Making a long term stock of bacteria

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Introduction

Whenever you successfully transform a bacterial culture with a plasmid or whenever you obtain a new bacterial strain you will want to make a long term stock of that bacteria. Bacteria can be stored for months and years if they are stored at -80C and in a high percentage of glycerol.

Materials

• 40% glycerol solution
• Day/overnight culture
• Cryogenic vials/1.5mL microfuge tube

Method

• Add 1 ml of 40% glycerol in H₂O to a cryogenic vial.
• Add 1 ml sample from the culture of bacteria to be stored.
• Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
  • Alternatively, pipet to mix.
• Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
• On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
• Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.

Notes

• While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.
• Certain antibiotics in the medium should be removed first as they are supposedly toxic over time, ex)Tetracycline. To do this, spin the culture down and resuspend in same volume of straight LB medium.

Specific Protocols

• Endy:Making a long term stock of bacteria
• Endy:Retrieving a Registry glycerol
• -80_Glycerol_Stocks
• Alm:Glycerol_stocks
• McClean:E._coli_Glycerol_Stocks
• Cfrench:glycerol
• Smolke:Protocols/Freezer_stocks