不同实验方法检测常用有机溶剂对细菌活性的影响及其安全使用限量

【目的】采用不同实验方法测定常用有机溶剂二甲基亚砜(DMSO)、丙酮和乙醇对细菌活性的影响,以指导抗菌类药物体外抑菌实验所用溶剂的选择和添加限量。【方法】采用常规体外抑菌实验方法(纸片扩散法、肉汤稀释法),并参照生长曲线法检测有机溶剂DMSO、丙酮和乙醇对大肠杆菌(Escherichia coli)及金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用,采用扫描电子显微镜(SEM)观察溶剂作用后的细菌形态变化。【结果】3种溶剂对E.coli和S.aureus抑菌率达到20%时,在肉汤稀释法下,DMSO、丙酮、乙醇的浓度(体积比)分别为1.00%、0.25%、2.00%和1.00%、1.00%、0.50%;在生长曲线法下,溶剂浓度(体积比)分别为0.50%、1.00%、0.50%和1.00%、0.50%、0.50%;而在纸片扩散法下,32%(体积比)DMSO和32%(体积比)乙醇对E.coli产生明显抑菌圈,但3种溶剂对S.aureus均无抑菌圈出现。3种方法比较后得出:当3种溶剂的抑菌率达到20%时,溶剂浓度(体积比)均低于0.5%,对细菌整体生长活性影响较小。SEM结果表明控制溶剂使用限量可有效减少其对E.coli生长过程的影响。【结论】相对于DMSO和丙酮,乙醇对微生物生长繁殖能力的影响更加明显;采用相同浓度有机溶剂时,液态条件下(肉汤稀释法和生长曲线法)微生物受到有机溶剂的影响较大。

Effects of common solvent concentrations on bacterial activities

Objective] To investigate the effects of common organic solvents of dimethyl sulfoxide (DMSO), acetone and ethanol on the activities of bacteria for guiding the selection of solvent and addition of its dosage in the antibacterial test in vitro. Methods] Consulting the results of growth curve method, conventional antibacterial tests (disk diffusion method and broth dilution method) were used to determine inhibitory effects of DMSO, acetone and ethanol on the bacterial strains of Escherichia coli and Staphylococcus aureus. The changes of cell morphology after treated by these solvents were studied by scanning electron microscope (SEM). Results] When the solvent inhibitory effects on E. coli and S. aureus were 20%, the concentration (V/V) of solvent DMSO, acetone, ethanol were 1.00%, 0.25%, 2.00% and 1.00%, 1.00%, 0.50% in the broth dilution method, respectively. For the growth curve method, the concentration (V/V) of DMSO, acetone, ethanol were 0.50%, 1.00%, 0.50% and 1.00%, 0.50%, 0.50% when 20% inhibitory effects on E. coli and S. aureus were reached, respectively. 32% (V/V) DMSO and 32% (V/V) ethanol showed obvious inhibitory rings for E. coli, but no inhibitory rings appeared for S. aureus in the same concentrations of studied solvents with disk diffusion method. To compare three methods, the inhibitory effects of DMSO, acetone and ethanol were 20% while their concentrations (V/V) all were lower than 0.5%; under this condition, effects on the viability of bacteria were slight. SEM showed that limited solvent concentrations could decrease their effects on the growth of E. coli. Conclusion] To compare with DMSO and acetone, ethanol showed a stronger effect on growth and reproduction of microorganisms. After treated with the same concentration of medicinal solvent, liquid testing condition, such as broth dilution method and growth curve method, induced the stronger effect on the cells.