You can find the very detailed procedure in the Clinical and Laboratory Standards Institute (CLSI) publications.

Within the ASM publications, you can also find the procedure in chapter 7 of the 5th edition (2023) of the Clinical Microbiology Procedures Handbook.

Liji S S Article Antibiotic susceptibility testing using minimum inhibitory c...

1. CLSI (Clinical and Laboratory Standards Institute) Guidelines

The CLSI recommends performing MIC testing using a microdilution broth method. A series of two-fold dilutions of the beta-lactam antibiotics (including ampicillin) are prepared in a cation-adjusted Mueller-Hinton broth (CAMHB). The bacterial suspension is standardized to 0.5 McFarland turbidity and further diluted to achieve a final inoculum density of approximately 5×1055 \times 10^55×105 CFU/mL in the wells. Plates are incubated at 35°C for 16-20 hours under aerobic conditions. The MIC is determined as the lowest concentration of the antibiotic that inhibits visible bacterial growth. This method ensures reproducibility and standardization across laboratories (CLSI, 2023).

Reference: CLSI, 2023. Performance Standards for Antimicrobial Susceptibility Testing. 33rd ed. CLSI Document M100.

2. BSAC (British Society for Antimicrobial Chemotherapy) Guidelines

The BSAC protocol is similar to CLSI but emphasizes using Iso-Sensitest broth instead of CAMHB to account for the nutritional differences that may affect bacterial growth and antibiotic activity. A standard inoculum, adjusted to 5×1055 \times 10^55×105 CFU/mL, is added to the wells containing two-fold dilutions of beta-lactams. The plates are incubated at 35°C for 18-20 hours. The MIC is the lowest antibiotic concentration with no visible growth, and results are interpreted using BSAC-specific breakpoints (BSAC, 2023).

Reference: BSAC, 2023. Methods for Antimicrobial Susceptibility Testing. Version 15.

3. EUCAST (European Committee on Antimicrobial Susceptibility Testing) Guidelines

EUCAST follows a similar microdilution procedure but strictly enforces its specific quality control ranges and breakpoints. CAMHB, supplemented with necessary ions (e.g., calcium and magnesium), is used for MIC determination. The bacterial inoculum is standardized to 5×1055 \times 10^55×105 CFU/mL, and the plates are incubated at 35°C for 18 ± 2 hours. EUCAST emphasizes precise adjustments of inoculum density to prevent variability and ensure accurate results. MICs are interpreted against EUCAST-defined clinical breakpoints for beta-lactams (EUCAST, 2024).

Reference: EUCAST, 2024. The European Committee on Antimicrobial Susceptibility Testing: MIC Determination Guidelines, Version 14.0.