ESBL Confirmatory Disk Method

1. Prepare a 0.5 McFarland dilution of the test isolate in 5 mL of broth or saline.

2. Spread with a cotton swab a lawn of this suspension onto Mueller Hinton Agar
plate.
3. Place ESBL confirmatory susceptibility disks on the plate. Use ceftazidime,
ceftazidime/clavulanate, cefotaxime, and cefotaxime/clavulanate.
4. Incubate overnight at 350C in ambient air for 16-24 hours.
5. Measure zones of inhibition. ESBL positive isolates should have a > 5mm
increase in zone diameter in either disk containing clavulanate when compared
with the drug alone.

Modification for isolates that produce AmpC:

To remove interference from possible AmpC beta-lactamase, add sterile

cloxacillin (I use cloxacillin sodium salt monohydrate (C9393-1G, Sigma-Aldrich, St.
Louis, MO) (AmpC inhibitor) to a calculated final concentration of 200 mcg/ml, to
molten sterile Mueller Hinton Agar and pour agar plates to a depth of around 4 mm. Let
plates cool, harden and dry before use. Plates can be stored packaged in a refrigerator for
up to 2 months, as the cloxacillin will become less potent over time. Set up ESBL
confirmatory disks as above and interpret accordingly.

Control Strains

ESBL negative Escherichia coli ATCC 25922 and ESBL positive Klebsiella pneumoniae
ATCC 700603