Fong:Cryogenic storage

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Overview

Cells can be stored long-term in the -80°C freezer or in liquid nitrogen. For important stocks, it is a good idea to maintain both, as liquid nitrogen stocks can survive power outages. There are freezer log books for both the -80 and the liquid N₂ dewar. Log placement of all cryovials in pencil in the log books as well as in your lab notebook. The addition of glycerol to frozen stocks prevents the formation of ice crystals which will rupture cells when frozen, but high glycerol concentrations can be bad for some cells, so be quick to transfer thawed stocks to fresh media.

For E. coli

We generally maintain E. coli in 15% glycerol frozen stocks. Sterile technique is critical to reliable culture maintenance.

Prepare 30% glycerol solution

Materials

• Glycerol
• ddH₂O
• 2 autoclaved bottles
• Stir bar and plate
• Vacuum pump and hose
• Disposable sterile filter to fit bottle

Methods

1. Fill one bottle roughly halfway with ddH₂O, add stir bar and place on stir plate. As water is stirred, slowly add the appropriate volume of glycerol (300mL / 1L total volume). Stir until dissolved.
2. Remove stir bar and fill remainder of bottle with ddH₂O. Cap and shake to dissolve further.
3. In sterile hood, filter glycerol solution into the other sterile autoclaved bottle. Cap tightly, and store at room temp.

Make and freeze stocks

Materials

• 1.5mL cryovials
• 0.5mL sterile 30% v/v glycerol solution
• 0.5mL E. coli culture in mid-log phase (usually ~0.6-1.0 A600)
• micropipette and tips

Methods

1. Add 0.5mL glycerol solution to a new cryovial.
2. Add 0.5mL bacteria culture to the glycerol solution and gently mix by pipetting. Freeze immediately in either -80°C freezer or liquid nitrogen. Viability of cells stored in the freezer can be improved by flash freezing in liquid nitrogen, but we generally find this to be unnecessary.
3. To revive E. coli cells, either thaw at room temperature and use entire stock to inoculate fresh media, or simply flake off ice from the still-frozen stock into fresh media. Return the frozen stock to storage before it has a chance to thaw.

For C. thermocellum

C. thermocellum stores happily at -80°C or in liquid nitrogen, but anaerobic technique must still be followed. Making several 2mL shorty vials with 1mL 2x glycerol solution is tedious but saves a lot of time because they can be stored long term and are ready any time for addition of 1mL cell culture. Feel free to contact Chris with any questions about this protocol.

Prepare glycerol vials

Materials

• 2ml Wheaton "shorty" vials (these are shipped with plain plastic caps, but fit stoppered Hungate tube caps
• Hungate-style screw on cap with black-butyl septum stopper
• Glycerol
• ddH₂O
• Needles, syringes, and sterile syringe filters
• Pure N₂ gas and vacuum to purge vials
• Ethanol burner or other flame

Methods

1. In a clean bottle, prepare 2x glycerol solution. We store C. thermocellum in 20% glycerol, so our 2x is 40%.
2. Distribute 1mL 2x glycerol to shorty vials and cap with Hungate stoppers and caps.
3. Evacuate and purge stoppered shorty vials at least 5 times according to the technique detailed in the Fong:Serum bottle anaerobic culture protocol, and remove needle with a positive pressure of about 5psi. Autoclave vials for ≥20min at 121°C.

Make and freeze stocks

Materials

• Shorty vial containing 2x glycerol solution
• Needle and syringe
• Ethanol burner or other flame
• C. thermocellum culture in mid-log phase

Methods

1. To a shorty vial containing 1mL of sterile 2x glycerol solution, use sterile technique to add 1mL of C. therm culture in mid-log phase. Freeze immediately in -80°C freezer or liquid nitrogen.
2. To revive cells, thaw at room temperature and transfer entire 2mL sample to fresh medium. Thawing can be sped up using a room temp. water bath or by rolling the vial in your hands.