Measurement of antimicrobial susceptibility by zones of inhibition

Understanding Zones of Inhibition

Zones of inhibition refer to clear circular areas around antimicrobial discs on agar plates where microbial growth has been prevented.
They are a representation of the effectiveness of an antimicrobial substance. The larger the zone, the more effective the antimicrobial agent is against the bacteria or microorganism.
These zones are usually measured in millimetres from the centre of the disc to the edge of the clear zone.

The concept of zones of inhibition is used in the disc diffusion method, a key technique for performing Antimicrobial Susceptibility Testing (AST).
This method helps to determine the sensitivity or resistance of pathogenic microorganisms to an antimicrobial substance. The larger the zone of inhibition, the more sensitive the microorganism is to the drug.

To perform a disc diffusion test, you start by spreading a sample of the bacteria over an agar plate to ensure uniform growth.
Then, discs impregnated with different antimicrobials are placed onto the agar surface. After incubation, the plates are examined for zones of inhibition.
The interpretation of the zones of inhibition is based on guidelines such as those provided by the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

It’s worth noting that several factors can affect the size of zones of inhibition, including the diffusion rate of the antimicrobial in agar, thickness of the agar medium, and incubation conditions.
Failure to standardise these conditions can lead to inaccurate interpretation of results.

While the disc diffusion method is relatively easy to perform and interpret, it is not without limitations.
It gives a qualitative result (i.e., sensitive, intermediate, or resistant) rather than a quantitative one (like the Minimum Inhibitory Concentration or MIC given by the broth dilution method).
Moreover, some antimicrobials do not diffuse well in agar and hence this method might not be suitable for all antimicrobial agents.