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Factors promoting in vitro excystation of Giardia muris cysts
Abstract:
Giardia muris cysts, isolated from mouse faeces, excysted routinely at levels greater than 90%, when induced in 1X Hanks' supplemented with 17 mM glutathione, 29 mM L-cysteine-HCl, and 50 mM NaHCO3 for 30 minutes at 35 degrees C, followed by washing and suspension in trypsin-Tyrode's solution at pH 8.0. Although trypsin was not required in this final step, it enhanced the escape of the trophozoites from their cysts. G. muris excystation was dependent upon the length of the induction period, pH, oxidation-reduction potential and temperature. Optimal induction conditions for excystation were: an induction period of 5 to 30 min; pH of 2; 120 mV oxidation-reduction potential; and a temperature around 35 degrees C. A gradual decline in excystation occurred as pH and oxidation-reduction potential were changed to 7 and 57 mV, respectively. There was a pronounced increase in excystation percentages with increasing temperatures between 0 and 37 degrees C. At 40 degrees C and above, the G. muris cysts showed signs of inactivation. The thermal death point of G. muris cysts was determined to be about 54 degrees C. G. muris cysts showed no polarity; however, the tail or posterior trophozoite portion always emerged through one end of the cyst first. Cytokinensis began within the first hour after excystation. This method always produced extremely active, normal-looking G. muris trophozoites.
Insights
Researchers optimized Giardia muris cyst excystation for laboratory studies. Key factors influencing excystation include induction period, pH, temperature, and redox potential, yielding highly active trophozoites.
Area of Science:
- Parasitology
- Microbiology
- Cell Biology
Background:
- Giardia muris is a protozoan parasite studied in laboratory models.
- Efficient excystation of G. muris is crucial for in vitro research.
Purpose of the Study:
- To establish a reliable method for excysting Giardia muris cysts.
- To identify optimal conditions for maximizing G. muris excystation rates.
Main Methods:
- Giardia muris cysts were induced using Hanks' solution with glutathione, L-cysteine-HCl, and NaHCO3.
- Excystation was optimized by varying induction time, pH, temperature, and oxidation-reduction potential.
- Trophozoite emergence and viability were assessed post-induction.
Main Results:
- Over 90% excystation was achieved under specific conditions.
- Optimal induction involved a 5-30 min period, pH 2, 120 mV redox potential, and 35°C.
- Temperatures above 40°C reduced excystation, with a thermal death point around 54°C.
Conclusions:
- A reproducible method for G. muris excystation was developed.
- Optimized conditions ensure the production of active, morphologically normal trophozoites.
- Understanding excystation parameters is vital for Giardia research.