Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1111 Ljubljana, Slovenia.
J Microbiol Methods. 2010 May;81(2):121-6. doi: 10.1016/j.mimet.2010.02.004. Epub 2010 Feb 18.
The aim of this study was to evaluate diffusion and dilution methods for determining the antibacterial activity of plant extracts and their mixtures. Several methods for measurement of the minimal inhibitory concentration (MIC) of a plant extract are available, but there is no standard procedure as there is for antibiotics. We tested different plant extracts, their mixtures and phenolic acids on selected gram-positive (Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes) and gram-negative bacteria (Escherichia coli O157:H7, Salmonella Infantis, Campylobacter jejuni, Campylobacter coli) with the disk diffusion, agar dilution, broth microdilution and macrodilution methods. The disk diffusion method was appropriate only as a preliminary screening test prior to quantitative MIC determination with dilution methods. A comparison of the results for MIC obtained by agar dilution and broth microdilution was possible only for gram-positive bacteria, and indicated the latter as the most accurate way of assessing the antimicrobial effect. The microdilution method with TTC (2,3,5-triphenyl tetrazolium chloride) or INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) to indicate the viability of aerobic bacteria was found to be the best alternative approach, while only ATP determination was appropriate for microaerophilic Campylobacter spp. Using survival curves the kinetics of bacterial inactivation on plant extract exposure was followed for 24h and in this way the MIC values determined by the microdilution method were confirmed as the concentrations of extracts that inhibited bacterial growth. We suggest evaluation of the antibacterial activity of plant extracts using the broth microdilution method as a fast screening method for MIC determination and the macrodilution method at selected MIC values to confirm bacterial inactivation. Campylobacter spp. showed a similar sensitivity to plant extracts as the tested gram-positive bacteria, but S. Infantis and E. coli O157:H7 were more resistant.
本研究旨在评估扩散和稀释方法，以确定植物提取物及其混合物的抗菌活性。有几种方法可用于测量植物提取物的最小抑菌浓度（MIC），但没有像抗生素那样的标准程序。我们用扩散环法、琼脂稀释法、肉汤微量稀释法和大剂量稀释法测试了不同的植物提取物、它们的混合物和酚酸对选定的革兰氏阳性（金黄色葡萄球菌、蜡状芽孢杆菌和单核细胞增生李斯特菌）和革兰氏阴性菌（大肠杆菌 O157:H7、婴儿沙门氏菌、空肠弯曲菌和大肠弯曲菌）的作用。扩散环法仅适用于定量 MIC 测定之前的初步筛选试验。只有在革兰氏阳性菌中，才能对琼脂稀释法和肉汤微量稀释法测定的 MIC 结果进行比较，这表明后者是评估抗菌效果的最准确方法。用 TTC（2,3,5-三苯基氯化四氮唑）或 INT（2-对碘苯基-3-对硝基苯基-5-苯基氯化四氮唑）进行微量稀释法来指示需氧菌的活力是一种较好的替代方法，而仅 ATP 测定适用于微需氧弯曲菌属。通过生存曲线，我们跟踪了植物提取物暴露 24 小时后细菌失活的动力学，通过这种方式，通过微量稀释法确定的 MIC 值被确认为抑制细菌生长的提取物浓度。我们建议使用肉汤微量稀释法作为快速筛选方法，以确定 MIC 值，并在选定的 MIC 值下使用大剂量稀释法，以确认细菌失活，从而评估植物提取物的抗菌活性。与测试的革兰氏阳性菌相比，弯曲菌属对植物提取物表现出相似的敏感性，但婴儿沙门氏菌和大肠杆菌 O157:H7 的耐药性更强。
