Institute of Integrative Biology (IBZ), ETH Zürich, Zürich, Switzerland.
PLoS One. 2012;7(11):e49046. doi: 10.1371/journal.pone.0049046. Epub 2012 Nov 14.
We here present an efficient, precise and reliable method to isolate and cultivate healthy and viable single Crithidia bombi cells from bumblebee faeces using flow cytometry. We report a precision of >99% in obtaining single trypanosomatid cells for further culture and analysis ("cloning"). In the study, we have investigated the use of different liquid media to cultivate C. bombi and present an optimal medium for obtaining viable clones from all tested, infected host donors. We show that this method can be applied to genotype a collection of clones from natural infections. Furthermore, we show how to cryo-preserve C. bombi cells to be revived to become infective clones after at least 4 years of storage. Considering the high prevalence of infections in natural populations, our method provides a powerful tool in studying the level and diversity of these infections, and thus enriches the current methodology for the studies of complex host-parasite interactions.
我们在这里展示了一种高效、精确和可靠的方法，可使用流式细胞术从熊蜂粪便中分离和培养健康且有活力的单个克里蒂亚氏原生动物细胞。我们报告了一种获得用于进一步培养和分析的单个原生动物细胞（“克隆”）的精度>99%。在研究中，我们调查了使用不同液体培养基来培养 C. bombi 的情况，并提出了一种从所有测试的感染宿主供体中获得有活力克隆的最佳培养基。我们表明，该方法可用于对来自自然感染的克隆集进行基因分型。此外，我们展示了如何冷冻保存 C. bombi 细胞，以便在至少 4 年的储存后恢复为感染性克隆。考虑到自然种群中感染的高流行率，我们的方法为研究这些感染的程度和多样性提供了强大的工具，从而丰富了当前用于研究复杂宿主-寄生虫相互作用的方法。
