Cryptosporidium Oocyst Purification Using Discontinuous Gradient Centrifugation

Abstract

Many laboratory studies in cryptosporidial research require a source of purified oocysts. Sources can include experimentally infected laboratory animals or from samples collected from naturally infected animals and from clinical cases of human cryptosporidiosis. Purification of oocysts can be accomplished with readily available laboratory equipment including tabletop centrifuges and microcentrifuges. Following purification, oocysts can be stored in antibiotic-supplemented buffers or in 2.5% aqueous potassium dichromate for over 6 months. Ultimately, oocyst viability and infectivity decline to less than 10% after 1 year, so if isolates are expected to be maintained, serial passage in a suitable host at ≤6-month intervals is recommended. Oocysts purified as described in this chapter are suitable for animal infection studies, cell culture studies, and a wide range of molecular biological studies, environmental studies, drug testing, and disinfection studies.

Keywords: Centrifugation; Cesium chloride; Cryptosporidium; Gradient; Isolation; Oocyst; Purification; Sheather’s solution; Sucrose.