Method for excystation of sporocysts
View Patent ↗A process to excystate protozoal sporocysts involves treatment of an infected tissue sample with a sodium hypochlorite solution to stimulate excystation of the sporocysts from the tissue. Thereafter, removal of the sodium hypochlorite solution and treatment of the sample with a cell culture media as the excystation fluid eliminates incubation and subsequent washing steps using expensive reagents. The excystation fluid contains substantially no chelating agents, proteins, enzymes or bile acids.
1. A method of counting protozoal sporozoites in a mammalian tissue sample comprising protozoal sporocysts, comprising:
a) exposing said mammalian tissue sample to a sodium hypochlorite solution having a sodium hypochlorite concentration in a range of about 15-25% v/v for a period of up to about 45 minutes;
b) removing said sodium hypochlorite from said mammalian tissue sample by rinsing said mammalian tissue sample with a saline solution;
c) exposing said rinsed mammalian tissue sample from step (b) to an excystation fluid to affect excystation of said protozoal sporocysts to release sporozoites, wherein said excystation takes place with substantially no incubation or washing; and
d) counting said sporozoites.
2. The method according to claim 1 , wherein said sample is exposed to said sodium hypochlorite solution at a temperature within the range of about 0 to 4° C.
3. The method according to claim 1 , wherein said sample is exposed to said sodium hypochlorite solution for a period not exceeding about 30 minutes.
4. The method according to claim 3 , wherein said sample is exposed to said sodium hypochlorite solution for about 15 to 30 minutes.
5. The method according to claim 1 , wherein said excystation fluid cdl culture media consists essentially of RPMI 1640 culture medium.
6. The method according to claim 1 , further comprising the formation of a pellet of said mammalian tissue sample after said exposure to said sodium hypochlorite.
7. The method according to claim 6 , wherein said pellet is formed via centrifugation.
8. The method according to claim 1 , wherein said sporozoites formed from said excystated sporocysts from said mammalian tissue sample are counted using tissue culture flasks.
9. The method according to claim 8 , wherein said mammalian tissue sample is transferred directly to said flasks prior to said counting, but after said exposure to excystation fluid to affect excystation.
10. The method according to claim 1 , wherein said tissue sample is pre-digested prior to said exposure to said sodium hypochlorite.
11. The method according to claim 10 , wherein said pre-digestion is undertaken using a blender.
12. The method according to claim 1 , wherein said exposure to said excystation fluid is conducted using cell culture media which contain substantially no chelating agents, digestive proteins, enzymes, or bile acids.