Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification
文献类型：期刊论文
| 作者 | Xia Shuang1,2; Cheng, Yingyin3; Zhu, Huan1,2; Liu, Guoxiang1; Hu, Zhengyu1 |
| 刊名 | JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
 |
| 出版日期 | 2013-03-01 |
| 卷号 | 23期号:3页码:289-296 |
| 关键词 | Cryptomonad fixative glutaraldehyde Lugol's solution morphology PCR amplification |
| ISSN号 | 1017-7825 |
| 通讯作者 | Liu, GX (reprint author), Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China. |
| 中文摘要 | 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. |
| 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. |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 类目[WOS] | Biotechnology & Applied Microbiology ; Microbiology |
| 研究领域[WOS] | Biotechnology & Applied Microbiology ; Microbiology |
| 关键词[WOS] | MOLECULAR PHYLOGENY ; ELECTRON-MICROSCOPY ; RDNA PHYLOGENY ; GLUTARALDEHYDE ; DNA ; CRYPTOPHYCEAE ; FIXATION ; PRESERVATION ; NUCLEAR ; SAMPLES |
| 收录类别 | SCI |
| 资助信息 | National Natural Science Foundation of China [30970501] |
| 语种 | 英语 |
| WOS记录号 | WOS:000316919500002 |
| 公开日期 | 2013-10-31 |
| 源URL | [http://ir.ihb.ac.cn/handle/342005/19353]  |
| 专题 | 水生生物研究所_藻类生物学及应用研究中心_期刊论文 |
| 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 |
| 推荐引用方式 GB/T 7714 | Xia Shuang,Cheng, Yingyin,Zhu, Huan,et al. Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification[J]. JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY,2013,23(3):289-296. |
| APA | Xia Shuang,Cheng, Yingyin,Zhu, Huan,Liu, Guoxiang,&Hu, Zhengyu.(2013).Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification.JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY,23(3),289-296. |
| MLA | Xia Shuang,et al."Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification".JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY 23.3(2013):289-296. |
入库方式： OAI收割
来源：水生生物研究所
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