DUAL STAINING OF NATURAL BACTERIOPLANKTON WITH 4',6-DIAMIDINO-2-PHENYLINDOLE AND FLUORESCENT OLIGONUCLEOTIDE PROBES TARGETING KINGDOM-LEVEL 16S RIBOSOMAL-RNA SEQUENCES
DUAL STAINING OF NATURAL BACTERIOPLANKTON WITH 4',6-DIAMIDINO-2-PHENYLINDOLE AND FLUORESCENT OLIGONUCLEOTIDE PROBES TARGETING KINGDOM-LEVEL 16S RIBOSOMAL-RNA SEQUENCES
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DOI：
10.1128/aem.58.7.2158-2163.1992
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发表时间：
1992-07-01
影响因子：
4.4
通讯作者：
STAHL, DA
中科院分区：
文献类型：
--
作者：
HICKS, RE;AMANN, RI;STAHL, DA
本文提出了一种在稀释的小型浮游细菌群落中定量测定真细菌细胞密度的方法。水样中的细胞用4 '，6-二脒基-2-苯基吲哚（DAPI）染色，转移到明胶包被的载玻片上，并与罗丹明标记的对王国级16 S rRNA序列特异的寡核苷酸探针杂交。在膜滤器上捕获的48 - 69%的细胞被转移到明胶包被的载玻片上。用真细菌探针可视化的DAPI染色细胞的数量从35%到67%不等。这些细胞中只有2 - 4%在与设计用于靶向真核16 S rRNA序列的探针杂交后也发出荧光。这些样品中0.1%至6%的浮游细菌是自发荧光的，可能被误认为是与荧光寡核苷酸探针杂交的细胞。双重染色允许精确估计转移的细胞明胶薄膜的效率，并可用于测量总浮游细菌的百分比，也与荧光寡核苷酸探针杂交，表明特定的系统发育组。
A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.