Bacterial viability and antibiotic susceptibility testing with SYTOX Green nucleic acid stain
Bacterial viability and antibiotic susceptibility testing with SYTOX Green nucleic acid stain
复制标题
利用 SYTOX Green 核酸染色法进行细菌活力和抗生素敏感性检测
DOI：
10.1128/aem.63.6.2421-2431.1997
复制
发表时间：
1997-06-01
影响因子：
4.4
通讯作者：
Millard, PJ
中科院分区：
文献类型：
--
作者：
Roth, BL;Poot, M;Millard, PJ
使用不穿透活细胞的荧光核酸染色剂来评估细菌质膜的完整性。SYTOX绿色核酸染色剂是一种不对称花青染料，带有三个正电荷，完全不含活的真核和原核细胞。SYTOX绿色染色剂与核酸的结合导致荧光发射增强>500倍（吸收和发射最大值分别在502和523 nm处），使质膜受损的细菌发出明亮的绿色荧光。SYTOX绿色染料很容易被氩离子激光的488 nm谱线激发。来自用SYTOX绿色染色剂标记的膜受损细菌的荧光信号通常比来自完整生物体的荧光信号亮>10倍。用SYTOX绿色染色剂标记的细菌悬浮液发出的绿色荧光与群体中透化细胞的分数成比例，通过显微镜、荧光测定法或流式细胞术对其进行定量。流式细胞术和荧光方法被用来量化β-内酰胺类抗生素对大肠杆菌细胞膜完整性的影响。通过流式细胞术对SYTOX绿色染色标记的活细胞和透化细胞的检测和区分明显优于基于碘化丙啶的检测。这些研究表明，用SYTOX绿色染色剂标记细菌是测量细菌活力和抗生素敏感性的常规方法的有效替代方法。
A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a >500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically >10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.