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

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 hyb...

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Bibliographic Details
Main Authors: Xiaoyan, Gao, Yunguang, Li, Huirong, Li, Wenli, Chen, Wei, Luo
Format: Article in Journal/Newspaper
Language:English
Published: Polar Research Institute of China - PRIC 2010
Subjects:
Fauna
Oceans
Arctic
Arctic Ocean
Advances in Polar Science
Polar Science
Online Access:http://library.arcticportal.org/2402/
http://library.arcticportal.org/2402/1/A2010-016.pdf
Description
Summary: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)10mg·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 6h. 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.

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