In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts

To purify C. parvum sporozoites, transfer the oocysts to a 15-milliliter tube, and resuspend the cells at a 1 x 10⁷ oocysts per milliliter excystation medium concentration. After 60 to 90 minutes at 37 degrees Celsius, mix 14 milliliters of an appropriate wash solution with the oocysts, and sediment the cells by centrifugation. Aspirate the supernatant carefully to avoid losing oocysts, and resuspend the sporozoite pellet at a 3 times 10 to the seventh oocysts per 1 to 2 milliliters of DMEM concentration.

To remove any remaining oocysts and shells, place a 10-milliliter syringe barrel equipped with a 47-milliliter filter holder apparatus fitted with a 3-micrometer pore-size polycarbonate filter into a 15-milliliter tube at 4 degrees Celsius. Add 7.5 milliliters of the sporozoite suspension to the filter assembly. When the entire volume has passed through the strainer by gravity, wash any remaining cells through the strainer with another 7.5 milliliters of DMEM.

When all of the wash has passed through the strainer, collect the filtered sporozoite suspension by centrifugation for 10 minutes, and label the pellet in 50 to 100 microliters of an appropriate organoid culture medium supplemented with 0.05% fast green dye and L-glutathione, betaine, L-cysteine, linoleic acid, and taurine-containing reducing buffer.