Application of tyramide signal amplification-fluorescence in situ hybridisation and flow cytometry to detection of Heterosigma akashiwo (Raphidophyceae) in natural waters

作者：Lee J; Katano T; Chang M; Han M S^*

来源：New Zealand Journal of Marine and Freshwater Research, 2012, 46(1): 137-148.

DOI：10.1080/00288330.2011.613404

• cell cycle analysis
• flow cytometer
• Heterosigma akashiwo
• accurate identification
• tyramide signal amplification-fluorescence in situ hybridisation

We developed protocols to analyse the cell cycle of Heterosigma akashiwo in the natural phytoplankton assemblage using tyramide signal amplification-fluorescence in situ hybridisation (TSA-FISH) and flow cytometry. We determined the optimum probe and formamide concentrations for the species-specific TSA-FISH probe to be 0.5 ng mu L-1 and 40%, respectively. The probe did not hybridise to non-target phytoplankton including Chattonella sp., which is closely related to Heterosigma and has high sequence similarity to it. We could successfully identify G1- and G2-phase cells and the diel cell cycle of H. akashiwo not only in the laboratory but also in the field after TSA-FISH. These results demonstrate that TSA-FISH for H. akashiwo in natural coastal waters could serve as a powerful tool for understanding their bloom mechanism using flow cytometry.

• 出版日期2012-3