Tryptic Soy Anaerobe Broth
Preparation
Tryptic Soy Anaerobe Broth
Step 1.
Add to flask
Tryptone
17
g
Phyton Peptone
3
g
Sodium Chloride
5
g
Potassium Phosphate, Dibasic
2.5
g
D-Glucose Anhydrous
2.5
g
Resazurin Sodium Salt
1
mg
Hemin Stock Solution (5 mg/mL)
1
mL
Step 2.
Autoclave at 121°C for 15 minutes.
Step 3.
While the media is still hot, begin sparging with N₂ to drive out oxygen.
Step 4.
Once the temperature drops safely to 45–50°C, add to flask
L-Cysteine Hydrochloride
0.5
g
Vitamin K Stock Solution (10 mg/mL)
50
μL
Step 5.
Dispense into anoxic Hungate tubes under an active N₂ gas blanket.
Nested recipe inputs
Used in step 1
Hemin Stock Solution (5 mg/mL)
Step 1.
Add to beaker
Hemin
0.5
g
Step 2.
Add NaOH. Stir or vortex vigorously until the powder dissolves completely into a dark, opaque fluid. Ensure no visible particles remain on the glass walls.
1M NaOH
10
mL
Step 3.
Bring to final volume using \(dH_{2}O\).
Step 4.
Autoclave the solution at 121°C for 15 minutes. (Hemin is heat-stable).
Store the sterilized solution in an amber glass bottle at 4°C.
Add 1 mL of this stock solution per 1 Liter of media to achieve a final concentration of 5 mg/L
Used in step 2
1M NaOH
Work in chemical hood and wear eye protection. NaOH is extermely caustic!
Step 1.
Add ~75% of the target volume in distilled water to a appropriately-sized beaker.
Step 2.
Gradually add NaOH pellets while stirring.
Sodium Hydroxide
2
g
The solution will heat up. This is normal.
Step 3.
After fully dissolved, transfer the solution to a falcon tube or media bottle.
Step 4.
Bring final volume to 50 mL with distilled water.
Used in step 4
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.