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.