Before you start
- Use pure overnight colonies.
- Use current CLSI or EUCAST breakpoint tables and quality-control strains.
Reagents and materials
Totals are for 4 isolates (+10% extra). Per-isolate amounts are in the middle column.
| Reagent | Per isolate | Total |
|---|---|---|
| Mueller–Hinton agar plate (4 mm depth) | 1 each | 1 each |
| Saline for 0.5 McFarland suspension | 2 mL | 2 mL |
| Antibiotic disks | 6 each | 6 each |
| Sterile swab | 1 each | 1 each |
Equipment
- 0.5 McFarland standard or densitometer
- Disk dispenser or forceps
- 35 °C incubator
- Ruler or calipers
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Prepare inoculum
Suspend colonies in saline to match a 0.5 McFarland standard (~1.5 × 10⁸ CFU/mL); use within 15 min.
Inoculate
Dip a swab, remove excess and swab the whole plate in three directions, rotating 60° each time.
Apply disks
Within 15 min, place disks firmly at least 24 mm apart.
Incubate
Incubate inverted at 35 ± 2 °C for 16–18 h.
Measure
Measure zone diameters in mm and interpret as susceptible, intermediate/susceptible-increased-exposure, or resistant using current breakpoints.
Critical points
- Inoculum density and agar depth strongly affect zone size.
- Run QC strains with known zone ranges each time.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Zones out of QC range | Check inoculum, disk storage, agar depth and incubation. |
Safety
Clinical isolates may require BSL-2 practices.
Further reading
- CLSI M02 Performance Standards for Antimicrobial Disk Susceptibility Tests; EUCAST disk diffusion method.
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.