Factors influencing optimization of diffusion assays for antibiotics

Agar nutrient content, cylinder charge volume, thickness (volume) of the agar layer, and incubation temperature were 4 factors varied to determine their effect(s) on the optimization of the cylinder-plate diffusion assay. Chlortetracycline was the pilot antibiotic and Bacillus cereus was used as the assay organism. Zones of inhibition were larger when the incubation temperature was lower than that which was commonly used and/or when the nutrient level was decreased; the zones were smaller when the incubation temperature was raised and/or when an increased nutrient level was used. The thickness (volume) of the assay layer played the most important role; the thinner the layer the less the effect the cylinder charge volume had on the zone diameter. The slopes of the response lines were minimally affected by cylinder charge volume. For a 7 mL assay layer per standard Petri plate, cylinder charge volumes ranging from 150 to 250 microL had little effect on zone diameter. The linearity of the response line was unaffected by assay layer thickness (volume), nutrient level, temperature of incubation, or cylinder charge volume. As long as the conditions for the assay were standardized, there were no discernible effects on recoveries or potencies.