how to make a glycerol stock

Daniel Parker

3 min read

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07-02-2025

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Meta Description: Learn how to create a glycerol stock, a crucial step in preserving valuable bacterial or cell cultures. This detailed guide provides a step-by-step process, crucial tips, and troubleshooting advice for successful long-term storage. Preserve your research with confidence!

Glycerol stocks are essential for long-term storage of bacterial and other cell cultures. They offer a convenient and reliable method to preserve valuable strains, safeguarding your research and preventing the need for repeated cultivations. This comprehensive guide will walk you through the process of making a glycerol stock, ensuring the viability of your cultures for years to come.

What is a Glycerol Stock?

A glycerol stock is a frozen suspension of bacterial cells or other microorganisms in a solution containing glycerol (typically 15-25%). Glycerol acts as a cryoprotectant, preventing the formation of ice crystals during freezing that can damage cell membranes and lead to cell death. This allows for long-term storage of your cultures at -80°C, maintaining their viability for extended periods. Properly prepared glycerol stocks are a cornerstone of microbiology labs and cell culture facilities worldwide.

Materials Needed

Before you begin, ensure you have all necessary materials gathered:

• Bacterial Culture: A healthy, actively growing culture in its log phase (exponential growth) is vital for successful stock preparation.
• Sterile Glycerol: A high-purity glycerol solution (typically 50% v/v sterile glycerol in water) is essential. Avoid using glycerol that has been previously frozen and thawed.
• Sterile Cryovials: These are small, screw-cap tubes specifically designed for cryogenic storage. They are crucial for maintaining sterility and preventing contamination.
• Microcentrifuge Tubes (optional): Useful for smaller aliquots.
• Pipettes and Tips: Sterile pipettes and tips are crucial for maintaining sterility throughout the process.
• Vortex Mixer: To mix the culture and glycerol solution thoroughly.
• -80°C Freezer: Essential for long-term storage.

Step-by-Step Procedure: How to Make a Glycerol Stock

1. Assess Your Culture: Ensure your bacterial culture is in the log phase of growth. This means it's actively dividing and will result in higher viability after freezing. You can check this by measuring the optical density (OD) of your culture using a spectrophotometer.

2. Prepare the Glycerol Solution: If you haven't already, prepare a sterile 50% (v/v) glycerol solution in distilled water. Autoclaving is the preferred sterilization method.

3. Mix the Culture and Glycerol: Using sterile techniques, add an equal volume of your bacterial culture to the 50% glycerol solution. For example, add 1 mL of culture to 1 mL of 50% glycerol solution. This results in a final glycerol concentration of approximately 25%, ideal for most microorganisms. Thoroughly mix the solution using a vortex mixer.

4. Aliquot the Mixture: Transfer the mixed solution into sterile cryovials using sterile pipettes. Fill cryovials to approximately 80-90% of their capacity, leaving room for expansion during freezing. Smaller aliquots (e.g., in microcentrifuge tubes) can be made for smaller experiments, reducing the risk of repeated freeze-thaw cycles.

5. Label the Cryovials: Clearly label each cryovial with the following information: * Bacterial strain name or identifier * Date of preparation * Your initials or lab identifier

6. Freeze the Glycerol Stocks: Slowly freeze the cryovials by placing them in a -80°C freezer. Rapid freezing can damage cells. Some labs utilize isopropyl alcohol-based freezing containers to control the rate of freezing.

7. Long-Term Storage: Store the glycerol stocks at -80°C for long-term preservation. Avoid repeated freeze-thaw cycles as these decrease cell viability.

Thawing and Reviving Your Glycerol Stock

To revive your bacterial culture from a glycerol stock:

1. Remove a cryovial from the -80°C freezer.
2. Thaw the cryovial quickly in a 37°C water bath, ensuring the vial is submerged until fully thawed. Avoid shaking vigorously.
3. Aseptically transfer the contents to a suitable growth medium.
4. Incubate under appropriate conditions for your specific microorganism.

Troubleshooting

• Low Viability After Thawing: This may be due to improper freezing, old cultures used for stock preparation, or poor handling of the cryovial. Ensure your culture was in log phase and that freezing was gradual.
• Contamination: Strict sterile technique is critical. Any contamination during preparation will compromise the entire stock.
• Cryovial Leakage: Use high-quality cryovials and ensure they are properly sealed.

By following these steps and maintaining stringent sterile techniques, you can successfully create and store glycerol stocks, ensuring the long-term viability of your valuable cultures. Remember that proper labeling and storage are crucial for accurate identification and maintaining the integrity of your microbial collection.