The Forsyth Institute; Harvard School of Dental Medicine.
The Forsyth Institute; Harvard School of Dental Medicine;
J Vis Exp. 2021 Apr 13(170). doi: 10.3791/62484.
Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 µm filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.
许多细菌物种无法通过标准方法在实验室中培养，这对研究地球上大多数微生物多样性构成了重大障碍。需要采用新方法来培养这些未培养的细菌，以便研究人员能够使用实验室中现有的强大工具有效地研究它们的生理学和生活方式。候选门辐射（CPR）是未培养细菌中最大的群体之一，约占地球上生物多样性的 15%。该组的第一个分离物是 Saccharibacteria 门的成员，'Nanosynbacter lyticus' 菌株 TM7x。TM7x 是一种异常小的细菌，作为共生体与细菌宿主 Schaalia odontolytica 菌株 XH001 直接接触而生活。利用异常小的细胞尺寸及其作为共生体的生活方式，我们开发了一种从牙菌斑中快速培养 Saccharibacteria 的方案。该方案将展示如何通过 0.2 µm 过滤器过滤牙菌斑悬浮液，然后浓缩收集的 Saccharibacteria 细胞并感染宿主生物的培养物。由此产生的共培养物可以像任何正常细菌培养物一样传代，并且可以通过 PCR 确认感染。由此产生的二元培养物可以在实验室中保存，并用于未来的实验。虽然存在污染的可能性，但可以通过进一步过滤和再感染宿主，或者通过平板接种二元培养物并筛选感染的菌落来纯化二元培养物。我们希望该方案可以扩展到其他样本类型和环境，从而培养更多 CPR 中的物种。
