how to make glycerol stock

3 min read 18-01-2025

Glycerol stocks are a crucial tool for long-term storage of microbial cultures like bacteria and yeast. This method ensures the viability of your valuable strains, preventing loss due to repeated subculturing. Creating a glycerol stock is a straightforward process, but accuracy is key to preserving your cultures effectively. This guide will walk you through making glycerol stocks, ensuring your strains remain viable for future experiments.

Materials You'll Need:

Before you begin, gather the necessary materials. Having everything prepared beforehand streamlines the process and minimizes the risk of contamination.

Sterile Glycerol: Use high-quality, sterile glycerol (typically 50% v/v in sterile water or broth). Contamination can ruin your stock, rendering it useless.
Bacterial Culture: Your actively growing bacterial or yeast culture. Ensure it’s in the log phase of growth for optimal viability. A fresh culture is vital for successful stock creation.
Sterile Cryovials: These small tubes are designed for cryogenic storage and prevent contamination. Properly labeling your cryovials is essential for easy identification.
Microcentrifuge Tubes: (Optional, but helpful for smaller volumes)
Pipettes and Pipette Tips: Sterile pipettes and tips are essential for maintaining sterility. Accurate measurement is crucial for the correct glycerol concentration.
Micropipettes: (For precise volume measurements)
Vortex Mixer: (Helpful for mixing the glycerol and culture)
Ice Bucket: Keeping the mixture cold during the process enhances viability.
Permanent Marker: For labeling your cryovials with the strain name, date, and any other relevant information.

Step-by-Step Guide to Making Glycerol Stock:

Following these steps carefully will result in a viable glycerol stock.

1. Prepare Your Culture:

Begin with a healthy, actively growing culture. The ideal growth phase is the log phase (exponential growth). This ensures a high cell density and better survival rates. Take an optical density (OD) reading if possible to confirm.

2. Mix the Glycerol and Culture:

A common ratio is 1:1, mixing equal volumes of your bacterial culture and sterile 50% glycerol solution. For instance, you might mix 1 mL of culture with 1 mL of glycerol. Smaller volumes (e.g., 0.5 mL of each) are also suitable depending on your needs. Use sterile pipettes to avoid contamination. Thoroughly mix using a vortex mixer to ensure even distribution.

3. Aliquot into Cryovials:

Transfer the mixed culture-glycerol solution into sterile cryovials. The volume per vial will depend on your needs. Typically, 0.5-1.0 mL per vial is sufficient. Avoid overfilling the cryovials.

4. Label Your Cryovials:

Use a permanent marker to clearly label each cryvial with the following information:

Strain Name or Identification Number: This ensures you know what you're working with.
Date of Preparation: Tracking the date is critical for monitoring stock viability.
Any other relevant information: This might include the source of the culture or any relevant experimental conditions.

5. Freeze the Glycerol Stock:

Gradually lower the temperature of your glycerol stock to preserve cell viability. One common method is slow freezing:

Place the cryovials in an isopropyl alcohol (IPA) freezing container within a -80°C freezer. This allows for controlled freezing, reducing ice crystal formation that damages cells. The IPA helps to ensure uniform cooling.

Alternative Freezing Method (Fast Freezing):

While slower freezing is preferred, direct placement into a -80°C freezer may be used if an IPA container is unavailable. However, this may reduce the viability of some strains.

6. Store Your Glycerol Stock:

Once frozen, store your glycerol stock at -80°C. This low temperature significantly slows down metabolic processes, keeping your culture viable for extended periods, often years. Avoid repeated freeze-thaw cycles, as they reduce cell viability.

Recovering Your Glycerol Stock:

To recover your culture, carefully thaw a vial in a 37°C water bath or at room temperature. Ensure that the vial is fully thawed and gently vortex to re-suspend the cells before streaking or inoculating into fresh growth media. Remember to work aseptically to avoid contamination.

Troubleshooting:

Poor Viability After Thawing: This could indicate contamination, improper freezing, or using an old culture. Review your steps and use a fresh, healthy culture for future stocks.
Contamination: Sterile techniques are vital to prevent contamination. Make sure all materials are sterile.

By following these steps, you'll have successfully created glycerol stocks of your valuable microbial cultures, ensuring their long-term viability and preservation. Remember, careful technique and proper labeling are paramount for success.