Optimization of fluorescence in situ hybridization (FISH) for the identification of two polar coccoid green algae species  

作　　者：高小艳 李运广 李会荣 陈雯莉 罗玮 

机构地区：[1]College of Life Science and Technology,State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University [2]Key Laboratory for Polar Science of the State Oceanic Administration,Polar Research Institute of China

出　　处：《Chinese Journal of Polar Science》2010年第2期167-179,共13页极地研究（英文版）

基　　金：supported by the National Natural Science Foundation of China(Grants No.40806073,40876097);the Polar Science Strategic Research Foundation of China and the Youth Foundation for Marine Science of SOA (Grants No.2008128)

摘　　要：Standard FISH protocols using fluorochrome-labeled oligonucleotide probes have been successfully applied for in situ detection.However,optimized protocols of FISH for specific eukaryotes in marine environments are often not developed.This study optimized the conditions of fluorescence in situ hybridization （FISH） by using two polar isolated microalgae.The modified conditions were as follows：（1） 10 mg·mL^（-1） lysozyme solution pretreatment at 37℃for 30 min;（2） the hybridization buffer including 20%formamide;（3） the hybridization condition was 47℃for 6 h.The cells enumerated by FISH were compared with those enumerated by flow cytometry（FCM） and DAPI to confirm the cell loss and hybridization efficiency.The optimized protocol was also successfully applied to Arctic Ocean samples,which were found to be dominated by Micromonas sp.The modified protocol showed a high relative efficiency and could be successfully applied for the detection of specific microbial eukaryotes in environmental samples.Standard FISH protocols using fluorochrome-labeled oligonucleotide probes have been successfully applied for in situ detection.However,optimized protocols of FISH for specific eukaryotes in marine environments are often not developed.This study optimized the conditions of fluorescence in situ hybridization （FISH） by using two polar isolated microalgae.The modified conditions were as follows：（1） 10 mg·mL^（-1） lysozyme solution pretreatment at 37℃for 30 min;（2） the hybridization buffer including 20%formamide;（3） the hybridization condition was 47℃for 6 h.The cells enumerated by FISH were compared with those enumerated by flow cytometry（FCM） and DAPI to confirm the cell loss and hybridization efficiency.The optimized protocol was also successfully applied to Arctic Ocean samples,which were found to be dominated by Micromonas sp.The modified protocol showed a high relative efficiency and could be successfully applied for the detection of specific microbial eukaryotes in environmental samples.

关 键 词：Fluorescence in situ hybridization（FISH） Chlorella vulgaris strain Lw2008/02 Micromonas sp.strain CCMP2099 

分 类 号：Q948.8[生物学—植物学]