How would an agar depth >4 mm affect the diameter of the zone of inhibition?

a. The agar depth is irrelevant to the outcome of this experiment.

b. The diameter of the zone of inhibition would be less than expected.

c. The diameter of the zone of inhibition would be greater than expected.

The diameter of the zone of inhibition will be less than expected (b)

Explanation:

In the preparation of agar plates, it is a standard procedure to pour agar into the plates to a depth of 4 mm which is an approximate agar volume of 25 ml for a 100 mm diameter plates, and 60 ml for a 150 mm diameter plate.

If agar is poured to get an agar depth of >4 mm, and antibiotic discs are placed on it, after incubation, the diameter of inhibition zone will be less than expected when compared to a plate with agar depth of 4 mm because more agar means more dilution medium for the antibiotic in the disc, hence reducing the concentration of the antibiotic in the medium this is so because the antibiotic sensitivity testing procedure effectively works with diffusion, and a thicker medium creates more diffusion gradient reducing the overall antibiotic concentration making the bacteria appear resistant (false resistant) compared to media of 4 mm thickness.

On the other hand, if the agar depth is < 4 mm thick, then the effective antibiotic concentration is increased hence creating a bigger zone of inhibition creating a false susceptible result.