Team:Cambridge/Protocols/Glycerol Stocks

Glycerol Stocks

Preparation of glycerol stocks of bacteria allows for long-term storage at -80°C without compromising viability of cells.

Theory

Freezing is an efficient way of storing bacteria. Glycerol allows to reduce the harmful effect of ice crystals of bacteria which can damage cells by dehydration caused by a localized increase in salt concentration leading to denaturation of proteins. Additionally, ice crystals can also puncture cellular membranes. 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.

Practice

• Preparation of glycerol stocks of bacteria

1. Prepare a liquid culture of bacteria in LB with antibiotic for selection. Use 100-fold dilution of an overnight culture of the strain of interest.
2. Incubate cells at 37° for 3-4 hours until the culture reaches the mid-log phase.
3. Transfer around 1ml of the culture into an Eppendorf tube and mix with an appropriate amount of glycerol to get a final concentration of 20%.
4. For high viabilitystore at -80°.

Tip:For precious strains, storage of two stock vials is recommended. Tip:Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria. Tip: When recovering a stored strain, it is advisable to check that the antibiotic markers have not been lost by streaking the strain onto an LB-agar plate containing the appropriate antibiotic(s).

Safety

All materials that come into contact with transgenic bacteria must be autoclaved.

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