Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria
Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria
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DOI：
10.3354/ame014113
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发表时间：
1998-02-13
影响因子：
1.4
通讯作者：
Fuhrman, JA
中科院分区：
文献类型：
--
作者：
Noble, RT;Fuhrman, JA
一种新的核酸染色剂SYBR绿色I可用于快速准确地测定各种海洋样品中的病毒和细菌丰度。我们用福尔马林保存的沿海水样本和深度剖面样本测试了这种染色（至800米）从19和190公里的离岸站点，通过在0.02 μ m孔径滤膜上过滤数ml，染色15分钟。细菌计数与吖啶橙子（AO）计数比较，病毒计数与透射电子显微镜（TEM）计数比较显示出很强的相关性。AO和SYBR绿色I的细菌计数难以区分，几乎完全相关（r（2）= 0.99）。病毒计数范围很广，从0.03到15 x 10（7）病毒ml（-1）。SYBR绿色I法的病毒计数平均高于TEM法，SYBR绿色I与TEM图的回归斜率为1.28。两者之间的相关性非常高，r（2）值为0.98。SYBR绿色I法的精密度与透射电镜法相同，变异系数为2.9%。SYBR绿色I染色的病毒和细菌染色强烈，易于与旧一代和新一代落射荧光显微镜下的其他颗粒区分开来。碎屑通常不染色，不像使用替代染料YoPro I时，因此这种方法可能适用于沉积物。SYBR绿色I染色的样品不需要干燥或加热，可在过滤前用福尔马林固定，最佳染色时间为15 min（总制备时间小于25 min），取样后可立即在海上轻松进行计数。这种方法可能有助于将病毒研究纳入大多数水生微生物学实验室。
A new nucleic acid stain, SYBR Green I, can be used for the rapid and accurate determination of viral and bacterial abundances in diverse marine samples. We tested this stain with formalin-preserved samples of coastal water and also from depth profiles (to 800 m) from sites 19 and 190 km offshore, by filtering a few mi onto 0.02 mu m pore-size filters and staining for 15 min. Comparison of bacterial counts to those made with acridine orange (AO) and virus counts with those made by transmission electron microscopy (TEM) showed very strong correlations. Bacterial counts with AO and SYBR Green I were indistinguishable and almost perfectly correlated (r(2) = 0.99). Virus counts ranged widely, from 0.03 to 15 x 10(7) virus ml(-1). Virus counts by SYBR Green I were on the average higher than those made by TEM, and a SYBR Green I versus TEM plot yielded a regression slope of 1.28. The correlation between the two was very high with an r(2) value of 0.98. The precision of the SYBR Green I method was the same as that for TEM, with coefficients of variation of 2.9%. SYBR Green I stained viruses and bacteria are intensely stained and easy to distinguish from other particles with both older and newer generation epifluorescence microscopes. Detritus is generally not stained, unlike when the alternative dye YoPro I is used, so this approach may be suitable for sediments. SYBR Green I stained samples need no desalting or heating, can be fixed with formalin prior to filtration, the optimal staining time is 15 min (resulting in a total preparation time of less than 25 min), and counts can be easily performed at sea immediately after sampling. This method may facilitate incorporation of viral research into most aquatic microbiology laboratories.