Modified agar diffusion bioassay for better quantification of Nisaplin^®

Abstract

Aims: 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 (0Á625-125 lg ml À1 ) was filled in wells (3Á5 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 3Á5 mm diameter had smaller standard deviation and higher predictive accuracy, compared to wells with 7 mm diameter. Based on Tukey's test, prediffusion 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.Conclusions: Wells with 3Á5 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 0Á625-125 lg ml À1 . 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.

Modeling development of inhibition zones in an agar diffusion bioassay

“…Good agreement ( R 2  = 0.984) was obtained between model-predicted and experimentally determined values as reported by Lalpuria et al. (2012). This model can be used as an alternative method to develop standard curves for antimicrobial assays, resulting in savings in time when determining unknown concentrations of antimicrobials.…”

“…(2012). The media for agar bioassay consisted of 0.8% nutrient broth, 0.75% Bacto™ agar, and 1% Tween 20.…”

“…A modified agar diffusion method for quantification of nisin was described by Lalpuria et al. (2012). They found that using 3.5 mm diameter wells and prediffusing the nisin solution at 4°C for 48 h followed by incubation at 30°C offered the best precision and sensitivity for nisin quantification, when Micrococcus luteus was used as the indicator organism.…”

Antilisterial Bacteriocin from Lactobacillus rhamnosus CJNU 0519 Presenting a Narrow Antimicrobial Spectrum

“…Nisaplin is approved for use in over 50 countries (Lalpuria et al ., 2013). Several bacteriocins including pediocin PA-1 are promising candidate for biopreservatives or antibiotic alternatives (Bali et al ., 2014; Cotter et al ., 2013; Mehta et al ., 2013).…”

Ginger extract as green biocide to control microbial corrosion of mild steel

“…The minimal inhibition concentration of GIE with EN2 was experimentally determined by the conventional agar diffusion assay, as described by Lalpuria et al (2012). Different concentrations of GIE (i.e.,10,15,20,25, and 30 ppm) were used for this assay.…”