In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts
Trascrizione
To isolate sporozoites from oocysts of Cryptosporidium parvum in vitro, take bleach-treated oocyst suspension.
Add excystation media containing sodium taurocholate and trypsin.
Sodium taurocholate weakens the glycocalyx layer and oocyst walls, while trypsin degrades oocyst wall proteins, resulting in oocyst excystation and sporozoite release.
Microscopically assess the excystation efficiency.
Centrifuge to pellet the sporozoites, intact oocysts, and oocyst shells.
Resuspend the pellet and pass it through a filtering assembly placed on ice to keep the sporozoites dormant.
Larger oocyst shells and unruptured oocysts are retained in the membrane filter, while small-sized sporozoites pass through the filter via gravity and are collected in the filtrate.
Centrifuge the filtrate.
Resuspend the sporozoite pellet in media containing a dye and reducing buffer constituents.
The dye aids in visualizing the sporozoite injection procedure, while the reducing buffer creates a gastrointestinal tract-like environment, enhancing sporozoite viability and infectivity.
The prepared sporozoites are ready for injection procedures.
