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Short answer
MHA agar is Mueller-Hinton agar, the standard solid medium for disc diffusion antimicrobial susceptibility testing. CLSI and EUCAST both recommend it for that purpose. What makes it useful is control: its pH, depth and cation and thymidine content all change inhibition zone sizes, so those parameters are specified and checked.
Definition and scope
“MHA agar” is a redundant phrase, because the A already stands for agar. The medium was developed in 1941 by John Howard Mueller and Jane Hinton, and in 1966 Bauer and colleagues described a standardised disc diffusion method built on it (Ahman, Matuschek and Kahlmeter, 2020). It contains beef extract and casein hydrolysate as sources of nitrogen, vitamins, carbon and amino acids, agar as the solidifying agent, and starch, which is added to absorb toxic substances. Unsupplemented, it supports most non-fastidious pathogens.
One manufacturer’s technical data sheet describes the original use as cultivating pathogenic Neisseria, with later work on sulfonamide resistance in gonococci leading to its role as a susceptibility testing medium. The same sheet gives its published formula as 2.000 g/L infusion solids, 17.500 g/L acid casein hydrolysate, 1.500 g/L starch and 17.000 g/L agar, with a final pH of 7.3 ± 0.1 at 25 °C. Formulas differ between brands.
How MHA is used in practice
The main use is disc diffusion, the method behind the Kirby-Bauer test. It is also used for agar dilution, and the basic formula is the same as in Mueller-Hinton broth, which is used for reference broth microdilution (see minimum inhibitory concentration by broth microdilution). Disc diffusion results are then interpreted against breakpoints such as those described in CLSI M100, and inoculum density is usually set against a turbidity reference such as the 0.5 McFarland standard.
The medium is sold as dehydrated powder and as pre-poured plates. For fastidious organisms, the EUCAST method adds 5% mechanically defibrinated horse blood and 20 mg/L beta-NAD to the base medium.
Parameters that move zone sizes
The 2020 EUCAST evaluation states that pH, agar depth, and the content of divalent cations and thymidine all affect inhibition zone formation. The HiMedia data sheet adds inoculum density and disc potency. The table lists each parameter and what is stated about it.
| Parameter | Stated effect or limit |
|---|---|
| pH | Acceptable range 7.2 to 7.4 in the EUCAST evaluation |
| Agar depth | Plates poured to 4 ± 0.5 mm; acceptable limits 3.5 to 4.5 mm |
| Thymine and thymidine | Inhibit sulfonamides and trimethoprim, so the medium is made with low levels |
| Calcium and magnesium | Interfere with aminoglycosides; high levels can also decrease tetracycline activity |
| Inoculum density | Affects zone size |
| Disc potency and storage | Disc storage affects potency |
Standards and brand variation
Manufacturers work to several documents. Table 1 of the EUCAST evaluation lists the following.
| Document | pH | Agar depth |
|---|---|---|
| ISO/TS 16782:2016 | 7.2 to 7.4 | 3.5 to 5.0 mm |
| WHO 28th report (1977) | 7.2 to 7.4 | 4.0 mm |
| DIN 58940-3 | 7.2 to 7.4 | 3.0 to 4.0 mm |
| FDA BAM, 8th ed. Rev A (1998) | 7.3 ± 0.2 | — |
The same evaluation tested 21 brands of dehydrated Mueller-Hinton medium from 17 manufacturers, one lot per brand. Four of the 21 brands fell outside the acceptable pH range, two above it and two below it, and none of those manufacturers recommended adjusting pH. Overall, 4.9% of readings (93 of 1,890) fell outside quality control ranges, mainly for trimethoprim-sulfamethoxazole (25 of 189), aminoglycosides (25 of 315) and fluoroquinolones (15 of 252). Six brands met the study’s “excellent” criterion of at least 99% of readings in range and at least 70% within ±1 mm of target. Cation content varied considerably: calcium 6.8 to 43 mg/L, magnesium 4.0 to 64 mg/L and zinc 0.36 to 7.8 mg/L.
The authors’ conclusion on responsibility is plain: the final responsibility for the correctness of susceptibility results rests with the laboratory.
Checklist for labs choosing and checking plates
- Record which standard the supplier states it follows, using the table above to compare pH and depth statements.
- Check agar depth against the limit your method specifies, since it affects zone formation.
- Check pH against 7.2 to 7.4 for the lot in use.
- Run the quality control strains used in the EUCAST evaluation, E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 29213 and E. faecalis ATCC 29212, and watch the drug classes most often out of range: trimethoprim-sulfamethoxazole, aminoglycosides and fluoroquinolones.
Where it matters for labs and supply decisions
Because lot-to-lot and brand-to-brand differences in pH and cations showed up in measured zone diameters, the medium is a controlled reagent rather than a commodity. Supply records that capture brand, lot and the quality control result for each lot make a later out-of-range finding traceable. A cumulative antibiogram is only as sound as the susceptibility results feeding it.
Common misunderstandings
Unsupplemented MHA grows every organism
It does not. It supports most non-fastidious pathogens. Fastidious organisms may not grow without supplementation, such as blood, and fastidious anaerobes may not grow at all.
It can be used on mixed cultures
The HiMedia data sheet recommends it for susceptibility testing of pure cultures only.
Any Mueller-Hinton plate gives the same zones
The 21-brand study found that results differed between brands, which is why quality control is run on each lot.
FAQ
What does MHA stand for?
Mueller-Hinton agar, named after John Howard Mueller and Jane Hinton, who developed it in 1941.
What is MHA agar used for?
Disc diffusion susceptibility testing, agar dilution, and, as the broth version of the same formula, reference broth microdilution.
Why is Mueller-Hinton agar used for susceptibility testing?
One manufacturer’s data sheet gives these reasons for its adoption by CLSI: batch-to-batch reproducibility, low levels of sulfonamide, trimethoprim and tetracycline inhibitors, support for most non-fastidious pathogens, and extensive performance data.
What agar depth and pH should the plates have?
The EUCAST evaluation used a pH of 7.2 to 7.4 and a depth of 4 ± 0.5 mm; the manufacturing standards in the table above give their own ranges.
Sources
- Ahman J, Matuschek E, Kahlmeter G. EUCAST evaluation of 21 brands of Mueller-Hinton dehydrated media for disc diffusion testing. Clin Microbiol Infect 2020. doi:10.1016/j.cmi.2020.01.018. https://www.eucast.org/fileadmin/eucast/pdf/general_documents/publications/CMI_1905.pdf, checked 29 October 2026.
- HiMedia Laboratories. Technical data, Mueller Hinton Agar No. 2 (M1084). https://www.himedialabs.com/media/TD/M1084.pdf, checked 29 October 2026. Single-manufacturer data; other brands differ.
