应用二醋酸酯荧光素与碘化丙啶的流式细胞术分析三种细菌的活性

Flow cytometry for viability analysis of three bacteria species with fluorescein diacetate vs propidium iodide staining

目的:探讨在流式细胞术中应用二醋酸酯荧光素(FDA)与碘化丙啶(PI)分析金黄色葡萄球菌、大肠埃希菌和结核分枝杆菌活性的性能。方法:选取对数生长期金黄色葡萄球菌、大肠埃希菌和结核分枝杆菌临床分离株各20株,制备1麦氏浊度菌悬液2支(约2 ml/支);1支于85℃水浴30 min灭活,获取死菌菌液;分别应用FDA(0.5μg/m L)、PI(5μg/m L)两种荧光染料对死、活菌菌液进行染色(两种染料的染色时间分别为30 min和15 min);以不染色死菌液作为对照,应用流式细胞术检测经两种染料染色死、活菌菌液及不染色死菌液的平均荧光强度(FMI),对比分析其FMI均值水平。结果:以FDA为染料,金黄色葡萄球菌、大肠埃希菌的无染料管、活菌管、死菌管FMI均值水平无差别,结核分枝杆菌活菌管FMI均值水平(129.61±8.69)明显高于死菌管(3.34±0.11)及无染料管(0.33±0.02);以PI为染料,结核分枝杆菌的无染料管、活菌管、死菌管FMI均值水平无差别,金黄色葡萄球菌、大肠埃希菌的死菌管FMI均值水平[(48.33±2.13)、(25.92±1.21)]明显高于活菌管[(1.87±0.12)、(2.14±0.07)]和无染料管[(1.14±0.09)、(1.12±0.04)]。结论:FDA能较好指示结核分枝杆菌活性,PI能较好指示金黄色葡萄球菌、大肠埃希菌的活性。

Objective： To analysis the viability of Staphylococcus aureus, Escherichia coli and Mycobacterium tuberculosis by using flow cytometry with fluorescein diacetate（ FDA） and propidium iodide（ PI）staining. Methods： Each 20 strains of Staphylococcus aureus,Escherichia coli and Mycobacterium tuberculosis during the logarithmic phase of growth were selected. Two tubes of bacterial suspension with Maxwell turbidity 1（ about 2 m L/tube） for each bacterium were prepared,with one for viable,and the other for dead bacteria（ deactivated in water bath at 85 degrees Celsius for 30 min）. The viable or dead bacteria were stained with FDA（ 0. 5 μg/m L） for 30 min and PI（ 5 μg/m L） for 15 min,respectively. Using unstained dead bacteria as control,the mean fluorescence intensity（ FMI） as measured by flow cytometry was compared between the viable and dead bacteria stained with the two dyes. Results： In terms of FDA staining,there was no difference in FMI of unstained tube,viable and dead bacteria between Staphylococcus aureus and Escherichia coli,whereas viable Mycobacterium showed significantly higher FMI（ 129. 61 ± 8. 69） than dead（ 3. 34 ± 0. 11） or unstained Mycobacterium（ 0. 33 ± 0. 02）. In terms of PI staining,there was no difference in FMI of unstained tube,viable and dead bacteria of Mycobacterium,whereas the dead bacteria of Staphylococcus aureus（ 48. 33 ± 2. 13） and E. coli（ 25. 92 ± 1. 21） showed significantly higher FMI compared with viable（ 1. 87 ± 0. 12） and（ 2. 14 ± 0. 07）and unstained（ 1. 14 ± 0. 09） and（ 1. 12 ± 0. 04） tubes of these bacteria,respectively. Conclusion： FDA can better indicate the viability of Mycobacterium tuberculosis,as PI can do for the viability of Staphylococcus aureus and Escherichia coli.