MTT方法评价微生物细胞活性的探讨

EVALUATION OF MTT METHOD FOR QUANTIFICATION OF MICROBIAL CELL VIABILITY IN MICROPLATES

对MTT比色法用于评价微生物细胞活性进行了探讨。本文以大肠杆菌为模式菌株,研究了不同浓度MTT、不同用量、在不同时间对试验结果OD570值的影响,结果表明细菌数在4.9×107—4.9×108个/mL范围内测出的OD570值与细菌浓度呈良好的正相关,0.5mg/mLMTT用量20μL,反应时间20min时效果最佳,其相关回归方程为y=0.1769x+0.03,R2=0.9983。

In the study of microbiology, the activity and the number of microbial cells are often determined to reflect the growth of microbial cells. The conventional methods usually include plate culture method, the cell number of automatic analyzer, turbidity, dry cell weight and biological fluorescence method, and so on. Among these conventional methods, the plate culture method is cumbersome, time-consuming and poor reproducibility, and most importantly, it can not truly reflect the growth of bacteria, furthermore, it is not suitable for bigger quantities experiment. For the other conventional methods, they even could not distinguish between live and dead cells, and the number and activity of cells are vulnerable to the medium and metabolites. In recent years, MTT colorimetric method is used to evaluate the microbial cell activity, which is more simple, rapid, sensitive and stable compared with the conventional methods. The principle is that the living cells can reduce MTT to a blue-violet matter by mitochondrial dehydrogenase in cells, the matter has the optical absorption value from 560 nm to 610 nm, and most reports indicate that blue-violet matter have the best optical absorption value at 570 nm. MTT colorimetric method was always used to detect the activity of immune cells and other animal cells, but it was rarely reported in the detection of the activity of the microbial cells. In this paper, MTT colorimetric method for quantification of microbial cell viability or cell growth in microplates was discussed. Escherichia coli was employed to determine the conditions of assay, which included the feasibility, reaction periods, microbial cell number, the concentration of MTT and so on. It was found that MTT had a high correlation between the values of OD570and the microbial cell number, which was from 4.9 × 10^7 cells/mL to 4.9 × 10^8 cells/mL The correlation was the best when bacteria was treated with MTT 0. 5 mg/ mL of 20μL for 20 min, and the correlation and regression equation was y = 0. 1769x ＋ 0.03 ,R2 = 0. 9983 ; when bacteria was treated with MTT 5 mg/mL of 20μL for 30 min, the correlation and regression equation was y = 0. 4032x ＋ 0. 0143 RE= 0. 998. MTT colorimetric method is economic, simple, fast, stable, high sensitive, and the results can show the number of live micro-organisms or cells activity clearly. Previous studies also showed that MTT colorimetric method was superior to other color, such as the TTC, INT, XTT, blade, etc. among evaluation methods of cell viability.