Department of Earth, Life and Environmental Sciences, University of Urbino Carlo Bo, Urbino, Italy.
J Microbiol Methods. 2011 Dec;87(3):309-15. doi: 10.1016/j.mimet.2011.09.003. Epub 2011 Sep 22.
Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.
流式细胞术和荧光原位杂交是鉴定和定量细菌细胞的常用方法。将细胞计数的快速性和多参数的准确性与寡核苷酸 FISH 探针的系统发育特异性相结合，被认为是水微生物学中一种强大的新兴工具。在本工作中，使用溶液杂交方案对大肠杆菌纯培养物和海洋细菌进行了测试，结果表明，第一种方法的杂交信号效率很高，而第二种方法的效率较低。其他实验是在建立的基于滤膜的 CARD-FISH 方案的基础上，在封闭系统中进行的，目的是提高细胞的脱离和检测效率。然后通过优化的洗脱程序，将杂交后的细胞从膜滤器上重新悬浮下来。杂交海洋细菌的细胞计数达到了总事件的 85.7%±18.1%。细胞图谱的质量表明，所描述的程序可能适用于对自然微生物群落中系统发育群的细胞计数定量分析。
