为了探索荧光原位杂交(FISH)技术,研究窖泥微生物群落结构特征多样性,进行了纯培养微生物及窖泥微生物的FISH检测。以Escherichia coli、Bacillus subtilis、Lactobacillus planetarium、Acetobacter rancens、Clostridiumacetobytylicum 5株不同种属的细菌为对象,研究了影响荧光原位杂交(FISH)技术对其定量表征的因素,并将该技术应用于窖泥微生物群落的研究。实验结果表明,E.coli及L.planetarium的定量表征受到生长期的影响,在对数和稳定期的菌体检出率>82%,衰退期的L.planetarium则明显减少;死菌体比活菌体的检出信号显著减弱;经硫酸铝处理,除去腐植酸的窖泥样品明显地提高了可辨力,加入窖泥中的E.coli,FISH检出量为光学显微镜计数量的46.89%。采用FISH技术可视化定量表征了窖泥中细菌和古菌的特征,有益于从细胞水平研究其微生物群落的关系。

In order to investigate the diversity of microbial community in pit mud by fluorescence in situ hybridization,detection of pure culture and samples were carried out.The influenced factors on FISH were studied based on five pure culture strains including Escherichia coli,Bacillus subtilis,Lactobacillus planetarium,Acetobacter rancens,Clostridium acetobytylicum.Then the microbial community in pit mud was further investigated by the modified method.The results showed that the quantitative characterization was influenced by growth phase.The detection rate was above 82% in logarithmic and stationary phases for E.coli and L.planetarium whereas was significantly less in death phase for L.planetarium.The detected fluorescentce signal of dead cells was rather weak than live cells.For E.coli added in pit mud samples,the resolution of FISH was improved significantly through removing humic acid by aluminum sulfate,the recovery was 46.89% of the direct microscopic somatic cell count.FISH could effectively characterize the bacteria and archaea community.Detection of bacteria and archaea visualized quantitatively by FISH is beneficial to study microbial community in cellular level.