Table of Contents

Introduction 

• Components of Mueller-Hinton Agar in the Quality Control and Assessment Process
• Preparation and Setup of Mueller-Hinton Agar for Accurate Quality Control
• Physical and Chemical Quality Control 
• Microbial Quality Control of Mueller-Hinton Agar and AST Quality Assessment Tests
• Storage and Expiration Date in 
• Common Problems and Troubleshooting in Quality Assessment of Mueller-Hinton Agar
• Specific Variants of Mueller-Hinton Medium and Their Quality Control
• Conclusion
• References in Quality Control and Assessment of Mueller-Hinton Medium
• Common Questions

Introduction

Mueller-Hinton agar is one of the most commonly used culture media in microbiology laboratories, primarily employed for antibiotic susceptibility testing (Antimicrobial Susceptibility Testing – AST). This medium, first introduced by Mueller and Hinton in 1941, is designed for culturing non-fastidious bacteria and enables precise evaluation of antibiotic effectiveness. The importance of quality control (QC) for this medium lies in its direct impact on AST results; any abnormality in its composition or performance can lead to incorrect interpretation of bacterial resistance or susceptibility.

According to the Clinical and Laboratory Standards Institute (CLSI) standards, Mueller-Hinton agar must comply with specific criteria to produce reproducible and valid results. This article covers all aspects of quality control for Mueller-Hinton medium, including components, preparation, physical and chemical parameters, microbial tests, storage, common problems, and specific variants. The information is compiled based on CLSI standards (such as M100, M02, and M07) and other reliable sources.

Quality control of Mueller-Hinton medium

Components of Mueller-Hinton agar

Mueller-Hinton agar (MHA) and broth (MHB) include basic nutrients that support bacterial growth without interfering with antibiotics. Standard components according to CLSI are:

• Beef Extract or Meat Infusion: 2 g/L, source of carbon and nitrogen.
• Casein Hydrolysate or Acicase: 17.5 g/L, provider of amino acids.
• Starch: 1.5 g/L, as a protector against toxic substances and energy source after hydrolysis.
• Agar: 17 g/L (only in MHA for solidifying the medium).

The final pH of the medium should be 7.3 ± 0.1 at 25°C. Levels of thymine and thymidine should be low to avoid interference with sulfonamide and trimethoprim tests, and concentrations of divalent cations such as calcium (Ca²⁺) and magnesium (Mg²⁺) should be controlled (typically 20-25 mg/L for Ca and 10-12.5 mg/L for Mg) to ensure accurate inhibition zone sizes.

Preparation and Setup of Mueller-Hinton agar for Accurate Quality Control

To prepare dehydrated medium, dissolve the specified amount (typically 38 g for MHA) in 1 L of distilled water and boil until fully dissolved. Then autoclave at 121°C for 15 minutes. After cooling to 45-50°C, pour the medium into sterile plates. The agar thickness should be 4 mm for proper antibiotic diffusion.

For specific variants if needed:

• MH with Blood (MH with 5% Blood): For fastidious bacteria like Streptococcus pneumoniae.
• MH-F (for fastidious organisms): With additional supplements for Haemophilus spp.

After preparation, pH control is essential; if pH is out of range, adjust with HCl or NaOH.

Physical and Chemical Quality Control

Physical Parameters in Quality Control of Mueller-Hinton Medium

• Appearance: The medium should be clear, without sediment, bubbles, or irregularities. Excessive moisture or dryness can affect results.
• pH: pH measurement after preparation is mandatory. Acceptable range: 7.2-7.4 at 25°C. Deviation can disrupt bacterial growth or antibiotic diffusion.
• Hardness: Agar should be firm but not overly stiff for proper disk placement.

Chemical Parameters in Quality Assessment of Mueller-Hinton Medium

• Cation Concentrations: Controlled with aminoglycoside disk diffusion tests on Pseudomonas aeruginosa ATCC 27853. Zones outside the range indicate improper Ca/Mg levels.
• Thymine/Thymidine Levels: Checked with SXT test on Enterococcus faecalis ATCC 29212; small zones indicate high levels.
• Inhibitors: The medium should not contain tetracycline or sulfonamide inhibitors.

According to ISO/TS 16782, dehydrated medium must meet performance criteria for lot acceptance.

Microbial Quality Control of Mueller-Hinton agar and AST Quality Assessmen Tests

Microbial quality control of Mueller-Hinton medium is a vital part of the quality assurance process, aimed at verifying the medium’s performance in supporting bacterial growth and producing accurate results in antibiotic susceptibility testing (AST). This control is performed using standard quality control strains (QC strains) provided by institutions like ATCC (American Type Culture Collection). These strains are non-fastidious bacteria with predictable behavior against specific antibiotics. Microbial quality control should be conducted for each new lot of medium, as well as weekly or daily (depending on laboratory workload) to identify any changes in medium performance.

According to CLSI standards (M02 and M07) and WHO guidelines, quality control should include disk diffusion tests and minimum inhibitory concentration (MIC) determination. Below, details on the method, strains, acceptable ranges, and common problems are explained. Additionally, the table provided by the user, based on revised WHO standards, is used for practical examples. Note that ranges may differ between CLSI and WHO standards, so laboratories should adhere to their chosen standard.

Standard Control Strains in Quality Control of Mueller-Hinton agar

Common QC strains for Mueller-Hinton medium include:

• Escherichia coli ATCC 25922: For controlling broad-spectrum antibiotics like beta-lactams, aminoglycosides, and fluoroquinolones.
• Staphylococcus aureus ATCC 25923: For controlling anti-staphylococcal antibiotics like penicillins and tetracyclines.
• Pseudomonas aeruginosa ATCC 27853: For controlling cation levels (such as Ca and Mg) and anti-pseudomonal antibiotics like gentamicin.
• Enterococcus faecalis ATCC 29212: For controlling sulfonamides and trimethoprim (SXT), and thymine/thymidine levels.
• Additional strains for specific variants: such as Haemophilus influenzae ATCC 49247 for MH-F, or Streptococcus pneumoniae ATCC 49619 for blood-supplemented medium.

In the provided table, Enterococcus faecalis ATCC 33186 is used, which is typically for high-level resistance tests (like High-Level Aminoglycoside Resistance), but is also mentioned in some WHO standards for SXT control.

Quality Control Test Method in Quality Assessment of Mueller-Hinton agar

1. Inoculum Preparation: Revive the QC strain from frozen stock (-70°C) and culture on non-selective medium (like Blood Agar). Then suspend colonies in sterile saline to achieve 0.5 McFarland turbidity (equivalent to 1.5 × 10^8 CFU/mL). This step should be done within 15 minutes to maintain bacterial viability.
2. Disk Diffusion Method (Kirby-Bauer): Inoculate the MHA plate evenly with a cotton swab (three 60-degree rotations for full coverage). Place antibiotic disks with sterile forceps (maximum 6 disks on a 150 mm plate). Incubate the plate at 35 ± 2°C for 16-18 hours (for most bacteria) or 20-24 hours (for some). Measure the inhibition zone diameter (IZD) with a caliper (from the back of the plate, including the complete inhibition zone).
3. MIC Method (Broth Microdilution or E-test): Prepare serial dilutions of antibiotics in MHB. Final inoculum is 5 × 10^5 CFU/mL. After incubation, the lowest concentration inhibiting visible growth is the MIC.
4. Control Frequency: For each new lot of medium or disks. Weekly for low-volume labs. Daily if more than 5 tests per day (per CLSI). Record results in a logbook and perform trend analysis to identify deviations.

If results are out of range, reject the medium or disk lot and repeat the test. If the issue persists, check external factors like temperature, humidity, or water quality.

Acceptable Ranges

Ranges are based on CLSI M100 standards (recent versions like 2020 or newer) and WHO. In CLSI standards, ranges are updated annually (e.g., in the 2025 version, changes for some antibiotics like ciprofloxacin for E. coli). The table below is extracted based on revised WHO standards. This table shows inhibition zone diameters (in mm) for specific disks:

Table Explanation:

• “-” indicates inapplicability of the antibiotic for the respective strain in quality control.
• For SXT (Sulfamethoxazole-Trimethoprim), low thymine/thymidine levels in the medium are essential to avoid falsely small zones.
• These ranges are per revised WHO guidelines, which may be optimized for laboratories in developing countries. Compared to CLSI M100 (2020), there are some differences; for example:
  • Tetracycline for S. aureus ATCC 25923 in CLSI: 24-30 mm (vs. 19-28 mm in WHO).
  • Trimethoprim-Sulfamethoxazole for E. coli ATCC 25922 in CLSI: 23-29 mm (vs. 24-32 mm in WHO).
  • Polymyxin B is typically controlled for P. aeruginosa in CLSI (13-19 mm), but listed for E. coli and S. aureus in this table.

For MIC, ranges are different (in μg/mL). For example, per CLSI 2020 (Table 5A):

• Ampicillin for E. coli ATCC 25922: 2-8 μg/mL.
• Gentamicin for P. aeruginosa ATCC 27853: 0.5-2 μg/mL.

Laboratories should use the latest standard version (such as CLSI M100-35th ed. in 2025) and update ranges accordingly.

Common Problems and Troubleshooting

Zones Smaller Than Expected: May be due to high cation levels (for aminoglycosides) or thymidine (for SXT). Solution: Replace medium lot or adjust cations with CaCl2/MgCl2.

Larger Zones: Low cation levels or high pH. Solution: Chemical control of the medium.

Poor or No Growth: Contamination, improper pH, or dried medium. Solution: Sterility test and adjust pH to 7.2-7.4.

Recurrent Deviations: Check disk quality (expiration date, storage at -20°C), incubator, or inoculation technique.

Note: For fastidious strains, use MH-F and perform QC with specific strains like H. influenzae.

By following these steps, microbial quality control ensures that AST results are accurate and reliable, which is critical for patient treatment. Documenting all tests and reporting deviations to the laboratory supervisor is mandatory.

Storage and Expiration Date

• Storage: Store plates in the refrigerator (2-8°C) away from direct light. Dry medium at 10-30°C and away from moisture.
• Expiration Date: Typically 6 weeks for MHB and up to the labeled date for MHA. Avoid dried or contaminated media.
• Note: After opening, tightly close the lid to prevent moisture absorption.

Common Problems and Troubleshooting in Quality Assessment of Mueller-Hinto agar

• Problem: Small Zones with Aminoglycosides – Cause: High Ca/Mg levels; Solution: Adjust cations or replace lot.
• Problem: Poor Growth – Cause: Improper pH or contamination; Solution: Control pH and sterility.
• Problem: Large Zones with SXT – Cause: Low thymine levels; Solution: Test with control strain.
• General Problems: Bubbles (due to insufficient boiling), excess moisture (prolonged incubation), or contamination (sterility control). Common errors include improper inoculum adjustment, unsuitable incubation temperature, or early/late zone reading.

Specific Variants of Mueller-Hinton agar and Their Quality Control

• MH-F (Mueller-Hinton Fastidious): For fastidious bacteria like Haemophilus, with added NAD and horse blood.
• MH with Blood and NAD: For Streptococcus and Haemophilus, with separate QC control.
• MH for Fungi: For antifungal tests, with control for amphotericin B and itraconazole.

These variants require specific QC, such as using ATCC 49247 strains for Haemophilus.

Conclusion

Quality control of Mueller-Hinton medium is foundational for AST accuracy and should be performed according to CLSI standards. Adhering to all aspects from preparation to storage ensures valid and reliable results. Laboratory experts should regularly document QC and take corrective actions in case of deviations.

References in Quality Control and Assessment of Mueller-Hinton agar

• CLSI. Performance Standards for Antimicrobial Susceptibility Testing. M100, 30th ed. 2020.
• CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. M07.
• CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests. M02.
• ISO/TS 16782: Criteria for acceptable lots of dehydrated Mueller-Hinton agar and broth.

Common Questions

1. What is quality control of Mueller-Hinton agar?

Quality control of Mueller-Hinton medium is a process to ensure proper performance in antibiotic susceptibility tests, including physical, chemical, and microbial checks according to CLSI and WHO standards.

2. Why is quality control of Mueller-Hinton agar important?

This control ensures AST results are accurate and prevents misinterpretation of bacterial resistance, directly impacting patient treatment.

3. What are the main components of Mueller-Hinton agar in quality control?

Components include beef extract (2 g/L), casein hydrolysate (17.5 g/L), starch (1.5 g/L), and agar (17 g/L for MHA), with pH 7.3 ± 0.1.

4. How do we prepare Mueller-Hinton medium for quality control?

Dissolve 38 g of dry powder in 1 L of water, boil, autoclave, and pour into plates with 4 mm thickness, then adjust pH.

5. What are the physical parameters in quality control of Mueller-Hinton medium?

Including clear appearance without sediment, pH 7.2-7.4, and appropriate agar hardness for disk placement.

6. What are the standard strains for microbial quality control of Mueller-Hinton medium?

Strains like E. coli ATCC 25922, S. aureus ATCC 25923, P. aeruginosa ATCC 27853, and E. faecalis ATCC 29212.

7. What is the frequency of quality control for Mueller-Hinton medium?

For each new lot, weekly for low volume, and daily for more than 5 tests per day per CLSI.

8. What are common problems in quality control of Mueller-Hinton medium?

Small or large zones, poor growth due to improper pH, cation levels, or contamination.

9. What are specific variants of Mueller-Hinton medium for quality control?

MH-F for fastidious bacteria, MH with blood for Streptococcus, and MH for fungi with specific QC.

10. What are the main standards for quality control of Mueller-Hinton medium?

CLSI standards like M100, M02, M07, and ISO/TS 16782 for lot acceptance.

11. How do we check cation levels in quality control of Mueller-Hinton medium?

With aminoglycoside disk diffusion tests on P. aeruginosa ATCC 27853 and checking inhibition zones.

12. What is the expiration date of Mueller-Hinton medium in quality control?

Typically 6 weeks for MHB and up to the labeled date for MHA, with refrigerator storage.