Optimized routine flow cytometric enumeration of heterotrophic flagellates using SYBR Green I
Optimized routine flow cytometric enumeration of heterotrophic flagellates using SYBR Green I
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DOI：
10.4319/lom.2011.9.329
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发表时间：
2011-08-01
影响因子：
2.7
通讯作者：
Zubkov, Mikhail V.
中科院分区：
文献类型：
--
作者：
Christaki, Urania;Courties, Claude;Zubkov, Mikhail V.
异养鞭毛虫（HF）是水生生态系统中细菌的主要消耗者，在数量和生物量上占主导地位。Zubkov等人（2007）描述了一种基于dna染色的流式细胞术（FC）方法来枚举HF，但尚未被广泛采用。我们对该方法及其局限性进行了广泛的测试，使用了广泛的样本类型，并尝试了几种固定和保存方法。我们评估了该方法的一些步骤的简化，寻求精度和细胞图中HF与细菌和浮游植物的区分质量之间的最佳折衷。我们发现，在不使用注射泵增强机器改造的情况下，流速为120-220 μ L min(-1)，运行时间为8-10 min，即使在低于10(2)cells mL（-1）的值下也可以枚举HF。SYBR Green I的最终浓度为1:10 000，在室温下黑暗中染色时间为10分钟，足以染色和检测HF。从新鲜分析的样品中获得的细胞数量与以前在液体氮中冷冻的细胞数量没有显著差异。FC和epifluorescence microscopy （EpiM）的结果一致，并且FC在重复样品之间产生的变异性比EpiM低。我们遇到的一个限制是，在存在大型细菌和/或细菌聚集体的情况下，计数是困难的。然而，在没有细菌聚集物的样本中，Bact/HF比为bb0 1000， HF可以很好地枚举。
Heterotrophic flagellates (HF) are the major consumers of bacteria in aquatic ecosystems and dominate heterotrophic nanoplankton in numbers and in biomass. A DNA-staining based flow cytometry (FC) protocol to enumerate HF was described by Zubkov et al. (2007), but has not yet been widely adopted. We tested extensively the method and its limitations using a wide range of sample types and trying several fixation and conservation alternatives. We evaluated simplification of some steps of the method, seeking the best compromise between precision and the quality of distinction of HF from bacteria and phytoplankton in the cytograms. We found that a flow rate of 120-220 mu L min(-1) without using a syringe-pump enhanced machine modification, and running times of 8-10 min allowed enumeration of HF even at values below 10(2) cells mL(-1). SYBR Green I, at final concentrations of 1: 10000 and a minimum staining time of 10 min at room temperature in the dark, was adequate for staining and detecting HF. No significant differences were found between cell numbers obtained from freshly analyzed samples and those previously frozen in liquid-N. FC and epifluorescence microscopy (EpiM) were in good agreement and FC yielded lower variability between replicate samples than EpiM. One limitation we encountered was that, in the presence of large bacteria and/or bacterial aggregates, enumeration was difficult. However, in absence of bacterial aggregates samples with Bact/HF ratios >1000, HF could be well-enumerated.