365. THERMOANAEROBACTER MEDIUM II

NH₄Cl 1.0 g

NaCl 0.1 g

MgCl₂ × 6 H₂O 0.1 g

CaCl₂ × 2 H₂O 0.05 g

K₂HPO₄ × 3 H₂O 0.4 g

Trace element solution (see below) 10.0 ml

Yeast extract 0.75 g

Na-resazurin solution (0.1% w/v) 0.5 ml

NaHCO₃ 2.6 g

Cellobiose 4.0 g

Vitamin solution (see below) 10.0 ml

Na₂S × 9 H₂O 0.25 g

Distilled water 1000.0 ml

Trace element solution:

Nitrilotriacetic acid 1.5 g

MgSO₄ × 7 H₂O 3.0 g

MnSO₄ × H₂O 0.5 g

NaCl 1.0 g

FeSO₄ × 7 H₂O 0.1 g

CoSO₄ × 7 H₂O 0.18 g

CaCl₂ × 2 H₂O 0.1 g

ZnSO₄ × 7 H₂O 0.18 g

CuSO₄ × 5 H₂O 0.01 g

KAl(SO₄)₂ × 12 H₂O 0.02 g

H₃BO₃ 0.01 g

Na₂MoO₄ × 2 H₂O 0.01 g

NiCl₂ × 6 H₂O 0.03 g

Na₂SeO₃ × 5 H₂O 0.3 mg

Na₂WO₄ × 2 H₂O 0.4 mg

Distilled water 1000.0 ml

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.

Vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine-HCl 10.0 mg

Thiamine-HCl × 2 H₂O 5.0 mg

Riboflavin 5.0 mg

Nicotinic acid 5.0 mg

D-Ca-pantothenate 5.0 mg

Vitamin B₁₂ 0.1 mg

p-Aminobenzoic acid 5.0 mg

Lipoic acid 5.0 mg

Distilled water 1000.0 ml

Dissolve ingredients (except bicarbonate, cellobiose, vitamins and sulfide), then sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 – 45 min to make it anoxic. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add cellobiose, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N₂ gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Stock solutions of cellobiose and vitamins should be sterilized by filtration. Adjust pH of complete medium to 7.0, if necessary.

 

Updated 11.08.2021