School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.
Bioimaging Facility, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.
FEMS Microbiol Lett. 2022 May 5;369(1). doi: 10.1093/femsle/fnac029.
Ammonia-oxidising archaea (AOA) are environmentally important microorganisms involved in the biogeochemical cycling of nitrogen. Routine cultivation of AOA is exclusively performed in liquid cultures and reports on their growth on solid medium are scarce. The ability to grow AOA on solid medium would be beneficial for not only the purification of enrichment cultures but also for developing genetic tools. The aim of this study was to develop a reliable method for growing individual colonies from AOA cultures on solid medium. Three phylogenetically distinct AOA strains were tested: 'Candidatus Nitrosocosmicus franklandus C13', Nitrososphaera viennensis EN76 and 'Candidatus Nitrosotalea sinensis Nd2'. Of the gelling agents tested, agar and Bacto-agar severely inhibited growth of all three strains. In contrast, both 'Ca. N. franklandus C13' and N. viennensis EN76 tolerated Phytagel™ while the acidophilic 'Ca. N. sinensis Nd2' was completely inhibited. Based on these observations, we developed a Liquid-Solid (LS) method that involves immobilising cells in Phytagel™ and overlaying with liquid medium. This approach resulted in the development of visible distinct colonies from 'Ca. N. franklandus C13' and N. viennensis EN76 cultures and lays the groundwork for the genetic manipulation of this group of microorganisms.
氨氧化古菌（AOA）是参与氮的生物地球化学循环的重要环境微生物。常规的 AOA 培养是在液体培养基中进行的，而关于它们在固体培养基上生长的报告则很少。能够在固体培养基上生长 AOA 不仅有益于富集培养物的纯化，还有助于开发遗传工具。本研究的目的是开发一种从固体培养基上的 AOA 培养物中分离单个菌落的可靠方法。本研究测试了 3 株具有不同系统发育特征的 AOA 菌株：“Candidatus Nitrosocosmicus franklandus C13”、Nitrososphaera viennensis EN76 和 “Candidatus Nitrosotalea sinensis Nd2”。在所测试的胶凝剂中，琼脂和 Bacto-agar 严重抑制了所有 3 株菌的生长。相比之下，“Ca. N. franklandus C13”和 N. viennensis EN76 均可耐受 Phytagel™，而嗜酸的 “Ca. N. sinensis Nd2”则完全被抑制。基于这些观察结果，我们开发了一种液体-固体（LS）方法，该方法涉及将细胞固定在 Phytagel™中并用液体培养基覆盖。这种方法使“Ca. N. franklandus C13”和 N. viennensis EN76 培养物中形成了可见的明显菌落，为该组微生物的遗传操作奠定了基础。
