Mueller Hinton Agar (MHA) Composition, Principle, Preparation

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Mueller Hinton Agar (MHA)

  • Mueller and Hinton came up with Mueller Hinton Agar (MHA) during 1941 to isolate of pathogenic Neisseria species.
  • Mueller Hinton Agar is an agar medium for microbiological growth that is used extensively to test susceptibility to antibiotics using the Kirby-Bauer disc diffusion.
  • MHA can be used for diffusion of antimicrobial agents imbedded on a disc of paper through an agar gel according to the procedure explained in CLSI Approved Standard. Zone diameters for each antimicrobial to determine resistance, intermediate, and sensitive microorganisms for the pathogenic category are provided on the Clinical and Laboratory Standards Institute (CLSI).
  • Mueller Hinton Agar, which contains sheep blood 5% and Mueller Hinton Agar with Hemoglobin are recommended for testing antimicrobial susceptibility of Streptococcus pneumoniae, as well as Haemophilus influenza.
  • MHA was selected by the CLSI due to a variety of reasons.
    • It provides good reproducibility from batch to batch for testing that is susceptible.
    • It is deficient in trimethoprim, sulfonamide, and tetracycline inhibitors.
    • It is a key factor in the growth of a majority of non-fastidious bacterial pathogens.
    • Many details and many years of information about its performance has been collected.

Why MHA is used for antibiotic susceptibility testing?

  • This is an inherently non-selective and non-differential medium. This means that the majority of organisms that are placed on it will expand.
  • It is a source of starch. Starch has been known to absorb toxins that are released by bacteria, which means that they do not interfere with antibiotics. Starch also aids in the speed of transmission of antibiotics via the agar.
  • It is an agar that is loose. This permits greater diffusion of antibiotics than other plates. This results in an increased inhibition zone.
  • MHA has acceptable reproducibility from batch to batch for susceptibility tests.
  • MHA is very low in trimethoprim, sulfonamide and inhibitors of tetracycline (i.e. the concentration of inhibitors thymidine as well as thymine is very low of thymine and thymidine in MHA).
  • Both the para-aminobenzoic acids (PABA) and the Thymine/Thymidine content within Mueller Hinton Agar is diminished to a minimum which significantly reduces the activation of trimethoprim and sulfonamides if the medium is used to determine the susceptibility of bacteria against these antibiotics.
  • Mueller Hinton Agar is not appropriate for testing using the disc diffusion technique with slow-growing organisms, anaerobes , and capnophiles.

Mueller Hinton Agar (MHA) Principle

Mueller Hinton Agar media is made up of Beef Extract Acid Hydrolysate made from Casein Starch, Beef Extract and Agar. Beef Extract as well as Acid Hydrlysate of Casein offer carbon, nitrogen, vitamins amino acids, sulphur and other important nutrients. Starch functions as a colloid and is used to absorb any toxic metabolites created. The hydrolysis of starch produces dextrose which acts as an energy source. Agar is the main agent for forming solids. The concentrations of tetracycline and sulfonamide inhibitors, such as thymidine magnesium, thymine and calcium ions are controlled in order to not hinder susceptibility testing, and to allow for a healthy growth.

It is essential to use an appropriate medium to test how susceptible microorganisms are trimethoprim and sulfonamides is vital. Sulfonamide-related inhibition can be demonstrated through para-aminobenzoic acid (PABA) along with its counterparts. The reduced activity of trimethoprim leading to smaller inhibition zones and less innerzonal colonies, is observed on an incompatible Mueller Hinton medium that has high levels of Thymidine. The PABA and the thymine/thymidine levels within Mueller Hinton Agar is decreased to a minimum, thereby significantly reducing the activation of trimethoprim and sulfonamides when the medium is used to determine the susceptibility of bacteria for these antimicrobials.

Composition of Mueller Hinton Agar (MHA)

Beef extract2.0
Acid hydrolysate of casein17.5

Final pH 7.3 +/- 0.1 at 25ºC.


Mueller Hinton Agar (MHA) Preparation

  1. Take a suitable amount of dehydrated Mueller-Hinton powder (follow the directions of the manufacturer on the bottle) and then place it in an ounce flask.
  2. Incorporate 1 Liter distillate water, then swirl it around to distribute the powder.
  3. Place on a hot surface with a magnetic stirring device (or other heating equipment) and heat until the powder is completely dissolved (bring to a moderate boil). Don’t boil too vigorously and don’t apply the heat directly without stirring, since the medium could burn.
  4. Remove agar from the heat and then disperse the desired amount into the containers of your choice (e.g. Dispense 250-ml volumes in 500-ml Erlenmeyer flasks).
  5. Remove the cover from containers (e.g. Insert stopper inside the flask’s mouth).
  6. Autoclave at 121°C for 15 minutes.
  7. Let cool in the 48degC water bath.
  8. Arrange sterilized Petri plate on a level surface, to provide the same depth.
  9. To test disk diffusion For disk diffusion tests, pour precisely calculated volumes of molten Agar in plates*.
    1. 60 to 70 ml/150 mm plate
    2. 25-30 ml/100-mm plate
  10. Remove any bubbles that may have formed on the surface of the agar that is molten by swiftly (and with care) moving the torch of the Bunsen burner across the agar.
  11. Allow the plates to set at room temperature. Keep lids of plates slightly open.
  12. Examine the made MHA to make sure that the pH of the final product is 7.3 +-1 at 25degC.
  13. Be aware that if you have a pH <7.2 certain drugs may appear to decrease their potency (aminoglycosides and quinolones, macrolides, etc.) and other drugs could appear to exhibit excessive action (tetracycline). In the case of pH >7.4 The opposite result can occur.
  14. Keep plates in storage at temperatures between 2 and 8 degrees Celsius in sealed, tightly sealed containers.

*Note: The right depth of Mueller-Hinton to conduct disk diffusion tests is vital. In the event of not dispensing accurate volumes, it can result in agar which is not thick enough (often giving false-susceptible result) as well as too heavy (often producing false-resistant results).

Zone of inhibition is observed around the antibiotics
Zone of inhibition is observed around the antibiotics | Image Source:

Sheep blood (5%)-supplemented Mueller-Hinton agar

  1. Prepare 1 liter Mueller-Hinton agar in the manner described in the previous paragraph.
  2. Add 50 ml of defibrinated sheep’s blood to melt and then cool (48degC) Agar.
  3. Swirl gently to mix, then serve plates according to the instructions above.
  4. Keep plates in storage at temperatures between 2 and 8 degrees Celsius in sealed, tightly sealed containers.

Quality Control of Mueller Hinton Agar (MHA)

If a new lot of media is being prepared, make the certain checks to assure the high-quality of the prepared media.

  • Tests for fertility,
  • Measurement of pH
  • Fill measurements (volume depth, volume, etc.)
  • Checks on performance

Try your Mueller Hinton agar by using the strains listed below at least once a week to ensure that the disks and media work as intended. Examine the inhibition zone and compare it to CLSI specified limits. The susceptibility test results must be within the ranges defined by CLSI.

Quality Control strain(s) to perform test for performance test Mueller Hinton Agar

  • Escherichia coli ATCC 25922
  • Staphylococcus aureus ATCC 25923
  • Pseudomonas aeruginosa ATCC 27853
  • Enterococcus Faecalis ATCC 29212
  • Streptococcus pneumoniae ATCC 49619 (for Mueller-Hinton agar that contains 5 % sheep blood)

Modifications of Muller Hinton agar

Mueller Hinton Agar medium that is supplemented with 5 percent sheep blood is suggested for determining the susceptibility to antimicrobials

  • Streptococcus pneumoniae
  • Neisseria meningitidis

Haemophilus test medium (HTM) is the most commonly used medium used for antimicrobial susceptibility test for H. influenzae with the altered Kirby Bauer disk diffusion method. The medium of HTM is composed of the three components: thymidine free MHA supplemented by 15 mg/ml NAD, mg/ml bovine Hemin, as well as 5 mg/ml of yeast extract.

Uses of Mueller Hinton Agar (MHA)

  • The primary use for Mueller Hinton Agar can be used susceptibility testing for antimicrobials. It has been a standard medium used in tests using the Bauer Kirby method, and its efficiency is defined through the NCCLS.
  • It is also employed in the cultivation process of Neisseria.
  • It is outlined in the FDA Bacteriological Analytical Manual for tests on food and other procedures that are typically performed on facultative and aerobic anaerobic bacteria.

Limitations of Mueller Hinton Agar (MHA)

  • Many factors affect the outcomes: the size of the inoculum as well as growth rate in the medium formulation, pH and so on. The strictest adherence to protocols is essential to guarantee the accuracy of results.
  • The amount of inoculum may affect the size of the zone. A high concentration of inoculum can lead to smaller zones. Alternatively, too little inoculum could result in larger zones.
  • Organisms that are invasive may not thrive on this medium and might require the supplementation of blood.
  • Anarobes that are meticulous may not develop on this type of fabric.
  • Disk diffusion shouldn’t be used to treat obligatory anaerobes, slow-growing organisms, or capnophiles. This method was designed to be standardized for facultative organisms and aerobes with rapid growth.
  • Inactivation of the drug can be caused by the lengthy incubation periods needed by slow growers.
  • The amount of divalent cations particularly magnesium and calcium, can affect the results of aminoglycoside as well as tetracycline and colistin tests using P. Aeruginosa isolates.


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