Hicks R E, Amann R I, Stahl D A
Department of Biology, University of Minnesota, Duluth 55812.
Appl Environ Microbiol. 1992 Jul;58(7):2158-63. doi: 10.1128/aem.58.7.2158-2163.1992.
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.
本文介绍了一种在小型浮游细菌稀释群落中定量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，转移到涂有明胶的载玻片上，并用罗丹明标记的针对界级16S rRNA序列的寡核苷酸探针进行杂交。膜滤器上捕获的细胞中有48%至69%转移到了涂有明胶的载玻片上。用真细菌探针观察到的DAPI染色细胞数量在35%至67%之间。与设计用于靶向真核16S rRNA序列的探针杂交后，这些细胞中只有2%至4%也发出荧光。这些样品中0.1%至6%的浮游细菌具有自发荧光，可能被误认为是与荧光寡核苷酸探针杂交的细胞。双重染色可以精确估计细胞转移到明胶膜上的效率，并可用于测量与荧光寡核苷酸探针杂交的浮游细菌总数的百分比，从而表明特定的系统发育群体。
