Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech Republic.
Department of Chemistry, Faculty of Science, Humanities and Education, Technical University of Liberec, Liberec, Czech Republic.
J Microbiol Methods. 2024 Sep;224:107010. doi: 10.1016/j.mimet.2024.107010. Epub 2024 Aug 2.
The quantification of cellular metabolic activity via MTT assay has become a widespread practice in eukaryotic cell studies and is progressively extending to bacterial cell investigations. This study pioneers the application of MTT assay to evaluate the metabolic activity of biofilm-forming cells within bacterial biofilms on nanofibrous materials. The biofilm formation of Staphylococcus aureus and Escherichia coli on nanomaterials electrospun from polycaprolactone (PCL), polylactic acid (PLA), and polyamide (PA) was examined. Various parameters of the MTT assay were systematically investigated, including (i) the dissolution time of the formed formazan, (ii) the addition of glucose, and (iii) the optimal wavelength for spectrophotometric determination. Based on interim findings, a refined protocol suitable for application to nanofibrous materials was devised. We recommend 2 h of the dissolution, the application of glucose, and spectrophotometric measurement at 595 nm to obtain reliable data. Comparative analysis with the reference CFU counting protocol revealed similar trends for both tested bacteria and all tested nanomaterials. The proposed MTT protocol emerges as a suitable method for assessing the metabolic activity of bacterial biofilms on PCL, PLA, and PA nanofibrous materials.
通过 MTT 测定法来量化细胞代谢活性已经成为真核细胞研究中的一种普遍做法，并逐渐扩展到细菌细胞研究中。本研究开创了将 MTT 测定法应用于评估纳米纤维材料上细菌生物膜中形成生物膜的细胞代谢活性的先河。研究了金黄色葡萄球菌和大肠杆菌在聚己内酯（PCL）、聚乳酸（PLA）和聚酰胺（PA）纳米材料上的生物膜形成情况。系统研究了 MTT 测定法的各种参数，包括（i）形成的甲臜的溶解时间，（ii）葡萄糖的添加，以及（iii）分光光度法测定的最佳波长。基于中期研究结果，设计了一种适用于纳米纤维材料的改良方案。我们建议将溶解时间设定为 2 小时，添加葡萄糖，并在 595nm 处进行分光光度测量，以获得可靠的数据。与参考 CFU 计数方案的比较分析表明，两种测试细菌和所有测试纳米材料都呈现出相似的趋势。因此，所提出的 MTT 方案可作为评估 PCL、PLA 和 PA 纳米纤维材料上细菌生物膜代谢活性的一种合适方法。
