Glycerol stock serves as a fundamental method for preserving biological samples, particularly microbial cultures, over extended periods. This technique involves suspending cells in a solution containing glycerol, which acts as a cryoprotectant. Its primary goal is to maintain sample viability and genetic integrity during freezing and long-term storage.
Why Make Glycerol Stock?
Creating glycerol stock offers significant advantages for long-term preservation. Glycerol’s cryoprotective properties shield cells from damage during freezing. Without a cryoprotectant, ice crystals form inside and outside cells, physically rupturing membranes and organelles, leading to cell death.
Glycerol lowers the solution’s freezing point, preventing large, destructive ice crystals. It permeates cells, reducing free water for ice formation and minimizing cellular dehydration. This protective action helps maintain cell viability, ensuring the biological sample remains stable and retrievable for future experiments and archiving unique strains.
What You Will Need
To prepare glycerol stock, gather specific materials and equipment, ensuring sterility. You will need molecular biology grade glycerol, typically a 50% solution diluted with sterile water or growth media. This concentration is important for effective cryoprotection. The biological sample, such as an overnight bacterial culture, is also required.
Sterile cryovials or screw-cap tubes are essential containers, designed to withstand ultra-low temperatures and prevent leakage. Screw-cap tubes are preferred over snap-caps, which can open unexpectedly at very cold temperatures. Other items include sterile pipettes and tips for aseptic liquid transfer, and a freezer capable of -80°C for long-term storage. All materials must be sterile to prevent sample contamination.
Step-by-Step Preparation
Begin by preparing your glycerol solution; a common approach is to create a 50% glycerol solution by mixing 100% glycerol with sterile water. Some protocols may use an 80% glycerol solution, which is then mixed with the culture to achieve a final concentration of 15-25% glycerol. This concentration range is widely used for effective cryopreservation.
Next, ensure your biological sample, such as a bacterial culture, has reached an appropriate growth phase, typically an overnight culture. This ensures a sufficient density of viable cells for storage. Aseptically transfer the biological sample and the prepared glycerol solution into sterile cryovials. A common ratio involves combining equal volumes, for instance, 500 µL of the overnight culture with 500 µL of the 50% glycerol solution, resulting in a final glycerol concentration of 25%.
Gently mix the contents of the cryovial by inverting the tube several times or by gentle vortexing. This ensures the glycerol is evenly distributed throughout the cell suspension without causing cell lysis. Avoid vigorous shaking, which can damage the cells. Thorough mixing is indicated by the solution appearing uniform with no visible layers.
Once mixed, label each cryovial clearly with essential information such as the sample name, date, and any relevant identifiers. Labeling directly onto the tube with a permanent marker before freezing is recommended, as adhesive labels can detach and frozen tubes are difficult to write on. This meticulous labeling prevents sample mix-ups and ensures proper identification during retrieval. Immediately after labeling, the prepared glycerol stocks are ready for freezing.
Storing and Using Your Stock
Proper storage of glycerol stock is essential for maintaining sample integrity over many years. Glycerol stocks should be stored at ultra-low temperatures, ideally in a -80°C freezer. This consistent low temperature prevents damaging ice crystals and ensures long-term viability. While some sources suggest -20°C for shorter periods, -80°C is the standard for extended preservation.
Effective organization and clear labeling of the cryovials within the freezer are important for easy retrieval. Each tube should be plainly marked with the sample name, date, and any other relevant information. This systematic approach helps prevent loss or misidentification of valuable samples. When a sample is needed, retrieve the cryovial from the freezer and use a sterile loop or pipette tip to scrape a small amount of the frozen material from the surface.
Avoid completely thawing the entire glycerol stock during retrieval. Repeated freeze-thaw cycles can significantly reduce cell viability and compromise the sample’s integrity. The scraped frozen material can then be used to inoculate fresh growth media or streaked onto an agar plate to culture the cells. Promptly return the remaining frozen glycerol stock to the -80°C freezer to preserve its longevity.