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Maintenance of Acanthamoeba culbertsoni by cryopreservation
R Alejandre-Aguilar1, L Calvo-Méndez, B Nogueda-Torres
1Departamento de Parasitología, ENCB-IPN, México DF, México.
Abstract:
In order to determine the viability of trophozoites of Acanthamoeba culbertsoni under cryopreservation conditions, cultures in serum-casein-glucose-yeast extract medium were subject to 5%, 7.5%, and 10% concentrations of dimethylsulfoxide (DMSO). With the methodology followed, the percentages of recovery varied between 75.6% and 86.6% with DMSO at 10%, between 54.5% and 73.5% with DMSO at 7.5%, and between 43.6% and 68.5% with DMSO at 5%. The amebae were kept in liquid nitrogen for 30-210 days. The highest viability of trophozoites was founded when DMSO was used at a final concentration of 10% and an equilibrium temperature of 4 C. Gross cultural or morphological changes were not noted in trophozoites thawed from frozen suspensions.
Insights
Cryopreservation of Acanthamoeba culbertsoni trophozoites is viable using dimethylsulfoxide (DMSO). Optimal recovery rates were achieved with 10% DMSO and a 4°C equilibrium temperature, with no observed morphological changes post-thaw.
Area of Science:
- Microbiology
- Cell Biology
- Cryobiology
Background:
- Acanthamoeba culbertsoni is an opportunistic pathogen.
- Cryopreservation is crucial for maintaining microbial cultures.
- Understanding cryoprotective agent efficacy is vital for Acanthamoeba research.
Purpose of the Study:
- To evaluate the viability of Acanthamoeba culbertsoni trophozoites after cryopreservation.
- To determine the optimal concentration of dimethylsulfoxide (DMSO) for Acanthamoeba cryopreservation.
- To assess the impact of cryopreservation on Acanthamoeba morphology and culture characteristics.
Main Methods:
- Cultures of Acanthamoeba culbertsoni trophozoites were prepared in a serum-casein-glucose-yeast extract medium.
- Trophozoites were exposed to varying concentrations of dimethylsulfoxide (DMSO): 5%, 7.5%, and 10%.
- Cryopreservation was performed using liquid nitrogen for durations of 30 to 210 days, with an equilibrium temperature of 4°C.
Main Results:
- Recovery percentages varied significantly with DMSO concentration: 43.6%-68.5% for 5% DMSO, 54.5%-73.5% for 7.5% DMSO, and 75.6%-86.6% for 10% DMSO.
- The highest trophozoite viability was observed at a final DMSO concentration of 10% and an equilibrium temperature of 4°C.
- No gross cultural or morphological alterations were detected in Acanthamoeba culbertsoni trophozoites post-thaw.
Conclusions:
- Dimethylsulfoxide (DMSO) at a 10% concentration is effective for cryopreserving Acanthamoeba culbertsoni trophozoites.
- Cryopreservation using 10% DMSO and a 4°C equilibrium temperature ensures high viability and maintains morphological integrity.
- This study establishes a reliable method for long-term storage of Acanthamoeba culbertsoni cultures.