An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy
An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy
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DOI：
10.1111/j.1462-2920.2005.00767.x
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发表时间：
2005-07-01
影响因子：
5.1
通讯作者：
Simon, M
中科院分区：
文献类型：
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作者：
Lunau, M;Lemke, A;Simon, M
我们提出了一种通过 10% (v/v) 甲醇和超声处理有效分离颗粒相关细菌的新程序，该程序特别适合具有高颗粒负载和沉积物的样品。我们还通过一步应用高度 dsDNA 特异性荧光染料 SybrGreen I 和光学明亮的封固剂（聚乙烯醇 4-88，“moviol”）来优化样品制备。与其他方法相比，新方案可以更快、更轻松且毒性更低地处理样品。细胞直接在黑色 Nuclepore 滤光片上染色，并显示出低背景的强荧光信号。针对 10% 甲醇溶液的温度 (35° C)、超声处理和离心对分离程序进行了优化。与焦磷酸盐和 Tween-80 的分离程序相比，新方法的应用对包括沉积物在内的各种类型的海洋样品总是产生更高数量和/或更高分数的颗粒相关细胞。使用 moviol-SybrGreen I 溶液对样品进行染色和封固，可以对具有高浓度 SPM 的样品中的细菌进行准确且高度可重复的计数。与福尔马林固定相比，戊二醛固定细菌产生更亮的荧光。由于 SybrGreen I 对 dsDNA 具有高特异性和明亮的荧光，操作快速简便，并且可以在 -20°C 下保存染色样品至少几个月而不损失任何荧光强度，因此新开发的方法也是水生细菌 DAPI 染色的一种有吸引力的替代方法。
We present a new procedure for effectively detaching particle-associated bacteria by 10% (v/v) methanol and sonication which is particularly suitable for samples with a high particle load and sediments. We also optimized the sample preparation by applying the highly dsDNA-specific fluorescent stain SybrGreen I together with an optically brilliant mounting medium (polyvinylalcohol 4-88, 'moviol') in one step. The new protocol allows a much faster, easy and less toxic handling of samples as compared to other methods. Cells are stained directly on a black Nuclepore filter and show an intensive fluorescence signal with low background. The detachment procedure was optimized with respect to the temperature of the 10% methanol solution (35° C), ultrasonication and centrifugation. The application of the new method in comparison with detachment procedures with pyrophosphate and Tween-80 with various types of marine samples including sediments always yielded higher numbers and/or higher fractions of particle-associated cells. Staining and mounting the samples with the moviol-SybrGreen I solution allowed an accurate and highly reproduceable enumeration of bacteria also in samples with high concentrations of SPM. Fixation of bacteria by glutardialdehyde resulted in a brighter fluorescence as compared to fixation by formalin. Because of the high specificity to dsDNA and bright fluorescence of SybrGreen I, the fast and easy handling and the possibility to store stained samples for at least several months at -20° C without any loss in fluorescence intensity, the newly developed method is also an attractive alternative to DAPI staining of aquatic bacteria.