PREPARATIONS
0.2% Resazurin Solution Glycerol Solution
Make in ddH2O 12 ml Glycerol
Store at 4˚C 28 ml ddH2O
2 drops of 0.2% Resazurin solution
PROTOCOLS
Preparation of Glycerol Vials
1. Steam glycerol solution in a 160 ml serum bottle and let cool while gassing with N2. Flush and evacuate bottle.
2. Flush and evacuate 40 12.5 ml serum bottles with N2.
3. Transfer 1 ml of solution to each vial via syringe.
4. Flush and evacuate all vials repeatedly (_ h).
5. Autoclave 30 min.
Freezing Cells
1. Grow up a fresh culture of bacteria to high density.
2. Add a drop or two of an appropriate reducing agent (such as sodium sulfide) to vials until the resazurin turns colorless. Wrap label tape completely around vial.
3. Using aseptic technique, add 1 ml of culture by syringe to each vial. Quickly place vial into a dry ice/ethanol bath.
4. When cultures are frozen, put in labeled boxes in 80˚C freezer.
materials
20 mM Phosphate buffer pH 7
Resazurine solution (500 mg/l)
Glycerol
~100 mM titanium citrate solution (filter‐sterilized)
10 ml serum bottles(Sigma))
Butyl stoppers and crimp caps
Method
Prepare 100 ml phosphate solution
Add resazurine (final concentration 0.5 mg/l)
Bring the solution to a full boil and cool down under N2 stream
Add 100 ml glycerol; this makes a 50% (v/v) glycerol solution
Fill the anaerobic vials with 4 ml of the glycerol solution
Stopper and cap them
Change gas phase to 100% N2
or 80/20% N2/CO2; fill with 0.2 atm overpressure
Autoclave the vials
Inject 3 – 4 ml of a late log phase bacterial culture to the sterilized glycerol solution
using a 5ml syringe. Final concentration of the glycerol should be 25 – 30 %
Using 1 ml syringe, inject aprox. 3 drops of filter sterilized titanium citrate solution
to each vial to reduce the stocks
Freeze them at –80c
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