CLSI M23S3-2023
Procedure for Confrming the Acceptability of Mueller-Hinton Agar Sources for Subsequent Use in CLSI and/or EUCAST Studies to Establish Disk Difusion Quality Control Ranges
- Standard No.
- CLSI M23S3-2023
- Release Date
- 2023
- Published By
- Clinical and Laboratory Standards Institute IX / CLSI
- Introduction
Analysis of the Core Content of the M23S3 Standard
The CLSI M23S3 standard document, "Procedure for Confirming the Acceptability of Mueller-Hinton Agar Sources for Subsequent Use in CLSI and/or EUCAST Studies to Establish Disk Diffusion Quality Control Ranges," provides microbiology laboratories worldwide with a standardized procedure for confirming the acceptability of Mueller-Hinton agar sources. This standard is fully consistent with EUCAST SOP 13.0 and demonstrates the deep collaboration between CLSI and EUCAST in the field of antimicrobial susceptibility testing.
Background of Standard Development and Technological Evolution
With the growing global threat of antimicrobial resistance, the importance of standardized antimicrobial susceptibility testing methods has become increasingly prominent. As the primary culture medium for disk diffusion testing, the quality and stability of Mueller-Hinton agar directly impacts the accuracy and comparability of test results. Differences in composition between MHAs produced by different manufacturers can lead to systematic variation and affect the establishment of quality control ranges.
Through years of research, a joint CLSI and EUCAST working group has recognized that variability in MHA composition is the most common cause of systematic variation in disc diffusion test results. Therefore, the two groups jointly developed this supplementary procedure to confirm the reliability of the MHA source before conducting full quality control studies.
Detailed Explanation of Key Technical Requirements
2.1 Quality Control Strains and Clinical Isolate Requirements
The standard requires the use of standard QC strains representative of the target strains and containing at least two isolates with elevated MIC values and/or reduced inhibition zone diameters. The recommended number is 4-6 strains, depending on the number of target species for the antimicrobial drug.
2.2 Disc Requirements
Commercially produced discs must be used and should include products from at least two different manufacturers. If discs from two manufacturers are not available, two different lots from the same manufacturer may be used. The paper strip batches used in the study should be used in subsequent complete QC studies whenever possible.
2.3 Culture Media Requirements
Mueller-Hinton Agar must conform to international standards and demonstrate acceptable performance for QC strains. Plates prepared using MHA powder from at least four different manufacturers are required, including at least two that represent widely used MHA manufacturers in major markets.
Component Type Minimum Requirements Alternative Solutions Key Quality Control Points QC Strains 4-6 Standard Strains Includes WT and NWT Isolates Inhibition Zone>10mm Antimicrobial Drug Discs 2 Different Manufacturers 2 Lots from the Same Manufacturer Includes Control Drug MHA Medium 4 Different Manufacturers 2 Lots from the Same Manufacturer Compliant with ISO/TS 16782
Operational Procedures and Technical Points
3.1 Measuring and Recording Inhibition Zone Diameters
Perform the disc diffusion test according to CLSI M02 and EUCAST recommended methods. Each QC strain and clinical isolate should be tested in triplicate using a single bacterial suspension. All four MHA plates should be inoculated with the same bacterial suspension, and at least two test discs and one control disc should be placed on each plate.
The inhibition zone should be measured to the nearest millimeter and read by the same person. If colonies are observed within the inhibition zone, the diameters with and without colonies should be recorded. For inhibition zones that are difficult to read (e.g., blurred edges, double zones), measurements should be recorded by two independent readers.
3.2 Data Analysis
For each QC strain-antimicrobial drug combination, calculate the mean, median, standard deviation, and range for each tested MHA source. If the mean or median inhibition zone diameters for the three media are not within ±1 mm, three additional tests should be performed using the same disk-organism-media combination.
3.3 Data Review and MHA Selection
Upon completion of the study, the results should be submitted to the CLSI-EUCAST Joint Working Group for review. MHA sources that demonstrate mean or median inhibition zone diameters within ±1 mm for the test agent are preferred. If this criterion cannot be met, MHA sources that are within ±2 mm may be selected.
Implementation Recommendations and Quality Control
Media Performance Validation
Appendix A provides detailed guidance for establishing acceptable performance for Mueller-Hinton Agar. The QC strain-antimicrobial agent combinations in Table A1 are recommended for indirect monitoring of media components (pH, cations, thymidine) and other undefined characteristics.
Key monitoring combinations include: Pseudomonas aeruginosa ATCC 27853 with gentamicin (monitoring pH and Ca²⁺/Mg²⁺), ciprofloxacin (monitoring pH), and meropenem (monitoring Zn²⁺); Staphylococcus aureus ATCC strain with erythromycin, penicillin, and tetracycline; and Enterococcus faecalis ATCC strain with trimethoprim-sulfamethoxazole (monitoring thymidine).
Acceptable Result Criteria
According to the example in Appendix B, acceptable results must meet the following requirements: the maximum range between a single disk batch and a single culture medium batch is 3 mm; the QC results for the control drug are within the specified range; and for the test drug, the mean or median inhibition zone diameter for at least three culture media is within ±1 mm.
Value and Future Prospects of Standard Application
The release of the M23S3 standard marks a new phase in the collaboration between CLSI and EUCAST in the standardization of antimicrobial susceptibility testing. Although this procedure is not a mandatory requirement, it is highly recommended as a supplementary quality check and can significantly improve the reliability and reproducibility of quality control studies.
As the global antimicrobial resistance surveillance network continues to improve, standardized culture medium quality control procedures will play an increasingly important role in ensuring data comparability and monitoring accuracy. Laboratories should actively adopt this procedure to improve the quality assurance level of antimicrobial susceptibility testing.
