噻唑蓝法和刃天青法检测植物成分对两种病原细菌的抑菌活性

Methythiazol tetrazolium and resazurin assays for detectionof antibacterialactivities of plant constituents against two bacterial pathogens

通过比较茄青枯拉尔氏菌和柑橘黄单胞菌柑橘致病变种菌量、噻唑蓝和刃天青的浓度、抑菌剂与菌体作用时间等因素对吸光值的影响,优化噻唑蓝和刃天青法检测抑菌剂抑菌活性的条件,即:细菌初始浓度为105～106CFU/mL,噻唑蓝和刃天青浓度分别为0.5mg/mL和0.02mg/mL,抑菌剂作用时间16～24 h。运用优化的噻唑蓝和刃天青法检测了植物提取物和分离物对2种细菌的抑菌活性,2种方法得到的结果一致。以茄青枯拉尔氏菌为靶标菌,对于无色化合物EE-1和土黄色化合物EE-2,2种方法测定的最低抑菌浓度相同,分别为10μg/mL和20μg/mL。表明噻唑蓝和刃天青法能快速、准确地测定微量植物成分的抑菌活性。

Optimized conditions of methythiazol tetrazolium and resazurin assays for detection of antibacterial activities of bactericides were established by evaluating influences of the concentration of Ralstonia solanaceamrn and Xanthomonas citri pv. cirri, the concentration of methythiazol tetrazolium and resazurin, and reaction time of bactericide with bacteria on the optical density. The results showed that optimized conditions of methythiazol tetrazolium and resazurin assays were that bacterial concentration was 10s - 10s CFU/mL, methythiazol tetrazolium concentration was 0.5 mg/mL, resazurin concentration was 0. 02 mg/mL and reaction time of bactericide was 16 - 24 h. The antibacterial activities of some plant extracts and separating constituents against the two bacterial pathogens were detected by methythiazol tetrazolium and resazurin methods, and the results were consistent. The minimal inhibitory concentration of colorless compound EE-1 and khaki compound EE-2 against R. solanacearum were respectively 10 μg/mL and 20 μg/mL by using the two methods, which showed the same result. It could be concluded that antibacterial activity of micro-quantitative plant constituents could be tested fast and precisely by using methythiazol tetrazolium and resazurin assays.