Günther S, Hübschmann T, Rudolf M, Eschenhagen M, Röske I, Harms H, Müller S
UFZ, Centre for Environmental Research Leipzig, Department of Environmental Microbiology, Permoserstrasse 15, 04318 Leipzig, Germany.
J Microbiol Methods. 2008 Sep;75(1):127-34. doi: 10.1016/j.mimet.2008.05.017. Epub 2008 May 24.
Analysis of environmental bacteria on the single cell level often requires fixation to store the cells and to keep them in a state as near life-like as possible. Fixation procedures should furthermore counteract the increase of autofluorescence, cell clogging, and distortion of surface characteristics. Additionally, they should meet the specific fixation demands of both aerobically and anaerobically grown bacteria. A fixation method was developed based on metal solutions in combination with sodium azide. The fixation efficiencies of aluminium, barium, bismuth, cobalt, molybdenum, nickel, and tungsten salts were evaluated by flow cytometric measurement of the DNA contents as a bacterial population/community stability marker. Statistical equivalence testing was involved to permit highly reliable flow cytometric pattern evaluation. Investigations were carried out with pure cultures representing environmentally important metabolic and respiratory pathways as controls and with activated sludge as an example for highly diverse bacterial communities. A mixture of 5 mM barium chloride and nickel chloride, each and 10% sodium azide was found to be a suitable fixative for all tested bacteria. The described method provided good sample stability for at least 9 days.
在单细胞水平上分析环境细菌通常需要进行固定，以保存细胞并使其尽可能保持接近生命状态。此外，固定程序应抑制自发荧光的增加、细胞堵塞以及表面特征的扭曲。此外，它们还应满足需氧和厌氧生长细菌的特定固定要求。基于金属溶液与叠氮化钠相结合开发了一种固定方法。通过流式细胞术测量DNA含量作为细菌群体/群落稳定性标记，评估了铝、钡、铋、钴、钼、镍和钨盐的固定效率。采用统计等效性检验以实现高度可靠的流式细胞术模式评估。以代表环境中重要代谢和呼吸途径的纯培养物作为对照，并以活性污泥作为高度多样化细菌群落的示例进行了研究。发现5 mM氯化钡和氯化镍的混合物以及10%的叠氮化钠对所有测试细菌都是合适的固定剂。所描述的方法可提供至少9天的良好样品稳定性。
