Modified agar diffusion bioassay for better quantification of Nisaplin®



Minal Lalpuria, FDA/CFSAN/OFS/FPET/LACF, 5100 Paint Branch Parkway, College Park, MD 20740, USA. E-mail:



To investigate the effect of different well sizes and pre-diffusion times at 4°C, on the sensitivity, accuracy and precision of nisin quantification by agar diffusion bioassay.

Methods and results

Nisin solution (0·625–125 μg ml−1) was filled in wells (3·5 mm or 7 mm diameter) made on agar plates inoculated with Micrococcus luteus, followed by pre-diffusion (0, 24, 48 or 72 h), incubation and measurement of inhibition zone. Regression analysis indicated that wells with 3·5 mm diameter had smaller standard deviation and higher predictive accuracy, compared to wells with 7 mm diameter. Based on Tukey's test, pre-diffusion resulted in significantly different inhibition zones at different nisin concentrations. Pre-diffusion also improved sensitivity of the assay. Different regression models were considered to explore the relationship between inhibition zone and nisin concentration for different pre-diffusion times. A spline model was determined to be the best-fit model, and 48 h was the best pre-diffusion time.


Wells with 3·5 mm diameter demonstrated higher accuracy for nisin quantification compared to wells with 7 mm diameter. 48 h was the best pre-diffusion time for nisin concentration in the range 0·625–125 μg ml−1.

Significance and impact of the study

The findings from this study will be helpful in quantifying nisin and compounds with antimicrobial properties accurately over a wide range of concentrations using agar diffusion bioassay.