To store E. coli cells in glycerol for long-term preservation, follow these steps:
Procedure for Glycerol Stock Preparation
This method creates a glycerol stock that protects the cells during freezing and thawing. Glycerol acts as a cryoprotectant, preventing ice crystal formation that can damage cell membranes.
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Prepare the Glycerol Solution: Add 0.15 mL of sterile 100% glycerol to a sterile 2 mL screw-cap vial. Sterilization can be achieved by autoclaving the glycerol solution beforehand. This results in a final glycerol concentration of approximately 15% after mixing with the bacterial culture.
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Prepare the Bacterial Culture: Obtain 0.85 mL of a logarithmic-phase E. coli culture. This ensures the cells are actively growing and more viable for storage.
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Mix the Glycerol and Culture: Add the 0.85 mL of E. coli culture to the 0.15 mL glycerol solution in the vial.
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Thorough Mixing: Vortex the vial vigorously to ensure even mixing of the bacterial culture and the glycerol. This is crucial for consistent protection during freezing.
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Freezing: Immediately freeze the vial in either ethanol-dry ice bath or liquid nitrogen.
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Storage: Store the frozen glycerol stock at -70°C for long-term preservation.
Why Use Glycerol?
Glycerol is used because it's a cryoprotectant. When water freezes, it forms ice crystals. These ice crystals can puncture and damage cell membranes, leading to cell death. Glycerol prevents the formation of large ice crystals, thereby protecting the cells during freezing and thawing. The optimal concentration generally ranges from 10-20%.
Considerations
- Sterility: Ensure all materials are sterile to prevent contamination of the E. coli stock.
- Phase of Growth: Using a log-phase culture ensures that the cells are healthy and actively dividing, which increases their chances of survival during the freezing process.
- Rapid Freezing: Rapid freezing in ethanol-dry ice or liquid nitrogen minimizes ice crystal formation.
- Proper Labeling: Label the vial clearly with the E. coli strain, date, and any relevant information.
By following these steps, you can effectively store E. coli cells in glycerol, ensuring their viability for future use.
