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

1. Pick a single colony of the clone off a plate and grow an overnight in the appropriate selectable liquid medium (3-5ml).
2. Add 0.5 ml of 40% glycerol in H₂O to a cryogenic vial.
3. Add 0,5 ml sample from the culture of bacteria to be stored.
4. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
   • Alternatively, pipet to mix.
5. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
6. Freeze glycerol stock in liquid nitrogen and store in a -80C freezer.
   • This will also be a good time to record the strain information and record the location.

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