Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
J Microbiol Biotechnol. 2013 Mar;23(3):289-96. doi: 10.4014/jmb.1203.03057.
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.
隐藻是单细胞、双鞭毛藻类。通常，由于隐藻的脆弱性质，它们的细胞不能很好地保存，因此应该开发一种改进的方法来从自然栖息地中鉴定隐藻。在这项研究中，我们尝试使用几种细胞学固定剂，包括戊二醛、甲醛及其组合，来保存从各种水域采集的现场样本，并测试了目前使用的固定剂卢戈氏液进行比较。结果表明，在所测试的固定剂中，戊二醛对样本的保存效果最好，最佳戊二醛浓度为 2%。戊二醛很好地保存了细胞形态。细胞保持其原始颜色、体积和形状，并且可以清楚地观察到重要的分类特征，如沟/咽复合体、喷出体以及鞭毛，而这些细胞器在卢戈氏液保存的样本中经常消失。测试的渗透压调节剂和缓冲液不能显著提高细胞密度。统计计算表明，在固定程序 43 天后，用 2%戊二醛保存的样本中的细胞密度保持稳定。此外，通过用液氮研磨从戊二醛保存的样本中提取 DNA，并通过 PCR 扩增 18S rDNA 序列。该序列与参考序列几乎完全相同，系统发育分析表明其与同一生物的序列非常接近。总之，本研究表明，2%无缓冲戊二醛无需渗透压调节即可保存隐藻细胞，可用于光镜和基于 DNA 序列的系统发育分析鉴定。
