Department of Biology, Faculty of Education, Hirosaki University, Hirosaki 036-8560, Japan.
Department of Biology, Faculty of Education, Hirosaki University, Hirosaki 036-8560, Japan.
J Microbiol Methods. 2021 Jul;186:106254. doi: 10.1016/j.mimet.2021.106254. Epub 2021 May 28.
Paramecium bursaria is a ciliate that harbors Chlorella-like unicellular green algae as endosymbionts. The relationship between the host P. bursaria and the endosymbiotic Chlorella is facultative; therefore, both partners can be cultured independently and re-combined to re-establish symbiosis, making this system suitable for studying algal endosymbiosis. However, despite many previous studies, cultivation of endosymbiotic Chlorella remains difficult, particularly on agar plates. Here we describe a simple agar plate method for efficiently isolating and culturing cells of the endosymbiotic alga Chlorella variabilis from an individual P. bursaria cell, by co-culturing them with yeast Saccharomyces cerevisiae. The co-culture with the yeast significantly improved the colony-forming efficiency of the alga on agar. Growth assays suggest that the main role of the co-cultured yeast cells is not to provide nutrients for the algal cells, but to protect the algal cells from some environmental stresses on the agar surface. Using the algal cells grown on the plates and a set of specially designed primers, direct colony PCR can be performed for screening of multiple endosymbiont clones isolated from a single host ciliate. These methods may provide a useful tool for studying endosymbiotic Chlorella species within P. bursaria and various other protists.
泡囊虫是一种纤毛虫，其体内共生有类似于绿藻的单细胞绿藻。宿主泡囊虫和内共生绿藻之间的关系是兼性的；因此，两者都可以独立培养并重新组合以重新建立共生关系，这使得该系统适合研究藻类内共生。然而，尽管有许多先前的研究，内共生绿藻的培养仍然很困难，特别是在琼脂平板上。在这里，我们描述了一种简单的琼脂平板方法，通过与酵母酿酒酵母共培养，从单个泡囊虫细胞中有效地分离和培养内共生绿藻 Chlorella variabilis 的细胞。与酵母的共培养显著提高了藻类在琼脂上的集落形成效率。生长实验表明，共培养酵母细胞的主要作用不是为藻类细胞提供营养，而是保护藻类细胞免受琼脂表面某些环境压力的影响。使用在平板上生长的藻类细胞和一组专门设计的引物，可以进行直接的菌落 PCR，用于筛选从单个宿主纤毛虫中分离出的多个内共生克隆。这些方法可能为研究泡囊虫内共生绿藻和其他各种原生动物中的内共生绿藻物种提供有用的工具。
