Mueller Hinton With Chocolate

Cat. no. E20 Mueller Hinton with Chocolate, 15x100mm Plate, 24ml 10 plates/bag


Hardy Diagnostics Mueller Hinton with Chocolate is an enriched medium recommended for the isolation and cultivation of fastidious microorganisms, particularly Haemophilus species.


In the 1960s, Bauer, Kirby, et al. developed Mueller Hinton medium for use in the disk diffusion procedure for determining susceptibility testing of bacteria to antibiotic and chemotherapeutic agents. (2) The medium was also developed for the cultivation of pathogenic Neisseria spp. (7) Since fastidious microorganisms grew poorly, the medium was eventually supplemented with 1% hemoglobin and adopted for testing of Haemophilus influenzae . Consequently, Mueller Hinton with Chocolate was formerly recommended by the Clinical Laboratory Standards Institute (CLSI) for susceptibility testing of H. influenzae . (6,9) More recent methods of disk diffusion testing have replaced Mueller Hinton with Chocolate with Haemophilus Test Medium (HTM), Cat. no. G33 or H07. However, Mueller Hinton with Chocolate is still recommended by CLSI for antimicrobial susceptibility testing of bacteria isolated from animals. (10)

Mueller Hinton with Chocolate contains beef extract and casein which provide nitrogenous components, amino acids, vitamins and minerals to support microbial growth. Starch is added to neutralize toxic fatty acids present in the agar. Hemoglobin provides hemin (X-factor) for the growth of Haemophilus spp. The medium is also supplemented with KoEnzyme enrichment, a chemically defined supplement that provides NAD (V-factor), amino acids, vitamins, dextrose, ferric ions, and coenzymes to promote the growth of pathogenic Neisseria species.


Ingredients per liter of deionized water:*

Acid Hydrolysate of Casein 17.5gm
Hemoglobin 10.0gm
Beef Extract 2.0gm
Starch 1.5gm
KoEnzyme Enrichments 10.0ml
Agar 17.0gm

Final pH 7.3 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.



Specimen Collection: Consult listed references for information on specimen collection. (2-5,8,10) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation.

Method of Use:

1. Allow plates to acclimate to room temperature prior to use.

2. Streak the specimen to obtain isolated colonies. Alternatively, if the specimen is obtained from a swab, roll the swab over a small portion of the agar surface and streak to obtain isolated colonies.

3. Incubate plates at 35ºC. for 18-24 hours, and up to 72 hours, in an aerobic atmosphere enriched with 5-10% CO 2 .


Observe plates for the growth of isolated colonies within the streak zone. The growth of Haemophilus spp. may appear as small (1.0mm), moist, pearly colonies with a characteristic "mousy" odor.



Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Haemophilus influenzae
ATCC ® 10211
A 24hr 35°C CO 2 ** Growth

** Atmosphere of incubation is enriched with 5-10% CO 2 .


Physical Appearance

Mueller Hinton with Chocolate should appear opaque, smooth and brown in color.


1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Bauer, A.W., W.M.M. Kirby, et al. 1966. Am. J. Clin. Pathol.; 45:493-496.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4.  Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5.  Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. Methods for Dilution Antimicrobial Susceptibility Test For Bacteria That Grow Aerobically, M7-current edition. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.

7. Mueller, J.H. and J. Hinton. 1941. A protein-free medium for primary isolation of the Gonococcus and Meningococcus. Proc. Soc. Exp. Diol. and Med.; 48:330-333.

8. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

9. Performance Standards for Antimicrobial Disk Susceptibility Tests. M2-A. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

10. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard. M31-A. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

11. <Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

ATCC is a registered trademark of the American Type Culture Collection.