MTT方法评价微生物细胞活性的探讨
EVALUATION OFM TTM ETHOD FOR QUANTIFICATION OFM ICROBIAL CELL VIABIL ITY INM ICROPLATES
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摘要: 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。Abstract: In the study of microbiology, the activity and the number of microbial cells are often determined to reflect the growth ofmicrobial cells. The conventionalmethods usually include plate culturemethod, the cell numberof automatic analyzer, turbidity, dry cellweight and biological fluorescence method, and so on1 Among these conventionalmethods, 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 experiment1 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 metabolites1 In recent years, MTT colorimetric method is used to evaluate the microbial cell activity, which ismore simple, rapid, sensitive and stable compared with the conventionalmethods1 The principle is that the livingcells can reduce MTT to a blue-violetmatter bymitochondrial dehydrogenase in cells, the matter has the optical absorption value from 560 nm to 610 nm, and most reports indicate that blue2violet matter have the best optical absorption value at 570 nm. MTT colorimetricmethod was always used to detect the activity of immune cells and other animal cells, but itwas rarely reported in the detection of the activity of themicrobial cells1 In this paper, MTT colorimetricmethod for quantification of microbial cell viability or cell growth in microplateswas discussed1 Escherichia coliwas employed to determine theconditions of assay, which included the feasibility, reaction periods, microbial cell number, the concentration ofMTT andso on1 Itwas found thatMTT had a high correlation between the values of OD570 and the microbial cell number, which wasfrom 4.9 ×107 cells/mL to 4.19 ×108 cells/mL. The correlation was the bestwhen 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 R2= 0.998.MTT colorimetricmethod is economic, simple, fast, stable, high sensitive, and the results can showthe numberof live micro-organisms or cells activity clearly. Previous studies also showed that MTT colorimetricmethod wassuperior to other color, such as the TTC, INT, XTT, blade, etc1 among evaluation methods of cell viability.
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Keywords:
- MTT colorimetry /
- Escherichia coli /
- Viability
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