The cylinder-plate (cup-plate) diffusion assay is a standard method for determining antibiotic potency used worldwide in pharmaceutical quality control.
Method Principle
Agar Diffusion
When an antibiotic solution is placed in cylinders on an inoculated agar surface, the antibiotic diffuses into the agar. If the test organism is susceptible, a clear zone forms where bacterial growth is inhibited.
Zone Diameter Relationship
The diameter of the inhibition zone is proportional to the logarithm of the antibiotic concentration, allowing potency calculation.
Procedure Overview
Preparation
- Prepare base layer of sterile agar
- Add seed layer inoculated with test organism
- Allow to solidify
- Place sterile cylinders on agar surface
- Add standard and sample solutions to cylinders
Incubation
Plates are incubated at appropriate temperature (usually 35-37°C) for specified time (typically 16-18 hours).
Reading
Zone diameters are measured using an Antibiotic Zone Reader for accuracy and precision.
Critical Parameters
Test Organism Selection
Specific organisms are specified for each antibiotic type:
- Micrococcus luteus for penicillins
- Bacillus subtilis for many antibiotics
- Staphylococcus aureus for certain assays
Medium Preparation
Exact composition, pH, and depth of agar layers affect diffusion and zone formation.
Standard Curves
Multiple concentration levels of reference standard establish the dose-response relationship.
Pharmacopeial Compliance
Methods align with:
- USP <81> Antibiotics—Microbial Assays
- BP Appendix XIV
- EP 2.7.2
- JP antibiotic assay methods
Zone Reading Technology
The Antibiotic Zone Reader provides:
- High resolution measurement (0.01 mm)
- Uniform illumination
- Reduced operator variability
- Digital recording of results