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

【摘要】 <正>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.更多还原

【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℃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.更多还原