Autofluorescent characteristics of Candidatus Brocadia fulgida and the consequences for FISH and microscopic detection |
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| Institution: | 1. State Key Joint Laboratory of Environmental Simulation and Pollution Control, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. Anping Hongjia Environmental Protection Technology Co. LTD, China;4. Laboratory of Water Pollution Control Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China;5. Institute of Energy, Jiangxi Academy of Sciences, Nanchang 330096, China;1. Key Laboratory of Environment Remediation and Ecological Health, Ministry of Education, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, China;2. Wenzhou Institute of Eco-environmental Sciences, Wenzhou, China;3. Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou, China;1. Department of Environmental Engineering, College of Environmental & Resource Sciences, Zhejiang University, China;2. Department of Environmental Science, College of Resource and Environmental Science, Chongqing University, China;3. Advanced Environmental Biotechnology Centre, Nanyang Environment & Water Research Institute, Nanyang Technological University, Singapore;4. School of Environmental and Natural Resources, Zhejiang University of Science & Technology, China;5. School of Energy & Environmental Science, Yunnan Normal University, China |
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| Abstract: | An enrichment culture of Candidatus Brocadia fulgida was identified by three independent methods: analysis of autofluorescence using different microscope filter blocks and a fluorescence spectrometer, fluorescence in situ hybridization (FISH) with anammox-specific probes and partial sequencing of the 16S rDNA, hydrazine synthase hzsA and hydrazine oxidoreductase hzo. The filter block BV-2A (400–440, 470 LP, Nikon) was suitable for preliminary detection of Ca. B. fulgida. An excitation-emission matrix revealed three pairs of excitation-emission maxima: 288–330 nm, 288–478 nm and 417–478 nm. Several autofluorescent cell clusters could not be stained with DAPI or by FISH, suggesting empty but intact cells (ghost cells) or inhibited permeability. Successful staining of autofluorescent cells with the FISH probes Ban162 and Bfu613, even at higher formamide concentrations, suggested insufficient specificity of Ban162. Under certain conditions, Ca. B. fulgida lost its autofluorescence, which reduced the reliability of autofluorescence for identification and detection. Non-fluorescent Ca. Brocadia cells could not be stained with Ban162, but with Bfu613 at higher formamide concentrations, suggesting a dependency between both parameters. The phylogenetic analysis showed only good taxonomical clustering of the 16S rDNA and hzsA. In conclusion, careful consideration of autofluorescent characteristics is recommended when analysing and presenting FISH observations of Ca. B. fulgida to avoid misinterpretations and misidentifications. |
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| Keywords: | 16S rDNA FISH |
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