MTT-分光光度法快速测定地衣芽孢杆菌发酵液活菌数的研究

Research on rapid determination of viable bacteria count in fermentation broth of Bacillus licheniformis by MTT-spectrophotometry

试验以地衣芽孢杆菌发酵液为研究对象,旨在建立1种快速、稳定、灵敏度高的活菌计数方法,并探讨MTT-分光光度法用于活菌计数的可行性。试验筛选显色反应终止液最适浓度、最适反应波长、最适反应时间、最适菌液浓度等,考察不同培养基、培养液保存后和其他干扰物对测定结果的影响,并对比MTT-分光光度法与稀释平板法测定发酵菌液中地衣芽孢杆菌活菌数结果的关系。结果显示,在最大吸收波长550 nm处测定OD值,反应时间为1 h,反应终止液为1.0 mol/L盐酸,MTT添加量为0.2 mL,新鲜的地衣芽孢杆菌发酵液OD值>0.04。菌液浓度为10^(8)~10^(9)CFU/mL时,OD550 nm值与稀释平板法得到的活菌数具有很好的线性对应关系,当菌液浓度<10^(8)CFU/mL时对应关系不再成立。研究表明,活菌量>10^(8)CFU/mL时,MTT-分光光度法不受地衣芽孢杆菌生长阶段的影响,可准确计量地衣芽孢杆菌发酵液活菌量相对应的吸光度,从而快速、准确地检测活菌数。

The aim of the study was to establish a rapid,stable and highly sensitive viable counting method for Bacillus licheniformis fermentation broth,and to explore the feasibility of MTT-spectrophotometry for viable counting.The optimum concentration,reaction wavelength,reaction time and concentration of the chromogenic stopping solution were screened.The influence of different media,culture solution and other disturbing substances on the determination results was investigated.The relationship between the MTT-spectrophotometry and dilution plate method in the determination of the viable bacteria number of Bacillus licheniformis in the fermentation bacterial solution was compared.The results showed that the OD value was determined at the maximum absorption wavelength of 550 nm,the reaction time was 1 h,the reaction termination liquid was 1.0 mol/L hydrochloric acid,the amount of MTT was 0.2 mL,and the OD value of the fresh Bacillus licheniformis fermentation liquid was>0.04.When the concentration of bacterial solution was 10^(8)~10^(9)CFU/mL,the OD550 nm value had a good linear correlation with the number of viable bacteria obtained by dilution plate method.When the concentration of bacterial solution was less than 108 CFU/mL,the correlation was no longer valid.The study indicates that MTT-spectrophotometry is not affected by the growth stage of Bacillus licheniformis when the viable bacteria amount was>10^(8)CFU/mL,and could accurately measure the absorbance corresponding to the viable bacteria amount of Bacillus licheniformis fermentation liquid,so as to detect the viable bacteria number quickly and accurately.