How do you freeze bacterial cells with glycerol?
Bacterial Glycerol Stocks
- Put 0.5ml bacterial culture in a sterile eppendorf tube.
- Add 0.5ml of sterile 80% (v/v) glycerol soution.
- Freeze on dry ice or directly into –70oc .
- Store at –70oC. Cells are best for about 4-6 months, but will probably work ok for a whole year.
What is the role of glycerol in cryopreservation?
It has been shown previously that glycerol can reduce the water crystallization process, inhibit the growth of ice crystal, and reduce damage to the cell structure and function [38]. However, the viability of cells cryopreserved in glycerol alone was also lower than that of cells cryopreserved in 10% DMSO + 90% FBS.
How do you freeze bacterial cells?
Freeze-drying: Bacteria can be freeze-dried by suspending log-phase cells in a lyophilization medium and then freeze- drying the suspension. Not all bacteria can be successfully freeze-dried. Certain strains might not survive the process or die rapidly once freeze-dried.
How is glycerol sterilized?
Sterilize the glycerol in hot air oven at 160C for 60 mins. Alternatively aqueous solutions of glycerol can be either autoclaved(usually the case) or filter sterilized.
How does glycerol prevent freezing?
Glycerol as a cryoprotectant depresses the freezing point of bacterial cells, enhancing supercooling. It does so by forming strong hydrogen bonds with water molecules, competing with water-water hydrogen bonding. This disrupts the crystal lattice formation of ice unless the temperature is significantly lowered.
What does freezing do to bacteria?
Freezing does not kill germs and bacteria. Instead, it essentially puts them into hibernation. They are inactive while the food is frozen and will “wake up” as soon as the food thaws. And as the food thaws, so will the moisture, which means the bacteria will have the moisture it needs to survive.
How are microorganisms preserved by deep freezing?
Many bacteria can be preserved very effectively by freeze drying. By freezing the cells in a medium that contains a lyoprotectant (usually sucrose) and then pulling the water out using a vacuum (sublimation), cells can be effectively preserved.
How cryoprotectant protect cells from freezing injury?
Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity.
Can we sterilize glycerol?
Yes, I have sterilised glycerol in the autoclave in a glassware. It works fine!
Where do you store glycerol?
Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as long as it is kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life.