Establishing, Maintaining, and Cryopreserving In Vitro Cultures of the Asexual Stages of Babesia divergens.

Human babesiosis is caused by four Babesia spp., which are intraerythrocytic Apicomplexan zoonotic parasites that, in the last 50 years, have contributed to establishing the parasitic infection as a significant transfusion-transmitted infectious threat and an escalating public-health problem. Of the four species that infect humans, B. divergens was the first to be grown in vitro, via continuous culture in human RBCs. In this chapter, we have comprehensively outlined the basic and necessary tools, protocols, and methodology to successfully grow and study the parasite. Details of in vitro culturing using human serum and Albumax, cryopreservation and thawing, subculturing, maintenance of cultures over extended times, calculation of parasite load using the traditional light microscopy method, and flow cytometry have been elaborated upon. This chapter can serve as standard operating procedures (SOP) for laboratories working on Babesia research and those that want to establish in vitro culturing of B. divergens field isolates, as well as related parasites.