Related Experiment Videos
Simple method for cryopreservation of an anaerobic rumen fungus using ethylene glycol and rumen fluid
M Sakurada1, Y Tsuzuki, D P Morgavi
1Animal Science Division, Faculty of Agriculture, Miyazaki University, Japan.
Abstract:
The cryopreservation of an anaerobic rumen fungus, Piromyces communis OTS1, was examined at -84 degrees C using dimethyl sulfoxide, propylene glycol or ethylene glycol as cryoprotectants. Ethylene glycol was the most effective agent, combining high survival and low toxicity, followed by dimethyl sulfoxide and propylene glycol. Cell-free rumen fluid in the cryopreservation medium decreased the toxicity of the cryoprotectant agents and also had a protective action per se. A survival of 80% after 1 year storage was obtained when samples with an initial zoospore density of 5 x 10(4) zoospores/ml were equilibrated for 15 min in medium containing 0.64 M ethylene glycol and 5% cell-free rumen fluid, then frozen with dry ice and stored at -84 degrees C.
Insights
Cryopreservation of the anaerobic rumen fungus Piromyces communis OTS1 at -84°C was optimized using ethylene glycol and cell-free rumen fluid. This method achieved 80% survival after one year, crucial for maintaining fungal cultures.
Area of Science:
- Microbiology
- Cryobiology
- Mycology
Background:
- Anaerobic rumen fungi are essential for plant biomass degradation in ruminant digestion.
- Effective cryopreservation methods are needed to maintain viable cultures of these microorganisms for research and application.
- Piromyces communis OTS1 is a significant anaerobic rumen fungus.
Purpose of the Study:
- To evaluate the efficacy of different cryoprotective agents (CPAs) for cryopreserving Piromyces communis OTS1 at -84°C.
- To determine the optimal conditions for cryopreservation, including CPA type, concentration, and the role of cell-free rumen fluid.
- To assess the long-term viability of Piromyces communis OTS1 after cryopreservation.
Main Methods:
- Cryopreservation of Piromyces communis OTS1 zoospores at -84°C.
- Testing dimethyl sulfoxide, propylene glycol, and ethylene glycol as cryoprotectants.
- Investigating the effect of cell-free rumen fluid on CPA toxicity and fungal survival.
- Optimizing CPA concentration and equilibration time.
- Assessing zoospore viability after 1 year of storage.
Main Results:
- Ethylene glycol demonstrated the highest effectiveness as a cryoprotectant, showing high survival rates and low toxicity.
- Dimethyl sulfoxide and propylene glycol were less effective compared to ethylene glycol.
- The addition of cell-free rumen fluid significantly reduced CPA toxicity and provided a protective effect.
- Optimal conditions involved 0.64 M ethylene glycol and 5% cell-free rumen fluid, yielding 80% survival after 1 year of storage at -84°C.
Conclusions:
- Ethylene glycol is a superior cryoprotectant for Piromyces communis OTS1 compared to other tested agents.
- Cell-free rumen fluid plays a critical role in enhancing cryopreservation success by mitigating CPA toxicity and offering protection.
- The developed cryopreservation protocol ensures high viability of Piromyces communis OTS1 for at least one year at -84°C.