Lunau Mirko, Lemke Andreas, Walther Katja, Martens-Habbena Willm, Simon Meinhard
Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg, D-26111 Oldenburg, Germany.
Environ Microbiol. 2005 Jul;7(7):961-8. doi: 10.1111/j.1462-2920.2005.00767.x.
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 degrees 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 degrees C without any loss in fluorescence intensity, the newly developed method is also an attractive alternative to DAPI staining of aquatic bacteria.
我们提出了一种新方法，通过10%（v/v）甲醇和超声处理有效地分离与颗粒相关的细菌，该方法特别适用于高颗粒负载和沉积物的样本。我们还通过一步应用高度特异性针对双链DNA的荧光染料SybrGreen I和光学增亮的封片剂（聚乙烯醇4-88，“moviol”）优化了样本制备。与其他方法相比，新方案允许对样本进行更快、更简便且毒性更低的处理。细胞直接在黑色核孔滤膜上染色，并显示出低背景的强烈荧光信号。针对10%甲醇溶液的温度（35摄氏度）、超声处理和离心对分离程序进行了优化。与使用焦磷酸盐和吐温80对包括沉积物在内的各种类型海洋样本进行分离程序相比，新方法的应用始终能产生更高数量和/或更高比例的与颗粒相关的细胞。用moviol-SybrGreen I溶液对样本进行染色和封片，即使在高浓度悬浮颗粒物的样本中也能对细菌进行准确且高度可重复的计数。与用福尔马林固定相比，用戊二醛固定细菌会产生更亮的荧光。由于SybrGreen I对双链DNA具有高特异性和明亮荧光，处理快速简便，且能够将染色后的样本在-20摄氏度下保存至少几个月而荧光强度无任何损失，因此新开发的方法也是对水生细菌进行DAPI染色的一种有吸引力的替代方法。
