Measuring the grazing losses of picoplankton: methodological improvements in the use of fluorescently labeled tracers combined with flow cytometry
Measuring the grazing losses of picoplankton: methodological improvements in the use of fluorescently labeled tracers combined with flow cytometry
复制标题
测量超微型浮游生物的放牧损失：使用荧光标记示踪剂与流式细胞术相结合的方法学改进
DOI：
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发表时间：
1999
期刊：
影响因子：
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通讯作者：
D. Vaqué
中科院分区：
文献类型：
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作者：
E. Vázquez;Francesc Peters;J. Gasol;D. Vaqué
生物标记示踪剂（FLT）常被用来估计皮吨级生物在食草动物中的损失率.这些示踪剂通常通过落射荧光显微镜计数，尽管流式细胞术是检测FITC（荧光素+异硫氰酸酯）或DTAF（5-（（4，6-二氯三嗪-2-基）氨基）-荧光素）染色细菌1示踪剂的可行替代方法。然而，用FLT测量的噬菌性几乎没有应用于海洋沃茨的常规监测，部分原因是示踪剂的制备耗时，以及与产生可检测的示踪剂变化率所需的长期孵育相关的其他问题。此外，这些长期孵育使样品对示踪剂携带的不必要的营养物添加特别敏感。在这里，我们提出了一些实验设计，以方便估计放牧率的细菌与这种技术。测试了两种细菌1菌株和2种荧光染料：大肠杆菌微细胞（0.065 pm 3）和缺陷假单胞菌（0.064 pm 3），用DTAF或FITC染色。此外，代替在染色方案期间通常使用焦磷酸盐缓冲液，建议使用碳酸盐-碳酸氢盐缓冲液和直接从固体培养基刮下的细胞，以避免与样品的磷富集阿索的问题，所述问题有时可能发生在贫营养水样品中。FITC或DTAF染色的示踪物可以用落射荧光显微镜或流式细胞术观察。然而，FITC或DTAF染色的缺陷短波单胞菌比染色的小细胞更容易用流式细胞仪分辨。在重碳酸盐缓冲液中制备并用FITC染色的缺陷短波单胞菌示踪剂的流式细胞术检测是用于估计海洋环境中细菌的放牧损失率的快速方案。
Fluorescently labeled tracers (FLT) are often used to estimate the loss rates of picoplank- ton to grazers. These tracers are commonly enumerated by epifluorescence microscopy, although flow cytometry is a viable alternative in the detection of FITC (fluorescein+isothiocyanate)- or DTAF (5-((4,6-dichlorotriazin-2-yl)amino)-fluorescein)-stained bacteria1 tracers. However, the bacterivory measured with FLT has hardly been applied to routine monitoring of oceanic waters, partly because of the time-consuming preparation of the tracers and other problems associated with the long-term incu- bations needed to generate detectable rates of tracer change. In addition, these long-term incubations make samples especially sensitive to the unwanted addition of nutrients carried over with the tracers. Here we present some experiments designed to ease the estimation of grazing rates on bacteria with this technique. Two bacteria1 strains and 2 fluorescent dyes were tested: Escherichia coli minicells (0.065 pm3) and Pseudomonas diminuta (0.064 pm3), stained with DTAF or with FITC. In addition, instead of the common use of pyrophosphate buffer during the staining protocol, the use of carbonate- bicarbonate buffer and cells scraped directly from solid media is suggested to avoid the problems asso- ciated with phosphorus enrichment of the sample that at times can occur in oligotrophic water samples. The FITC- or DTAF-stained tracers can be observed with either epifluorescence microscopy or flow cytometry. However, FITC- or DTAF-stained P. diminuta were more easily resolved with the flow cytometer than stained minicells. Flow cytometric detection of P. diminuta tracers, prepared in bicar- bonate-buffer and stained with FITC, is a fast protocol for the estimation of the grazing loss rates of bacteria in oceanic environments.
影响因子：
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作者：
J. Paul Robinson;W. O. Carter;P. Narayanan
通讯作者：
J. Paul Robinson;W. O. Carter;P. Narayanan