Methods of Antibiotic Assay

Antibiotic assays can be carried out in the form of agar diffusion assays, or as tube assays in liquid media.  

Antibiotic solutions placed in reservoirs need sterilising. Autoclaving can only be used for a small number of antibiotics, and many need to be sterilised by membrane filtration. The use of cups to hold the antibiotic has the advantage that non-sterile solutions may be used. 

The antibiotic should be allowed to diffuse for a short period after application to the plate. The longer it is allowed to diffuse, the larger the zone of inhibition becomes and the steeper the dose-response curve. If the diffusion is carried out at room temperature, growth may occur during the diffusion period, leading to an indeterminate zone. The pre-incubation diffusion should therefore be carried out in a refrigerator to minimise growth. The reduction in temperature reduces the diffusion rate, and the best conditions must be determined by trial and error. A diffusion period of several hours at 4 °C is usually adequate. 

Theoretically all doses of antibiotic should be applied simultaneously. When large plates with a number of reservoirs are used, a significant time may elapse between filling the first and last reservoirs. This influences the results by altering the time of diffusion. This problem can be reduced by using an appropriate pattern of sample distribution based on a Latin square or quasi-Latin square. 

A Latin square allows a large number of samples to be applied to a square plate in a pre-determined manner. Samples are arranged in a square e.g. 4x4, 6 X 6, or 8 X 8. In a true Latin square, each antibiotic application appears once in each row and once in each column. A 4 x 4 (2+2) plate would have sixteen applications to the plate at two levels for the standard and two for the test. Low and high dose levels would be in the ratio 1 2. Each low standard would appear four times, each high standard four times, each low sample four times, and each high sample four times. 

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The cup plate method of assay (51) is similar to the widely used method of antibiotic assay except that a vitamin Bts deficient medium is employed and zones of stimulation rather than zones of inhibition are measured. The method described in this section is that of Harrison, Lees, and Wood (15). 

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