Mueller Hinton Agar (MHA) –

Composition, Principle, Uses and

Preparation
Mueller and Hinton developed Mueller Hinton Agar (MHA) in 1941 for the
isolation of pathogenic Neisseria species. Nowadays, it is more commonly
used for the routine susceptibility testing of non-fastidious microorganism by
the Kirby-Bauer disk diffusion technique.

Composition of MHA
Ingredients In Gram/Litre

Beef Extract 2.00 gm

Acid Hydrolysate of Casein 17.50 gm

Starch 1.50 gm

Agar 17.00 gm
Distilled Water 1000 ml
Final pH 7.3 ± 0.1 at 25ºC

Principle of MHA
Mueller Hinton Media contains Beef Extract, Acid Hydrolysate of Casein, Starch
and Agar. Beef Extract and Acid Hydrolysate of Casein provide nitrogen,
vitamins, carbon, amino acids, sulphur and other essential nutrients. Starch is
added to absorb any toxic metabolites produced. Starch hydrolysis yields
dextrose, which serves as a source of energy. Agar is the solidifying agent.

Uses of MHA
1. The major use of Mueller Hinton Agar is for antimicrobial susceptibility
testing. It has become the standard medium for the Bauer Kirby method
and its performance is specified by the NCCLS.
2. It can be used to cultivate Neisseria
3. It is specified in FDA Bacteriological Analytical Manual for food testing,
and procedures commonly performed on aerobic and facultative
anaerobic bacteria.

Why MHA is used for antibiotic susceptibility

testing?
1. It is a non-selective, non-differential medium. This means that almost all
organisms plated on here will grow.
2. It contains starch. Starch is known to absorb toxins released from
bacteria, so that they cannot interfere with the antibiotics. It also
mediates the rate of diffusion of the antibiotics through the agar.
3. It is a loose agar. This allows for better diffusion of the antibiotics than
most other plates. A better diffusion leads to a truer zone of inhibition.
4. MHA shows acceptable batch-to-batch reproducibility for susceptibility
testing.
5. MHA is low in sulfonamide, trimethoprim, and tetracycline inhibitors (i.e.
concentration of inhibitors thymidine and thymine is low in MHA).
 6. Both the para-aminobenzoic acid (PABA) and thymine/thymidine
content in Mueller Hinton Agar are reduced to a minimum, thus
markedly reducing the inactivation of sulfonamides and trimethoprim
when the media is used for testing the susceptibility of bacterial isolates
to these antimicrobics.

Preparation of MHA
1. Suspend 38 gm of the medium in one liter of distilled 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. Pour cooled Mueller Hinton Agar into sterile petri dishes on a level,
horizontal surface to give uniform depth.
5. Allow to cool to room temperature.
6. Check for the final pH 7.3 ± 0.1 at 25ºC.
7. Store the plates at 2-8 ºC.

The Kirby-Bauer Disc Method

This method is also called the agar diffusion method or the disk diffusion method.

The procedure followed is simply that a filter disk impregnated with an antibiotic is

applied to the surface of an agar plate containing the organism to be tested and the plate

is incubated at 37°C for 24-48 hours. As the substance diffuses from the filter paper into

the agar, the concentration decreases as a function of the square of the distance of

diffusion. At some particular distance from each disk, the antibiotic is diluted to the point

that it no longer inhibits microbial growth. The effectiveness of a particular antibiotic is

shown by the presence of growth-inhibition zones. These zones of inhibition (ZOIs)

appear as clear areas surrounding the disk from which the substances with antimicrobial

activity diffused. The diameter of the ZOI can be measured with a ruler and the results of

such an experiment constitute an antibiogram.

The agar diffusion method uses commercially available filter paper disks, each
containing a defined concentration of a specific antibiotic. The relative effectiveness of

different antibiotics provides the basis for a sensitivity spectrum of the organism. This

information, together with various pharmacological considerations, is used in the

selection of an antibiotic for treatment.