Mueller Hinton Agar (MHA): Composition, preparation and uses

Mueller Hinton Agar
Placing antibiotic disc in Mueller-Hinton Agar Medium

Last updated on June 4th, 2020

Mueller-Hinton agar (MHA)  is the best medium to use for routine antimicrobial susceptibility testing using the Kirby-Bauer disc diffusion method for nonfastidious bacteria (both aerobe and facultative anaerobe). The use of media other than Mueller-Hinton agar may result in erroneous results.

Mueller Hinton Agar
Placing antibiotic disc in Mueller-Hinton agar medium

Mueller-Hinton agar is also the standard medium used for most broth dilution testing as the conditions of this medium (i.e. pH, cation concentration, and thymidine contents) are well maintained.

Mueller Hinton agar (MHA) can be purchased from commercial suppliers or can also be prepared from the dehydrated medium. Be sure to prepare the media according to the manufacturer’s directions.


Formula for Mueller-Hinton agar per liter of purified water

  • Beef extract: 2.0 g
  • Acid Hydrolysate of casein: 17.5 g
  • Starch: 1.5 g
  • Agar: 17.0 g

Preparation of  Mueller-Hinton agar

Muller Hinton Agar
  1. Suspend 38 g of medium (or the components listed above) in 1 liter of purified water.
  2. Mix thoroughly.
  3. Heat with frequent agitation and boil for 1 minute to completely dissolve the components.
  4. Autoclave at 121°C for 15 minutes.
  5. Cool to 45°C
  6. Pour cooled Mueller Hinton Agar into sterile Petri dishes on a level, horizontal surface to give uniform depth.
    Note: The plates must be poured to a depth of 4 mm (approximately 25 ml of liquid agar for 100-mm plates and 60 ml of liquid agar for 150-mm plates, but in any case to a measured depth of 4 mm).  Plates that are too shallow will produce false susceptible results as the antimicrobial compound will diffuse further than it should, creating larger zones of inhibition.  Conversely, plates poured to a depth >4 mm will result in false resistant results.
  7. Allow solidifying at room temperature.
  8. Check prepared MHA to ensure the final pH is 7.3 ±1 at 25°C.
    Note: If the pH is <7.2 certain drugs will appear to lose potency (aminoglycosides, quinolones, macrolides), while other agents may appear to have excessive activity (tetracycline). If the pH is >7.4, the opposite results may occur.
  9. Prepared media can be stored at 4 to 8°C. MHA is stable for approximately 70 days from the date of preparation.

Note: Mueller Hinton agar should be tested with known strains of organism at least weekly in order to verify that the media and disks are working as expected.

Why Mueller-Hinton agar is used in routine antibiotic susceptibility testing (AST)?

Antimicrobial Susceptibility testing
Antimicrobial Susceptibility testing using disk diffusion

Mueller-Hinton agar is the best medium for routine antibiotic susceptibility testing (AST) because of the following reasons:

  1. It shows acceptable batch-to-batch reproducibility for susceptibility testing
  2. It supports satisfactory growth of most nonfastidious pathogens
  3. It is low in sulfonamide, trimethoprim, and tetracycline inhibitors (i.e. concentration of inhibitors thymidine and thymine is low in MHA)
  4. A large body of data and experience has been collected concerning susceptibility tests performed with this medium.

Modifications of Muller Hinton agar

  1. Mueller Hinton agar medium supplemented with 5% sheep blood is recommended for determining the antimicrobial susceptibility of
    1. Streptococcus pneumoniae
    2. Neisseria meningitidis
  2. Haemophilus test medium (HTM) is the preferred medium for the antimicrobial susceptibility testing of H. influenzae using modified Kirby Bauer disk diffusion method. HTM medium consists of following ingredients: thymidine free MHA supplemented with 15 μg/ml NAD, 15 μg/ml bovine hemin, and 5 mg/ml yeast extract.
About Acharya Tankeshwar 466 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


    • Coster Nsindo, E.coli does not give specific colored colonies in MHA. Generally there are creamed color (you wont be able to differentiate it from other organisms in the MHA).

  1. Shall I incubate the Mueller Hinton agar plate with combidisc in inverted position or upright position in incubator?

  2. Hi, Can i know if there is any difference between Mueller Hinton II Agar and Mueller Hinton Agar? Many thanks!!

  3. Thank you for this. Please why is cation adjusted mueller hinton broth required for broth dilution antibiotics susceptibility testing?

  4. i don’t have any query but just to thank you for your clear and helpful blog. i am Medical Laboratory at Africa University

  5. sir how can i check antibiotic susceptibility on lactobacillus.Can I take it from curd.please give me full procedure for it sir.I will be very grateful

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