Glycerol is a simple polyol compound that serves as a highly effective stabilizing agent in biological applications. It is a viscous, clear, and non-toxic liquid that is completely miscible with water. The 50% glycerol solution is a specific, high-concentration stock preparation commonly utilized in laboratory settings. This solution’s primary role is to act as a cryoprotective agent. The goal of using this specific concentration is to prepare biological samples for safe, long-term storage at ultra-low temperatures, a process known as cryopreservation.
The Primary Application: Cryopreservation
The 50% glycerol stock solution is most often prepared for the long-term preservation of microbial cultures, such as bacterial and yeast stocks. In a common laboratory protocol, this stock is mixed in a one-to-one ratio with a concentrated liquid culture. This mixing typically yields a final glycerol concentration of 25% (v/v) in the freezing mixture, which is considered optimal for many bacterial strains like E. coli.
The cryoprotectant and ultra-low temperatures halt the metabolism and degradation of the biological sample. These glycerol stocks are then immediately frozen and stored at temperatures of -80°C in specialized freezers. The resulting storage state can maintain cell viability for many years, allowing researchers to reliably retrieve the exact strain or culture when needed.
While the 50% stock is fundamental for microbial preservation, glycerol is also used for cryopreserving other biological materials, including red blood cells, sperm cells, and certain types of mammalian cell lines. For these more sensitive cell types, the glycerol is often introduced gradually to achieve a final concentration between 5% and 15% before being plunged into liquid nitrogen at -196°C. Glycerol makes cryopreservation an indispensable tool in biobanking and research.
Mechanism of Cellular Protection
Glycerol functions as a cryoprotective agent by disrupting the physical and chemical processes that cause cellular damage during freezing. The primary threat to cells at low temperatures is the formation of large, sharp ice crystals, which can physically puncture cell membranes and organelles. Glycerol prevents this by interposing itself within the water’s hydrogen bond network.
As a solute, glycerol also lowers the freezing point of the solution that reduces the temperature at which ice begins to form. This action minimizes the amount of water that freezes outside the cell, reducing osmotic stress. When water moves out of the cell to form extracellular ice, the remaining solution around the cell becomes dangerously concentrated with salts.
The glycerol molecules are small enough to penetrate the cell membrane, where they replace water molecules through hydrogen bonding. This intracellular presence helps maintain the hydration and structural integrity of internal components, such as proteins and DNA. By stabilizing the cellular membranes and replacing the water, glycerol prevents excessive cellular dehydration and shrinkage, thereby mitigating the major causes of cell death during the freezing and thawing cycle.
Practical Handling and Storage
Handling 50% glycerol requires attention to sterility, as the solution is mixed directly with living cultures intended for long-term preservation. The 50% stock is typically prepared by diluting pure, concentrated glycerol with sterile water, followed by sterilization through autoclaving or filter sterilization. Because pure glycerol is highly viscous, heating the stock solution slightly on a magnetic stirrer can make it easier to pipette accurately during preparation.
The mixing technique is important for achieving a uniform cryoprotective environment. The 50% glycerol stock is mixed with the liquid culture, and the mixture must be thoroughly homogenized, often by gentle vortexing or inversion, to ensure the glycerol is evenly distributed throughout the cell suspension. This even distribution allows the cryoprotectant sufficient time to permeate the cells before the freezing process begins.
Once mixed, the stock should be frozen immediately at the designated storage temperature, usually -80°C, using cryo-designated vials with screw caps. It is important to minimize the time the sample spends outside the ultra-low temperature freezer, and a stock should never be allowed to fully thaw once frozen. Repeated freeze-thaw cycles significantly reduce the viability of the stored cells, making it necessary to only scrape a small amount of frozen material from the surface for inoculation.