Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists
Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists
复制标题
催化报告的沉积荧光原位杂交方案评估混合营养原生生物的吞噬作用
DOI：
10.1128/aem.71.11.7321-7326.2005
复制
发表时间：
2005
影响因子：
4.4
通讯作者：
E. Casamayor
中科院分区：
文献类型：
--
作者：
J. M. Medina;M. Felip;E. Casamayor
摘要我们描述了一种报道的催化沉积-荧光原位杂交(CARD-FISH)方法，该方法特别适合于评估混合营养型原生生物对原核生物的吞噬作用，因为它保持了细胞和质体的完整性，避免了细胞丢失和猎物的排出，并允许显示标记的猎物以对抗叶绿体的自发荧光。该方法包括Lugol‘s-甲醛-硫代硫酸盐固定、琼脂糖细胞附着、溶菌酶加去色素酶使细胞壁通透、Alexa-Fluor 488信号放大等步骤，使我们能够检测到几乎100%的浮游原核生物(细菌和古生菌)，并首次显示混合营养型原生生物摄取的古细胞。
ABSTRACT We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists.