Congratulations on successfully isolating and purifying your bacteriophages! Now, as you embark on the next phase of your phage journey, it is crucial to understand the importance of proper storage. Preserving the viability and potency of your phages is vital for ongoing experiments and future applications. Today, we delve into the art of phage storage, providing you with essential techniques to maintain the longevity and effectiveness of your phage stocks.
The Resilience of Phages
Phages, those incredible predators of bacteria, possess remarkable resilience to adverse conditions. They can survive for extended periods in various environments, including dust, water bodies (including sewage LoL), and food. Storing these phages in a manner that ensures their viability is within our reach. However, maintaining high-titer preparations for a long time presents a more significant challenge.
Various Methods of Phage Storage
Let's look at the different methods commonly used for phage storage and their implications:
- Freeze Drying
Freeze Drying, also known as lyophilization, is a technique that utilizes low pressure and sublimation to remove water from a material, transforming it from ice to a dried state. This process finds wide-ranging applications, from food manufacturing to medicines. For instance, Oligonucleotides (Primers) used in PCR reactions are commonly shipped as lyophilized products that need to be reconstituted before use.
Despite its numerous advantages for storing various materials, freeze drying is not an ideal method for preserving phages. It leads to a significant reduction in phage titers, making it generally unsuitable for their storage, especially when the intention is to preserve them for a short period. In such cases, alternative storage methods are preferred to maintain phage viability. However, for long-term preservation, some culture collections do opt for this approach.
Interestingly, the destabilization of phages during the freeze-drying process appears to occur primarily during the process itself rather than after the material has been freeze-dried. As a result, it is crucial to ensure high initial titers before lyophilization to compensate for the expected losses during the process. This approach can enhance the success of long-term preservation using freeze drying for phages. Freeze-drying requires excipients like sucrose or trehalose.
- Storage at Room Temperature
Certain phages show no significant decrease in titer when stored at room temperature for several weeks. However, it's essential to note that titers may eventually decline over extended periods. To ensure successful long-term preservation of phages, relying solely on room temperature storage is not recommended, particularly when maintaining high titers is of utmost importance. Environmental temperature fluctuations can impact the stability of the stored phages.
If you choose to store phages at room temperature, it is crucial to take precautions and avoid exposing the preparation to direct sunlight. Sunlight exposure can further negatively affect the phages, potentially compromising their viability and overall effectiveness. Therefore, it's prudent to implement appropriate storage methods and conditions to preserve the phages optimally and maintain their efficacy over time.
- Storage at 2°– 8°C (Refrigeration temperature)
Storing phages at refrigeration temperatures, typically between 2°C and 5°C, has gained popularity as an effective short-term storage method. When phages are kept in appropriate media under these conditions, their titers generally experience slower declines, with reductions ranging from 2% to 10% over a six-month period (based on my own research conducted over eight months). This method is well-suited for storing phages for up to a few months.
Several media options are commonly used for refrigeration storage, including SM buffer, Tryptic Soy Broth (TSB), and Phosphate-buffered saline (PBS). SM buffer provides a stable environment that prevents phage replication, ensuring the preservation of phage characteristics. On the other hand, TSB offers nutrients that can help maintain phage stability. However, it's essential to note that TSB, due to its higher nutrient content and support for bacterial growth, may be more susceptible to contamination compared to SM buffer. Careful handling and sterile techniques are important to prevent unwanted bacterial growth and maintain the integrity of the stored phages.
- Frozen Storage
The most prevalent and dependable approach for preserving phages over extended durations is frozen storage. Some individuals may choose to freeze freshly infected bacteria as part of this method. Regardless of the specific approach, the addition of cryoprotectants is essential. Cryoprotectants are chemical compounds that shield cells or tissues from damage caused by freezing.
By incorporating cryoprotective agents such as glycerol or dimethyl sulfoxide (DMSO) during freezing, the survival of phages can be significantly enhanced. These agents play a crucial role in safeguarding phage particles from the stresses of freezing and thawing, thereby improving their long-term viability. Several approaches can be utilized in this process:
- Quick/Snap Freezing: Phages can be rapidly frozen using liquid nitrogen or at -70°C, ensuring their stability and long-term viability. This method allows for a rapid reduction in temperature, minimizing potential damage to the phage particles.
- Gradual Freezing: Gradual freezing involves freezing phages at a controlled rate. While less commonly used than quick freezing, this method can be beneficial for certain phages that may be more sensitive to rapid temperature changes.
Phage stocks in suitable media, such as nutrient-rich broth or buffer solutions, can be stored at temperatures of at least -18°C for long-term preservation. Lower temperatures, such as -30°C, are preferable if available.
Best Practices and Recommendations
To ensure the highest chance of preserving your phages' viability, consider the following recommendations:
- Store new phage stocks at 2°– 8°C in addition to frozen conditions as a precautionary measure. This provides a backup option in case the phage is not stable to freezing.
- For long-term storage, freezing in appropriate media at temperatures below -18°C is recommended. This ensures optimal viability and stability over extended periods.
- Regularly monitor the viability of stored phage preparations, especially those not previously worked with. Periodic titration can help assess phage viability and adjust storage conditions if necessary.
By employing proper storage techniques, you can maintain the integrity and effectiveness of your phages, ensuring they remain potent allies in your scientific endeavors.
Remember, preserving the magic of phages unlocks a world of possibilities!
Happy Phage Storage!
