Wilkins-Chalgren Anaerobe Broth
Preparation
Wilkins-Chalgren Anaerobe Broth
Step 1.
Combine in flask
Tryptone
10
g
Peptone
10
g
Yeast Extract
5
g
Sodium Chloride
5
g
D-Glucose Anhydrous
1
g
L-Arginine
1
g
Sodium Pyrophosphate (Tetrasodium Pyrophosphate)
1
g
Hemin
5
mg
Resazurin Sodium Salt
1
mg
Step 2.
Bring to final volume with distilled water
Step 3.
Autoclave for 15 minutes at 121°C.
Step 4.
While the media is still hot, begin sparging with N₂ to drive out oxygen.
Step 5.
Once the temperature drops safely to 45–50°C, add to flask
Vitamin K Stock Solution (10 mg/mL)
50
μL
L-Cysteine Hydrochloride
0.5
g
Step 6.
Dispense into anoxic Hungate tubes under an active N₂ gas blanket.
Nested recipe inputs
Used in step 5
Vitamin K Stock Solution (10 mg/mL)
Step 1.
Add to falcon tube
Vitamin K
0.1
g
95% ETOH
10
mL
Step 2.
Vortex until the solution becomes completely clear and yellow.
Do not autoclave. Ethanol is self-sterilizing. If you dilute this further with water, you must pass it through a 0.22µm PTFE syringe filter (nylon or cellulose filters can absorb hydrophobic vitamins).
Storage: Store in a tightly sealed amber tube or wrapped in aluminum foil at -20°C. Vitamin K is highly light-sensitive and will degrade if exposed to light.
Usage: Add 0.05 mL (50 µL) of this stock solution per 1 Liter of media to achieve a final concentration of 0.5 mg/L.
Used in step 1
95% ETOH
Step 1.
Add to falcon tube
Ethanol, 200 proof
47.5
mL
(95 % v/v)
Step 2.
Bring final volume to 50 mL with distilled water.