IDENTIFICATION OF INDIVIDUAL PROKARYOTIC CELLS BY USING ENZYME-LABELED, RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES
IDENTIFICATION OF INDIVIDUAL PROKARYOTIC CELLS BY USING ENZYME-LABELED, RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES
复制标题
DOI：
10.1128/aem.58.9.3007-3011.1992
复制
发表时间：
1992-09-01
影响因子：
4.4
通讯作者：
SCHLEIFER, KH
中科院分区：
文献类型：
--
作者：
AMANN, RI;ZARDA, B;SCHLEIFER, KH
介绍了一种用显微镜检测和识别细菌和古细菌领域成员单个细胞的方法。rRNA 靶向寡核苷酸的 5'端用辣根过氧化物酶标记，并用于全细胞杂交。通过酶促底物二氨基联苯胺在细胞内形成沉淀，可观察到特异性结合的探针。将酶标记的探针渗透到革兰氏阴性细菌的整个固定细胞中需要用溶菌酶-乙二胺四乙酸进行预处理，而在杂交缓冲液中加入去垢剂可以提高一些古细菌细胞的渗透性。迄今为止，我们尚未实现酶标记探针对革兰氏阳性菌和酵母细胞的渗透。这种方法对于在背景荧光较强的环境中或在没有外荧光显微镜的情况下识别合适的原核细胞是非常有价值的。
A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.