培养基灭菌方式对细菌分离效率的影响

Effect of the medium sterilization on bacterial isolation efficiency

自然界蕴含大量未/难培养微生物,分离这些微生物对理论研究和资源开发具有重要意义。本研究使用高压灭菌和过滤除菌方式制备培养基,采用稀释涂布方法,从红树林灰泥样品中分离获得123株细菌,通过16S rRNA基因序列分析对其进行鉴定,进而探究培养基灭菌方式对细菌分离效果的影响。结果表明:过滤除菌培养基生长的单菌落数目(339±82)个显著多于高压灭菌培养基生长的单菌落数目(179±65)个;两种培养基分离细菌的群落结构在门、科和属分类水平上总体相似,但优势类群的数目和少数类群存在差异;过滤除菌培养基分离细菌的Shannon Wiener’s指数、均匀度、新种率、基因多样性均高于高压灭菌培养基,而其与近缘模式菌株相似度的平均值和中位数则低于高压灭菌培养基。因此,过滤除菌培养基分离获得细菌的多样性、均匀性和新颖性均高于高压灭菌培养基。本研究首次探究培养基灭菌方式对细菌分离效果的影响,具有更高分离效率的过滤除菌培养基为未/难培养微生物菌株资源获取提供了借鉴。

Nature contains a large number of uncultured microorganisms,isolating these microorganisms is of great significance for theoretical research and resource development.In this study,123 bacterial strains were isolated from the grey mud of the mangrove by spreading serial dilutions on media prepared by the autoclave sterilization(AS) and the filter sterilization(FS),and were identified by 16S rRNA gene sequence analysis.The results showed that the number of colonies on the FS media(339±82) was significantly higher than that on the AS media(179±65);the community composition of bacteria from the two media was similar at the phylum,family and genus levels,but their numbers and a few taxa were different.The Shannon Wiener’s index,evenness,proportion of new species and phylotype diversity of bacteria isolated from the FS media were higher than those of bacteria isolated from the FS media;while the average value and median of the similarity of bacteria from the FS media with the closest type strains were lower than those of bacteria from the AS media.Therefore,the diversity,evenness and novelty of bacteria obtained by the FS media were much higher than those of bacteria from the AS media.This study is the first time to explore the effect of the medium sterilization on bacterial isolation efficiency,and the filter sterilization with the higher isolation efficiency provides a clue for the acquisition of uncultured microbial strain.