IHB OpenIR  > 藻类生物学及应用研究中心  > 期刊论文
Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification
Xia Shuang1,2; Cheng, Yingyin3; Zhu, Huan1,2; Liu, Guoxiang1; Hu, Zhengyu1; Liu, GX (reprint author), Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
2013-03-01
Source PublicationJOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
ISSN1017-7825
Volume23Issue:3Pages:289-296
AbstractCryptomonads 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.; 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.
SubtypeArticle
KeywordCryptomonad Fixative Glutaraldehyde Lugol's Solution Morphology Pcr Amplification
Department[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
DOI10.4014/jmb.1203.03057
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
Funding OrganizationNational Natural Science Foundation of China [30970501] ; National Natural Science Foundation of China [30970501] ; National Natural Science Foundation of China [30970501] ; National Natural Science Foundation of China [30970501]
Indexed BySCI
Language英语
WOS Research AreaBiotechnology & Applied Microbiology ; Microbiology
WOS SubjectBiotechnology & Applied Microbiology ; Microbiology
WOS IDWOS:000316919500002
WOS KeywordMOLECULAR PHYLOGENY ; ELECTRON-MICROSCOPY ; RDNA PHYLOGENY ; GLUTARALDEHYDE ; DNA ; CRYPTOPHYCEAE ; FIXATION ; PRESERVATION ; NUCLEAR ; SAMPLES
Funding OrganizationNational Natural Science Foundation of China [30970501] ; National Natural Science Foundation of China [30970501] ; National Natural Science Foundation of China [30970501] ; National Natural Science Foundation of China [30970501]
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Cited Times:1[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/19353
Collection藻类生物学及应用研究中心_期刊论文
Corresponding AuthorLiu, GX (reprint author), Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
Affiliation1.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
Recommended Citation
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,&Liu, GX .(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.
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