What Is MH Broth?

Mueller Hinton (MH) Broth is a non-selective, non-differential liquid growth medium originally developed by Mueller and Hinton in 1941 for the cultivation of Neisseria species. Over subsequent decades it became the universally recommended medium for in vitro antimicrobial susceptibility testing (AST) by broth microdilution, as endorsed by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

The medium supports reliable, reproducible growth of the majority of clinically relevant non-fastidious aerobic and facultatively anaerobic bacteria, making it the cornerstone of minimum inhibitory concentration (MIC) determinations used to guide antibiotic therapy.

Composition and Preparation

The classical formulation of MH Broth contains three principal ingredients:

• Beef extract – provides amino acids, nucleotides, vitamins, and carbon sources essential for bacterial growth.
• Acid hydrolysate of casein (casein hydrolysate) – supplies a rich mixture of peptides and free amino acids to support rapid proliferation.
• Starch – absorbs toxic metabolic by-products that could otherwise inhibit bacterial growth and potentially interfere with antibiotic activity.

To prepare MH Broth, the dehydrated powder is dissolved in purified (preferably distilled or deionized) water at the concentration specified by the manufacturer, mixed until fully dissolved, and sterilized by autoclaving at 121 °C for 15 minutes. After autoclaving, the pH should be confirmed to be 7.2–7.4 at 25 °C. The prepared medium should be stored at 2–8 °C and used within the manufacturer's recommended shelf-life period.

Note: Exact gram-per-liter values for each component should be confirmed against the lot-specific Certificate of Analysis (CoA) provided with Aladdin Scientific SKU M1528409.

Applications in Antimicrobial Susceptibility Testing

MH Broth is the reference medium specified by CLSI (document M07) and EUCAST for broth microdilution MIC testing. In this method, two-fold serial dilutions of an antimicrobial agent are prepared in MH Broth within a 96-well microplate. A standardized bacterial inoculum, typically adjusted to a 0.5 McFarland turbidity standard (approximately 1–2 × 10⁸ CFU/mL, then further diluted to a final inoculum of approximately 5 × 10⁵ CFU/mL in the well), is added to each well. After incubation at 35–37 °C for 16–20 hours, the lowest concentration of antimicrobial agent that visibly inhibits bacterial growth is recorded as the MIC.

MH Broth is intentionally low in thymine and thymidine to prevent antagonism of sulfonamide and trimethoprim activity. The medium's calcium and magnesium cation concentrations are critically important for aminoglycoside and tetracycline MIC results; CLSI specifies that cation-adjusted Mueller Hinton Broth (CAMHB) with defined Ca²⁺ (20–25 mg/L) and Mg²⁺ (10–12.5 mg/L) levels be used for these drug classes.

For fastidious organisms such as Streptococcus pneumoniae and Haemophilus influenzae, supplemented versions (e.g., MH Broth with 2–5% lysed horse blood or Haemophilus Test Medium) are required per CLSI guidelines.

Quality Control and Troubleshooting

Consistent MIC results depend on strict adherence to quality control (QC) practices. CLSI and EUCAST each define QC reference strains and acceptable MIC ranges that must be met before patient or research results are reported. Commonly used QC organisms include Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212.

Common sources of error include:

• Incorrect pH – pH outside 7.2–7.4 can shift MIC values; always verify after autoclaving.
• Improper inoculum density – too high or too low a starting inoculum will produce falsely low or high MIC readings respectively.
• Cation imbalance – for aminoglycosides and tetracyclines, using non-cation-adjusted broth is a frequent source of discrepant results.
• Contamination – visually inspect prepared medium for turbidity or particulates before use.
• Storage violations – medium stored above 8 °C or used past expiry may show reduced performance.

Ordering MH Broth from Aladdin Scientific

Aladdin Scientific offers MH Broth (SKU: M1528409) for research and laboratory use. The product is formulated to meet the compositional requirements for antimicrobial susceptibility testing and is supplied with a Certificate of Analysis confirming key quality parameters including pH, appearance, and performance testing data.

Researchers and procurement teams should confirm that the specific lot meets their applicable regulatory or accreditation requirements. For volume pricing, custom pack sizes, or technical enquiries, contact Aladdin Scientific's technical support team. Ordering can be completed directly through the Aladdin Scientific website or via your institutional procurement portal.