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Intended Use
Mueller Hinton Agar
is used in antimicrobial susceptibility testing by the disk diffusion method. This formula
conforms to National Committee for Clinical Laboratory Standards (NCCLS).1
Product Summary and Explanation
Mueller Hinton Agar is based on the formula recommended by Mueller and Hinton2 for the primary isolation of
Neisseria species. Mueller and Hinton selected pea meal extract agar as a simple transparent medium
containing heat stable ingredients.3 During their modification, starch replaced the growth-promoting properties
of pea extract, acting as a “protective colloid” against toxic substances.
Bauer, Kirby, Sherris and Tuck4 recommended Mueller Hinton Agar for performing antibiotic susceptibility
tests using a single disk of high concentration. This unsupplemented medium has been selected by the
National Committee for Clinical Laboratory Standards (NCCLS)1 for several reasons:5 this medium is low in
sulfonamide, trimethoprim and tetracycline inhibitors, provides satisfactory growth of most non-fastidious
pathogens and demonstrates batch-to-batch reproducibility.
Mueller Hinton Agar is often abbreviated as M-H Agar, and complies with requirements of the World Health
Organization.5 Mueller Hinton Agar is specified in FDA Bacteriological Analytical Manual6 for food testing, and
procedures commonly performed on aerobic and facultatively anaerobic bacteria.7 A variety of supplements
can be added to Mueller Hinton Agar, including 5% defibrinated sheep or horse blood, 1% growth supplement
and 2% sodium chloride.
Principles of the Procedure
Beef Extract and Acid Hydrolysate of Casein provide nitrogen, vitamins, carbon, and amino acids in Mueller
Hinton Agar. Starch is added to absorb any toxic metabolites produced. Agar is the solidifying agent.
A suitable medium is essential for testing the susceptibility of microorganisms to sulfonamides and
trimethoprim. Antagonism to sulfonamide activity is demonstrated by para-aminobenzoic acid (PABA) and its
analogs. Reduced activity of trimethoprim, resulting in smaller growth inhibition zones and inner zonal growth,
is demonstrated on medium possessing high levels of thymide. The PABA and thymine/thymidine content of
Mueller Hinton Agar are reduced to a minimum, reducing the inactivation of sulfonamides and trimethoprim.
Formula / Liter
Beef Extract . 2 g
Acid Hydrolysate of Casein . 17.5 g
Starch. 1.5 g
Agar . 17 g
Final pH 7.3 ± 0.1 at 25°C
Formula may be adjusted and/or supplemented as required to meet performance specifications.
1. For Laboratory Use.
1. Suspend 38 g of the medium in one liter of purified water.
2. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
3. Autoclave at 121°C for 15 minutes. Cool to room temperature.
4. OPTIONAL: Supplement as appropriate. Pour cooled Mueller Hinton Agar into sterile petri dishes on a
level, horizontal surface to give uniform depth. Allow to cool to room temperature.
5. Check prepared Mueller Hinton Agar to ensure the final pH is 7.3 ± 0.1 at 25°C.
Quality Control Specifications
Dehydrated Appearance:
Powder is homogeneous, free flowing, and beige.
Prepared Appearance: Prepared medium is slightly opalescent with no significant precipitation, and light to
medium amber.
Expected Cultural Response: Prepare, inoculate and dispense antibiotic disks following the procedure
described by NCCLS.1,8,9 The cultures listed should have middle range zone sizes of the concentration tested.8
Response & Reactions
Enterococcus faecalis ATCC 29212 growth; zone diameters within published specifications growth; zone diameters within published specifications growth; zone diameters within published specifications Pseudomonas aeruginosa ATCC 27853 growth; zone diameters within published specifications Staphylococcus aureus ATCC 25923 growth; zone diameters within published specifications Staphylococcus aureus ATCC 43300 growth; zone diameters within published specifications The organisms listed are the minimum that should be used for quality control testing.
Test Procedure
For a complete discussion on antimicrobic susceptibility testing, refer to procedures outlined in appropriate
Refer to appropriate documents for correct zone sizes.
Store the sealed bottle containing the dehydrated medium at 2 - 30°C. Once opened and recapped, place the
container in a low humidity environment at the same storage temperature. Protect from moisture and light by
keeping container tightly closed.
Refer to the expiration date stamped on the container. The dehydrated medium should be discarded if it is
not free flowing, or if the appearance has changed from the original color. Expiry applies to medium in its
intact container when stored as directed.
Limitations of the Procedure
1. Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on this medium.
2. Numerous factors can affect results: inoculum size, rate of growth, medium formulation and pH. Strict adherence to protocol is
3. Drug inactivation may result from the prolonged incubation times required by slow growers.104. Variation in the concentration of divalent cations, primarily calcium and magnesium affects result of aminoglycoside, tetracycline, and colistin test with P. aeruginosa isolates.7 Packaging
Mueller Hinton Agar

National Committee for Clinical Laboratory Standards. 1997. Performance standards for antimicrobial disk susceptibility tests.
Approved standard M2-A6. National Committee for Clinical Laboratory Standards, Wayne, PA.
Mueller, J. H., and J. Hinton. 1941. A protein-free medium for primary isolation of gonococcus and meningococcus. Proc. Soc.
Exp. Biol. Med. 48:3330-333.
Gordon and Hine. 1916. Br. Med. J. 678.
Bauer, A. L., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk
method. Am. J. Clin. Pathol. 45:493-496.
World Health Organization. 1961. Standardization of methods for conducting microbic sensitivity tests. Technical Report Series
No. 210, Geneva.
Food and Drug Administration. Bacteriological analytical manual, 8th ed., AOAC International, Gaithersburg, MD.
Wood, G. L., and J. A. Washington. 1995. Antibacterial susceptibility tests: dilution and disk diffusion methods, p. 1327-1341. In
Murray, P.R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (eds.). Manual of clinical microbiology, 6th ed. American
Society for Microbiology, Washington, D.C.
National Committee for Clinical Laboratory Standards. 1996. Protocols for evaluating dehydrated; App. Standard. Wayne PA.
National Committee for Clinical Laboratory Standards. 1999. M100-S9. Performance Standards for Antimicrobial Susceptibility
Testing; Ninth Informational Supplement. Wayne, PA.
10. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures handbook, vol. 1, American Society for Microbiology, Washington, D.C.
Technical Information
Contact Acumedia Manufacturers, Inc. for Technical Service or questions involving dehydrated culture media preparation or
performance at (410)780-5120 or fax us at (410)780-5470.


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