Before you start
- Prepare a 0.5 McFarland suspension and dilute it so each well receives about 5 × 10⁵ CFU/mL.
Reagents and materials
Totals are for 4 rows (+10% extra). Per-isolate × antibiotic (one row) amounts are in the middle column.
| Reagent | Per isolate × antibiotic (one row) | Total |
|---|---|---|
| Cation-adjusted Mueller–Hinton broth50 µL × 11 wells | 0.6 mL | 0.6 mL |
| Antibiotic working solution (2× highest concentration) | 100 µL | 100 µL |
| Standardised inoculum (to ~5 × 10⁵ CFU/mL final) | 0.6 mL | 0.6 mL |
Equipment
- Sterile 96-well U- or flat-bottom plate
- Multichannel pipette
- 35 °C incubator
- Plate reader (optional)
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Add broth
Add 50 µL broth to wells 2–12 of each row.
Add antibiotic
Add 100 µL of 2× top concentration to well 1.
Serially dilute
Transfer 50 µL from well 1 to 2, mix, and continue to well 10; discard 50 µL from well 10. Well 11 = growth control, well 12 = sterility control.
Inoculate
Add 50 µL inoculum to wells 1–11 (not 12).
Incubate
Cover and incubate at 35 ± 2 °C for 16–20 h.
Read
The MIC is the lowest concentration with no visible growth. Check that the growth control is turbid and the sterility control clear.
Critical points
- Confirm the inoculum by plating a CFU count.
- Include quality-control strains with known MIC ranges.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Skipped wells | Repeat; check pipetting and mixing. |
| No growth in control | Check inoculum and medium. |
Safety
Work at the organism’s biosafety level.
Further reading
- CLSI M07 Methods for Dilution Antimicrobial Susceptibility Tests; Wiegand I et al. Agar and broth dilution methods to determine the MIC. Nature Protocols (2008).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.