There are different methods for the preservation of microbial cultures. Bacteria preservation in glycerol is one of the common method for maintaining bacterial strains over long periods of time. This method involves adding glycerol to a bacterial culture and freezing it at a low temperature (-70°C or lower) to create a stable stock which one can store for years without losing viability.
Glycerol as a good Cryoprotectant
Following are the key properties of glycerol that make it an effective cryoprotectant for bacterial preservation:
- Prevents ice crystal formation during freezing and thawing, which can damage bacterial cells.
- Penetrates cell membranes to protect the cells from damage.
- Has a low freezing point, which further helps prevent ice crystal formation.
- It can form hydrogen bonds with water molecules, which provides additional protection to cell membranes.
- The non-toxic nature of glycerol makes it compatible with a wide range of bacterial species.
- It is cost-effective and convenient for preserving bacterial cultures.
Steps of bacteria preservation in glycerol stocks
Sure, here are the detailed steps involved in bacterial culture storage in glycerol. It is important to follow strict aseptic techniques during bacterial culture storage in glycerol to avoid contamination and ensure the preservation of the bacterial cells.
Grow bacterial culture:
Grow the bacterial culture in a suitable medium until it reaches mid- to late-log phase, when the cells are most active and healthy. This ensures that the bacterial cells are in a robust physiological state before preservation.
Harvest bacterial culture
Harvest the bacterial culture by centrifugation or filtration to remove the culture medium and concentrate the bacterial cells. There should be no damage to the bacterial cells during this cell harvesting process.
Prepare glycerol solution
Prepare a solution of glycerol and a suitable buffer in the desired concentration (usually 15-50% glycerol). The buffer helps to maintain the pH and osmolarity of the solution and may contain nutrients to support bacterial growth. The glycerol solution should be sterile.
Resuspend bacterial cells in glycerol solution
Resuspend the concentrated bacterial cells in the glycerol solution. To prevent cell damage, it is important to add the glycerol solution slowly and gently during the resuspension step. This step ensures that the glycerol is distribution takesplce evenly throughout the bacterial culture, providing optimal protection during freezing and thawing.
Dispense into cryovials
Dispense the bacterial culture and glycerol solution into sterile cryovials or tubes, usually in 1-2 ml aliquots. The labels on the cryovials should include important information such as the bacterial strain name, preparation date, glycerol concentration, and other relevant details.
Freeze the culture
Freeze the bacterial culture at a temperature of -80°C or lower. The glycerol solution protects the cells during freezing and ensures that the cells remain viable after thawing. To prevent damage to the bacterial cells, it is important to freeze the culture slowly to avoid the formation of ice crystals. You can accomplish this by gradually decreasing the temperature of the culture over time or by using a controlled-rate freezer.
Store the glycerol stock
Store the frozen glycerol stock in a dedicated freezer, preferably with a backup power supply or alarm system. The freezer should be monitored regularly to ensure that it maintains the correct temperature.
Thaw the glycerol stock
Thaw the glycerol stock by removing a cryovial from the freezer and rapidly thawing it in a water bath at 37°C or by gentle agitation in a refrigerator at 4°C. The thawing step should be done quickly to minimize the time the bacterial cells spend in a compromised state.
Use the bacterial culture
Inoculate a small amount of the thawed bacterial culture into fresh growth medium to start a new culture or use the cells for downstream applications. The thawed bacterial culture should be handled carefully to avoid contamination and should be monitored for growth and viability.
Advantages of bacteria preservation in glycerol
Cryoprotection:
Glycerol protects the bacterial cells from damage during freezing and thawing by preventing the formation of ice crystals that can rupture cell membranes.
Long-term storage
Bacterial cultures stored in glycerol can be stored for many years at temperatures below -70°C without significant loss of viability or genetic stability.
Cost-effective
Glycerol is a relatively inexpensive and readily
Disadvantages of bacteria preservation in glycerol
Toxicity:
Glycerol can be toxic to some bacterial strains, especially if the concentration used is too high. It is important to determine the appropriate glycerol concentration for each bacterial strain to avoid toxicity.
Freezer failure:
If the freezer storing the glycerol-preserved cultures fails or experiences a power outage, the bacterial cultures may thaw and be damaged or destroyed.
Quality control
Proper labeling and tracking of glycerol-preserved cultures is crucial to ensure that they are used correctly and not contaminated or misused.
Limited shelf life:
Glycerol-preserved bacterial cultures must be stored at temperatures below -70°C to maintain their viability and genetic stability. If the freezer fails or the temperature rises above this threshold, the cultures may become unusable.