Disk Diffusion Susceptibility Method
(Kirby Bauer)
Clinical Microbiology Review 227
Organisms
Inoculum
Medium
Disks
Incubation
Modifications for fastidious
bacteria
Reading
Situations/actions
Rapidly growing aerobes & facultative anaerobes. Not for slow growers, anaerobes, or fastidious
organisms (except with modifications).18–24 hr pure culture. 1.5 × 10^8 CFU/mL. Compare to 0.5 McFarland standard. Too heavy = smaller
zones/false resistant. Too light = larger zones/false susceptible.Mueller-Hinton agar. Swab entire surface evenly to achieve solid lawn of growth.Store in refrig or freezer in sealed, desiccated container. Warm to RT before use. Check expiration
dates. Place within 15 min of inoculation. No more than 12 per 150-mm plate.Within 15 min of disk placement. Inverted. Ambient air. 35°C for 16–18 hr. <16 hr = ↑zone, false
susceptible. >18 hr = ↓zone, false resistant. (Exception: MRSA may require 24 hr.)Streptococcus:Use MH supplemented with 5% sheep blood. Incubate in 5%–7% CO 2. Haemophilus:
Use Haemophilustest medium (HTM). Incubate in 5%–7% CO 2.Routine: Measure zones from back side of plate using ruler or calipers. Media containing blood: Read
from top with lid removed.Sparse growth: Repeat with standardized inoculum. Mixed culture: Repeat with pure inoculum.
Colonies within zone: Subculture & retest. Slight growth in zone for trimethoprim & sulfonamides:
Disregard. Swarming of Proteusinto zone: Ignore.continued...