MIC testing (broth microdilution and gradient strip)

Principle

• The minimum inhibitory concentration (MIC) is the lowest antimicrobial concentration that prevents visible growth.
• Broth microdilution is the CLSI/EUCAST reference method; gradient strips give an MIC on agar.

Specimen

• Pure overnight colonies; inoculum standardized from a 0.5 McFarland suspension.

Reagents and equipment

• Cation-adjusted Mueller–Hinton broth (CAMHB) in microtiter panels with two-fold dilutions (e.g. 0.25, 0.5, 1, 2 µg/mL).
• Gradient strips (e.g. Etest) with a continuous antibiotic gradient and printed scale; Mueller–Hinton agar plates.

Procedure

• Broth microdilution: dilute the suspension so each well contains about 5 × 10^5 CFU/mL.
• Include a growth control (no drug) and a sterility control (no organism).
• Incubate at 35 ± 2 °C in ambient air for 16–20 h (longer for some organisms/agents).
• Gradient strip: swab MH agar as for disk diffusion, apply the strip with the scale facing up, incubate the same way.

Results

• Broth: MIC is the first well with no visible growth (button or turbidity); growth control must show good growth.
• Strip: read where the edge of the elliptical inhibition zone intersects the strip; values between marks are rounded up to the next two-fold dilution.
• Interpret MIC as S, SDD/I or R using current breakpoints; an MIC value cannot be compared directly between different drugs.

Quality control and pitfalls

• QC strains include S. aureus ATCC 29213, E. faecalis ATCC 29212, E. coli ATCC 25922 and P. aeruginosa ATCC 27853.
• Skipped wells, contamination or inadequate growth invalidate results.
• Trailing endpoints: read trimethoprim–sulfamethoxazole at about 80% inhibition.
• Colistin/polymyxin MICs should be done by broth microdilution; gradient strips and disks are unreliable.

Clinical use

• Needed for serious infections (endocarditis, meningitis), vancomycin MIC in staphylococci, penicillin in pneumococci, and for dose optimization.