极地研究

极地研究 ›› 2010, Vol. 21 ›› Issue (2-English): 167-179.DOI: 10.3724/SP.J.1085.2010.00167

• 研究论文 • 上一篇    下一篇

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

Gao Xiaoyan(高小艳)1,2, Li Yunguang(李运广)1*, Li Huirong(李会荣)2, Chen Wenli(陈雯莉)1 and LuoWei(罗玮)2*
  

  1. 1 College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China;
    2 Key Laboratory for Polar Science of the State Oceanic Administration, Polar Research Institute of China, Shanghai 200136, China
     
  • 收稿日期:2010-06-28 修回日期:2010-10-11 出版日期:1960-06-30 发布日期:1960-06-30
  • 通讯作者: Li Yunguang and Luo Wei

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

Gao Xiaoyan(高小艳)1,2, Li Yunguang(李运广)1*, Li Huirong(李会荣)2, Chen Wenli(陈雯莉)1 and LuoWei(罗玮)2*
  

  1. 1 College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China;
    2 Key Laboratory for Polar Science of the State Oceanic Administration, Polar Research Institute of China, Shanghai 200136, China
  • Received:2010-06-28 Revised:2010-10-11 Online:1960-06-30 Published:1960-06-30
  • Contact: Li Yunguang and Luo Wei

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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°C for 30 min; (2) the hybridization buffer including 20% formamide; (3) the hybridization condition was 47°C 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

Abstract:

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°C for 30 min; (2) the hybridization buffer including 20% formamide; (3) the hybridization condition was 47°C 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.

Key words: Fluorescence in situ hybridization (FISH), Chlorella vulgaris strain Lw2008/02, Micromonas sp. strain CCMP2099