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题名: Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification
作者: Xia Shuang1, 2; Cheng, Yingyin3; Zhu, Huan1, 2; Liu, Guoxiang1; Hu, Zhengyu1
通讯作者: Liu, GX (reprint author), Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
关键词: Cryptomonad fixative ; glutaraldehyde ; Lugol's solution ; morphology ; PCR amplification
刊名: JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
发表日期: 2013-03-01
DOI: 10.4014/jmb.1203.03057
卷: 23, 期:3, 页:289-296
收录类别: SCI
文章类型: Article
部门归属: [Xia Shuang; Zhu, Huan; Liu, Guoxiang; Hu, Zhengyu] Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China; [Xia Shuang; Zhu, Huan] Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China; [Cheng, Yingyin] Chinese Acad Sci, Inst Hydrobiol, Ctr Water Environm & Human Hlth, Wuhan 430072, Peoples R China
WOS标题词: Science & Technology ; Life Sciences & Biomedicine
资助者: National Natural Science Foundation of China [30970501]
类目[WOS]: Biotechnology & Applied Microbiology ; Microbiology
研究领域[WOS]: Biotechnology & Applied Microbiology ; Microbiology
摘要: Cryptomonads are unicellular, biflagellate algae. Generally, cryptomonad cells cannot be preserved well because of their fragile nature, and an improved methodology should be developed to identify cryptomonads from natural habitats. In this study, we tried using several cytological fixatives, including glutaraldehyde, formaldehyde, and their combinations to preserve field samples collected from various waters, and the currently used fixative, Lugol's solution was tested for comparison. Results showed that among the fixatives tested, glutaraldehyde preserved the samples best, and the optimal concentration of glutaraldehyde was 2%. The cell morphology was well preserved by glutaraldehyde. Cells kept their original color, volume, and shape, and important taxonomic features such as furrow/gullet complex, ejectosomes, as well as flagella could be observed clearly, whereas these organelles frequently disappeared in Lugol's solution preserved samples. The osmotic adjustments and buffers tested could not preserve cell density significantly higher. Statistical calculation showed the cell density in the samples preserved by 2% glutaraldehyde remained stable after 43 days of the fixation procedure. In addition, DNA was extracted from glutaraldehyde preserved samples by grinding with liquid nitrogen and the 18S rDNA sequence was amplified by PCR. The sequence was virtually identical to the reference sequence, and phylogenetic analyses showed very close relationship between it and sequences from the same organism. To sum up, the present study demonstrated that 2% unbuffered glutaraldehyde, without osmotic adjustments, can preserve cryptomonads cells for identification, in terms of both light microscopy and phylogenetic analyses based on DNA sequences.
英文摘要: Cryptomonads are unicellular, biflagellate algae. Generally, cryptomonad cells cannot be preserved well because of their fragile nature, and an improved methodology should be developed to identify cryptomonads from natural habitats. In this study, we tried using several cytological fixatives, including glutaraldehyde, formaldehyde, and their combinations to preserve field samples collected from various waters, and the currently used fixative, Lugol's solution was tested for comparison. Results showed that among the fixatives tested, glutaraldehyde preserved the samples best, and the optimal concentration of glutaraldehyde was 2%. The cell morphology was well preserved by glutaraldehyde. Cells kept their original color, volume, and shape, and important taxonomic features such as furrow/gullet complex, ejectosomes, as well as flagella could be observed clearly, whereas these organelles frequently disappeared in Lugol's solution preserved samples. The osmotic adjustments and buffers tested could not preserve cell density significantly higher. Statistical calculation showed the cell density in the samples preserved by 2% glutaraldehyde remained stable after 43 days of the fixation procedure. In addition, DNA was extracted from glutaraldehyde preserved samples by grinding with liquid nitrogen and the 18S rDNA sequence was amplified by PCR. The sequence was virtually identical to the reference sequence, and phylogenetic analyses showed very close relationship between it and sequences from the same organism. To sum up, the present study demonstrated that 2% unbuffered glutaraldehyde, without osmotic adjustments, can preserve cryptomonads cells for identification, in terms of both light microscopy and phylogenetic analyses based on DNA sequences.
关键词[WOS]: MOLECULAR PHYLOGENY ; ELECTRON-MICROSCOPY ; RDNA PHYLOGENY ; GLUTARALDEHYDE ; DNA ; CRYPTOPHYCEAE ; FIXATION ; PRESERVATION ; NUCLEAR ; SAMPLES
语种: 英语
WOS记录号: WOS:000316919500002
ISSN号: 1017-7825
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/342005/19353
Appears in Collections:藻类生物学及应用研究中心_期刊论文

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作者单位: 1.Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China
3.Chinese Acad Sci, Inst Hydrobiol, Ctr Water Environm & Human Hlth, Wuhan 430072, Peoples R China
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