What is Mueller Hinton Agar (MHA) ?

Mueller Hinton Agar is used to isolate pathogenic species of Neisseria. It was developed in 1941 by Mueller and Hinton. Today, it is primarily used to test non-fastidious microorganisms using the Kirby-Bauer disk diffusion method.

Various organisms tested in a Mueller Hinton agar plate

Image 1: Various organisms tested in a Mueller Hinton agar plate.
Picture Source: microbiologyinfo.com

Visible growth colonies in a Mueller Hinton agar

Image 2: Visible growth colonies in a Mueller Hinton agar.
Picture Source: wikimedia.org

Mueller Hinton Agar Principle

The composition of Mueller Hinton Agar includes the following:

  • Beef extract and acid hydrolysate of casein, which serves as the sources of nitrogen, amino acids, carbon, sulphur, and other important nutrients.
  • Starch – It also contains starch, which absorbs toxic metabolites. Once the starch is hydrolyzed, it yields into dextrose and acts as an energy source.
  • Agar – The solidifying agent is agar. (1, 2, 3, and 4)

What are the uses of Mueller Hinton Agar?

  • It is primarily used for testing the antimicrobial susceptibility of an organism
  • It aids in the cultivation of Neisseria.
  • It can be used for food testing, especially in procedures that involve aerobic and facultative anaerobic bacteria. (3, 4, and 5)

What makes Mueller Hinton Agar perfect for testing the susceptibility of antibiotic?

  • Mueller Hinton Agar medium is non-selective and non-differential. It allows the growth of almost all types of organisms.
  • The starch absorbs toxins released by the bacteria. Hence, the toxins won’t be able to affect the actions of antibiotics.
  • It helps regulate the diffusion rate of antibiotics in the medium.
  • Antibiotics are diffused evenly because Mueller Hinton agar is loose in characteristic. Proper diffusion is a must as it results in a true inhibition zone.
  • It is low level of sulfonamide, tetracycline inhibitors, and trimethoprim.
  • Mueller Hinton Agar allows the batch to batch reproducibility for testing susceptibility.
  • Mueller Hinton Agar has a low para-aminobenzoci acid and thymidine content thereby lessening the inactivation of trimethoprim and sulphonamides when testing for bacteria susceptibility. (4, 5, 6, and 7)
ingredients used in the preparation of Mueller Hinton Agar

Image 3:The ingredients used in the preparation of Mueller Hinton Agar.
Picture Source: 

How to prepare Mueller Hinton Agar?

  1. A 38mg of the medium should be suspended in a liter of water (distilled water).
  2. Bring to heat and let it boil for a minute; just enough for the medium to be dissolved completely.
  3. Autoclave for 15 minutes at the desired temperature (121 degree Celsius). Let it cool down at room temperature.
  4. Once the agar has cool down, pour into the sterile petri dish. Make sure that the agar is in uniform depth. Let it cool down at room temperature.
  5. The final pH level should be 7.3 ± 0.1 at 25ºC.
  6. Store the plate at a temperature of not less than 2 degree Celsius and not more than 8 degree Celsius. (3, 6, 7, 8, and 9)
Quality control is a must when performing susceptibility testing using Mueller Hinton agar

Image 4: Quality control is a must when performing susceptibility testing using Mueller Hinton agar.
Picture Source: 

What to keep in mind?

Too shallow plates would lead to a false positive result because the antimicrobial compound will be diffused even more than expected. As a result, it will create a large zone of inhibition. On the other hand, a too deep plate will result in a false resistant reading.

The pH level is important too. A pH level of below 7.2 could lead to some type of drugs to lose their potency. Some may even have excessive activity. An opposite result will occur if the pH level is higher than 7.4.

The prepared media should be stored at the right temperature. It is good to be used until 70 days from the time it is prepared. However, to find out if the media still works as expected, a known strain of organism should be tested on a weekly basis. (2, 5, 9, and 10)

Mueller Hinton Agar Quality Control

Positive control and the corresponding expected results

  • Escherichia coli – there should be good growth. The color of the growth colonies should be pale straw.
  • Staphylococcus aureus – There should be good growth and the color of colonies is cream.
  • Pseudomonas aeruginosa – A good growth of straw-colored colony is observed. (2, 4, and 8)

Negative control and the corresponding expected results

There is no significant change in the un-inoculated medium.

Are there any limitations?

  • Various factors can significantly affect the results. Hence, it is important to strictly adhere to the protocol.  Factors that may affect the result include:
    • Rate of growth
    • Size of inoculum
    • Formulation of the medium
    • pH level.
  • Inactivation of the drug may lead to prolonged incubation time needed by slow growers. (4, 8, and 10)

Mueller Hinton Agar modifications

For antimicrobial susceptibility of Streptococcus pneumonia and Neisseria meningitidis, Mueller Hinton agar is supplemented with sheep blood (5%). For testing the antimicrobial susceptibility of H. influenza, a Haemophilus test medium is preferred using Kirby disk diffusion technique. (4, 6, and 9)


  1. https://en.wikipedia.org/wiki/Mueller-Hinton_agar
  2. https://microbiologyinfo.com/mueller-hinton-agar-mha-composition-principle-uses-and-preparation/
  3. https://microbeonline.com/why-mueller-hinton-agar-is-used-in-routine-antibiotic-susceptibility-testing/
  4. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MuellerHintonMed.htm
  5. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=Cm0337&c=UK&lang=EN
  6. https://www.sciencedirect.com/topics/immunology-and-microbiology/mueller-hinton-agar
  7. https://microbenotes.com/mueller-hinton-agar-mha/
  8. https://foodsafety.neogen.com/en/ncm-mueller-hinton-agar-1
  9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC272882/
  10. https://jcm.asm.org/content/42/3/1288

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