Fluorometric assessment of Gram-negative bacterial permeabilization
Fluorometric assessment of Gram-negative bacterial permeabilization
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DOI：
10.1046/j.1365-2672.2000.00971.x
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发表时间：
2000-02-01
影响因子：
4
通讯作者：
Mattila-Sandholm, T
中科院分区：
文献类型：
--
作者：
Helander, IM;Mattila-Sandholm, T
吸收的荧光探针1-N-苯基萘胺（NPN），适用于自动荧光分光光度计，使微孔板的多孔阅读，应用于确定革兰氏阴性菌的渗透性变化。完整的外膜是一个渗透屏障，并排除疏水物质，如NPN，但一旦损坏，它可以允许NPN进入磷脂层，导致突出的荧光。以大肠杆菌O 157、铜绿假单胞菌和鼠伤寒沙门氏菌作为试验菌，以乙二胺四乙酸和六偏磷酸钠作为模型透化剂，获得了定量和高度可重复的NPN摄取水平，这些水平在试验菌之间具有特征性差异。此外，柠檬酸在毫摩尔浓度下显示为有效的透化剂，其作用部分地（Ps. aeruginosa，Salm. typhimurium）或几乎完全（E. coliO 157）被MgCl 2消除，表明部分作用是通过螯合作用发生的。柠檬酸钠诱导弱的NPN摄取，这是完全废除MgCl 2。总之，NPN摄取测定与自动荧光分光光度计作为一种方便的方法，在分析和定量的外部代理商，包括潜在的食品防腐剂，对革兰氏阴性菌的影响。
Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram-negative bacteria.