How to Prepare Bacterial Glycerol Stocks for Long-Term Preservation - Cryopreservation of Bacteria

Welcome to the world of microbiology! In this blog, we will dive into the essential techniques for preparing bacterial glycerol stocks. This method is key for long-term preservation, ensuring that your bacterial strains remain viable for future experiments and research. Let’s explore the importance of this technique, the necessary materials, and the step-by-step procedure to master this skill.

Table of Content

• Introduction
• Importance
• Objectives
• Principle
• Materials Required
• Procedure
• Precautions
• Conclusion

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Introduction

Bacterial glycerol stocks are vital for the long-term preservation of bacterial strains. This method allows researchers to maintain cell viability over extended periods, reducing the need for constant culturing and conserving valuable resources. In research labs, the ability to archive strains is critical for reproducibility and continuity in experiments.

But what exactly are bacterial glycerol stocks? Essentially, they are suspensions of bacterial cells in a glycerol solution that can be frozen for long-term storage. The addition of glycerol acts as a cryoprotectant, which helps prevent damage to the bacterial cells during the freezing process.

• Bacterial glycerol stocks enable long-term preservation of strains.
• Maintain cell viability over extended periods.
• Reduce the need for continuous culturing, saving resources.

The Importance of Cryopreservation

Cryopreservation is crucial in microbiology for several reasons:

• Long-term viability: Bacterial cultures can remain stable for years when stored correctly at -80°C.
• Reduced culturing needs: By preserving cultures in glycerol stocks, researchers can avoid the time and resource costs associated with continuous culturing.
• Maintaining strain integrity: Properly prepared glycerol stocks help ensure that the genetic and phenotypic characteristics of bacterial strains are preserved over time.

Objectives

• Extend the lifespan of bacterial cultures.
• Simplify long-term sample management.

Principle

The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years and -20°C for several months. Glycerol reduces the harmful effects of ice crystals on bacteria which cause dehydration and damage cells through a localised increase in salt concentration leading to the denaturation of proteins.

Materials Required for Glycerol Stock Preparation

Before diving into the procedure, it’s essential to gather all the necessary materials. Here’s what you’ll need:

• Endorf tubes (autoclaved or RNA-free)
• Micro pipette with sterile tips
• Nutrient broth
• 15% glycerol solution (prepared by mixing 15 mL of glycerol with 85 mL of distilled water and autoclaving)
• Inoculating loop
• Pure isolated bacterial colonies
• Laminar flow hood for sterile handling
• Freezer set to -20°C and -80°C for storage

Step-by-Step Procedure for Preparing Glycerol Stocks

Now that you have all the materials ready, let’s walk through the detailed procedure for preparing your bacterial glycerol stocks.

Step 1: Prepare Nutrient Broth

Begin by arranging your sterile endorf tubes in a stand. Pipette 500 µL of nutrient broth into each labeled endorf tube. If you’re confident in your sterile techniques, you can use the same pipette tip for all tubes, but changing tips is recommended for beginners to avoid contamination.

Step 2: Inoculate the Nutrient Broth

Next, sterilize your inoculating loop by heating it until it’s red hot. Allow it to cool by touching it to an area of the agar plate with no bacterial growth. Carefully pick a single isolated colony and transfer it into the nutrient broth by shaking the loop gently inside the tube. Remember to sterilize the loop after each transfer to maintain sterility.

Step 3: Incubate the Culture

After inoculating all your tubes, vortex them to mix thoroughly. Place the tubes in an incubator set at 37°C for 18 to 24 hours. This incubation period allows the bacteria to grow and multiply in the nutrient broth.

Step 4: Add Glycerol

Once the incubation is complete, it’s time to add glycerol to your bacterial cultures. Vortex each tube again to ensure an even mixture. Then, add 500 µL of the 15% glycerol solution to each tube. Mix thoroughly again using the vortex.

Step 5: Freezing the Samples

Now, it’s time for the crucial freezing step. Place the tubes in a -20°C freezer for 1 to 2 days. This initial freezing prepares the samples for long-term storage. After this period, transfer the tubes to a -80°C freezer. At this temperature, your samples can remain viable for many years.

Step 6: Labeling and Organizing

Proper labeling is essential for easy identification. Label each tube with details such as strain name, date, and any other relevant information. It’s also a good practice to maintain a record of your samples for future reference.

Precautions to Avoid Contamination

During the preparation of glycerol stocks, maintaining sterility is crucial. Here are some key precautions to keep in mind:

• Always work in a laminar flow hood to minimize contamination risks.
• Avoid touching sterile surfaces or tools with bare hands.
• Use sterilized inoculating loops and change pipette tips as needed.
• Ensure that all materials, including nutrient broth and glycerol, are autoclaved before use.

Conclusion

Preparing bacterial glycerol stocks is a fundamental skill in microbiology that ensures the longevity and viability of bacterial cultures. By following the outlined steps and taking necessary precautions, you can efficiently preserve your bacterial strains for future research and experiments.