A rapid and accurate 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay for quantification of bacteriocins with nisin as an example.
The objective of this study is to propose a more accurate and faster MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay (MCA) for quantitative measurement of polypeptide bacteriocins in solutions with nisin as an example. After an initial incubation of nisin and indicator bacterium Micrococcus luteus NCIB 8166 in tubes, MTT was added for another incubation period. After that, nisin was quantified by estimating the number of viable bacteria based on measuring the amount of purple formazan produced by cleavage of yellow tetrazolium salt MTT. Then MCA was compared to a standard agar diffusion assay (ADA). The results suggested a high correlation coefficient (r(2)=0.975+/-0.004) between optical density (OD) and the inhibitory effect of nisin on a bacterial strain Micrococcus luteus NCIB 8166 at a range of 0.125-32 IU/ml. The MCA described in this study was very quick. Quantification of nisin took only 7-8 h and the detection limit was at the level of 0.125 IU/ml when compared to 12 IU/ml and 24-28 h for ADA. The MCA provides an accurate and rapid method for quantification of nisin in solutions and is expected to be used for quantification of other antimicrobial substances.
Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310029, China.
Fang
F
Wang
Li-ting
LT
Cao
Song-hua
SH
Hu
D001430:Bacteriocins / Q000032:analysis* / Q000378:metabolism; D003124:Colorimetry / Q000379:methods*; D005779:Immunodiffusion; D016982:Micrococcus luteus / Q000378:metabolism; D009561:Nisin; D012044:Regression Analysis; D013778:Tetrazolium Salts / Q000032:analysis*; D013844:Thiazoles / Q000032:analysis*; D013997:Time Factors
D001430:Bacteriocins; D013778:Tetrazolium Salts; D013844:Thiazoles; D009561:Nisin; C022616:thiazolyl blue