For antibiotic sensitivity tests and for the primary isolation of gonococci and meningococci, and other pathogens from clinical samples
FORMULA IN g/l
Acid Casein Peptone (H)
Final pH 7.4 ± 0.2 at 25ºC
Suspend 38 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into appropriate containers and sterilize in autoclave at 121°C for 15 minutes. Cool to 45 or 50°C and add defibrinated blood if desired. The blood mixture should be chocolated by heating to 80°C for 10 minutes if Neisseria development is desired. DO NOT OVERHEAT. To remelt the cold medium, heat as briefly as possible. The prepared medium should be stored at 8-15°C. The color of the prepared medium is amber, slightly opalescent.
The dehydrated medium should be homogeneous, free-flowing and cream in color. If there are any physical changes, discard the medium.
MUELLER HINTON AGAR II is used for antimicrobial susceptibility testing. It has become the standard medium for the Bauer-Kirby method and its performance is specified by NCCLS (National Committee for Clinical Laboratory Standards). Mueller Hinton Agar II can be used to cultivate gonococci and meningococci specimens. The medium can be used with complete confidence because it is a rich medium able to grow fastidious organisms. The use of a medium with suitable growth characteristics is essential to test susceptibility of microorganisms to antibiotics. It is also recommended for testing most commonly encountered aerobic and facultative anaerobic bacteria.
Beef infusion and Acid Casein peptone (H) provide nitrogen, vitamins, minerals and amino acids essential for growth. The starch absorbs any toxic metabolites produced. Bacteriological agar is the solidifying agent.
Inoculate and incubate at 35 ± 2°C for 18 – 24 hours.
The medium complies with the requirement of NCCLS and is manufactured carefully, choosing the raw materials to contain low concentrations of thymine and thymidine, as well as appropriate levels of calcium and magnesium ions.
The following results were obtained in the performance of the medium from type cultures after incubation at a temperature of 35 ± 2ºC and observed after 18-24 hours.
Polymyxin B 300 µg
Escherichia coli ATCC 25922
Staphylococcus aureus ATCC 25923
Enterococcus faecalis ATCC 29212
Pseudomonas aeruginosa ATCC 27853
Diameter halo in mm according to NCCLS
Mueller and Hinton A. Protein-Free Medium for Primary Isolation of the Gonococcus and Meningococcus. Proc. Soc. Exp. Biol. and Med. 48:330. 1941. Harris and Coleman Diagnostic.
Procedures and Reagents. 4th Edition APH, Inc. New York, 1963.
National Committee for Clinical Laboratory Standards. 1993.
Atlas, R.M. 1993 Handbook of microbiological media. CRC Press, Boca Raton. Fl.
Once opened keep powdered medium closed to avoid hydration.