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 excystating protozoal sporocysts from a tissue sample infected with a protozoa comprising
a) exposing said infected tissue sample to sodium hypochlorite;
b) removing said sodium hypochlorite from said sample, and
treating the sample with a cell culture medium to promote excystation of said protozoal sporocysts to affect excystation, wherein said cell culture medium contains no chelating agent, protein digesting enzymes and bile acids.
2. A method according to claim 1 , wherein the protozoa is of the genus Sarcocystis.
3. A method according to claim 2 , wherein the protozoa Sarcocystis is of species neurona.
4. A method according to claim 1 , wherein the sodium hypochlorite solution is a bleach solution of concentrations greater than 1% v/v sodium hypochlorite and water.
5. A method according to claim 4 , wherein the sodium hypochlorite solution concentration is about 2.6% to 50% v/v sodium hypochlorite and water.
6. A method according to claim 5 , wherein the hypochlorite solution is about 20% v/v sodium hypochlorite and water.
7. A method according to claim 6 , wherein the sample is exposed to the sodium hypochlorite solution occurs at a temperature greater than about −15° C.
8. A method according to claim 7 , wherein the temperature is about 0° C. to 4° C.
9. A method according to claim 8 , wherein the sample is exposed to the sodium hypochlorite solution for a period of more than about 1 minute.
10. A method according to claim 9 , wherein said exposure period is about 10 to 45 minutes.
11. A method according to claim 10 , wherein said exposure period is about 30 minutes.
12. A method according to claim 1 , wherein the step (b) removal of sodium hypochlorite solution is completed by washing the sample with an appropriate isotonic solution.
13. A method according to claim 12 , wherein the isotonic solution is a normal saline solution.
14. A method according to claim 13 , wherein the saline solution is prepared from about 8 grams of sodium chloride per liter of water.
15. A method according to claim 14 , wherein the saline solution has a molarity of approximately 7.3 moles/L.
16. A method according to claim 15 , wherein the temperature of the saline solution is greater than about −15° C.
17. A method according to claim 16 , wherein the saline solution is about 0° C. to 10° C.
18. A method according to claim 17 , wherein the saline solution is about 4° C.
19. A method of claim 1 , wherein said cell culture media is RPMI 1640 medium.