当前位置: X-MOL 学术Diversity › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom
Diversity ( IF 3.029 ) Pub Date : 2020-10-03 , DOI: 10.3390/d12100382
Yulia Zakharova , Artem Marchenkov , Nadezhda Volokitina , Aleksey Morozov , Yelena Likhoshway , Mikhail Grachev

Multiple ecological and genetic studies of diatom algae require an axenic culture. However, algae-associated bacterial biofilms often form in diatom-produced mucus, both during creation of monoclonal cultures from single cells and during biomass growth, and they may be difficult to remove. In this work, we describe a protocol for removing associated bacteria from a monoclonal culture of Ulnaria danica isolated from Lake Baikal. The axenization strategy involves selecting the latent phase of diatom growth, multiple washes to remove extracellular polymeric substances and bacterial cells, filter deposition, and treatment with antibiotics that are not toxic for diatoms. The absence of bacteria during these stages was controlled by light microscopy with Alcian blue staining for mucus, epifluorescent microscopy with DAPI (4′,6-diamino-2-phenylindole) staining for bacterial DNA, and scanning electron microscopy of the diatom cell surface. High-throughput sequencing of a 16S rRNA fragment, amplified with universal bacterial primers, from total DNA of a final culture failed to detect any bacterial contamination, confirming successful axenization. A detailed comparative description of all stages of our protocol may prove useful in developing axenic cultures of other diatoms for various ecological and genetic studies.

中文翻译:

从栽培硅藻中去除卫星细菌的策略

硅藻藻类的多种生态学和遗传学研究都需要树胶培养。然而,与藻类相关的细菌生物膜通常在硅藻生产的粘液中形成,无论是在从单细胞创建单克隆培养物还是在生物质生长过程中,都可能难以去除。在这项工作中,我们描述了从丹参单株培养中去除相关细菌的方案与贝加尔湖隔离。氧化处理的策略包括选择硅藻生长的潜伏期,多次洗涤以去除细胞外聚合物和细菌细胞,过滤沉积以及使用对硅藻无毒的抗生素进行处理。在这些阶段中细菌的不存在可以通过光学显微镜用粘液的Alcian蓝染色,用DAPI(4',6-二氨基-2-苯基吲哚)染色的落射荧光显微镜和硅藻细胞表面的扫描电子显微镜来控制。从最终培养物的总DNA中用通用细菌引物扩增的16S rRNA片段的高通量测序未能检测到任何细菌污染,从而证实了成功的轴突化。
更新日期:2020-10-04
down
wechat
bug