-12 Thymine and thymidine levels of raw materials are determined using the disc diffusion procedure with trimethoprim-sulfamethoxazole (SXT) discs and Enterococcus faecalis ATCC 33186 and/or 29212. Calcium and magnesium levels are controlled by testing raw materials and supplementing with sources of calcium and/or magnesium as required to produce correct zone diameters with aminoglycoside antibiotics and Pseudomonas aeruginosa ATCC 27853.¹³

See "Quality Control Procedures."

Control cultures should be included each time a susceptibility test is performed or weekly if satisfactory performance can be documented according to the CLSI standard.¹ The correct zone diameters will be found in M100-S16 (M2).

For in vitro Diagnostic Use.

If excessive moisture is observed, invert the bottom over an off-set lid and allow to air dry in order to prevent formation of a seal between the top and bottom of the plate during incubation.

Observe aseptic techniques and established precautions against microbiological hazards throughout all procedures. After use, prepared plates, specimen containers and other contaminated materials must be sterilized by autoclaving before discarding.