364. CALDANAEROBACTER MEDIUM

KCl 0.33 g

MgCl₂ × 6 H₂O 0.52 g

CaCl₂ × 2 H₂O 0.29 g

NH₄Cl 0.33 g

KH₂PO₄ 0.33 g

Trace element solution SL-4 (see below) 10.0 ml

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

NaHCO₃ 2.5 g

D-glucose 2.0 g

Yeast extract 0.05 g

Vitamin solution 1 (see below) 9.0 ml

Seven vitamins solution (see below) 1.0 ml

Na₂S × 9 H₂O 0.3 g

Distilled water 1000.0 ml

Trace element solution SL-4:

Na₂-EDTA 0.5 g

FeSO₄ × 7 H₂O 0.2 g

ZnSO₄ × 7 H₂O 0.1 g

MnCl₂ × 4 H₂O 0.03 g

H₃BO₃ 0.3 g

CoCl₂ × 6 H₂O 0.2 g

CuCl₂ × 2 H₂O 0.01 g

NiCl₂ × 6 H₂O 0.02 g

Na₂MoO₄ × 2 H₂O 0.03 g

Distilled water 1000.0 ml

First dissolve EDTA in distilled water and adjust pH to 7.0 using 2 N NaOH; then add other compounds.

Vitamin solution 1:

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

Seven vitamins solution:

Vitamin B₁₂ 100.0 mg

p-Aminobenzoic acid 80.0 mg

D(+)-Biotin 20.0 mg

Nicotinic acid 200.0 mg

Calcium pantothenate 100.0 mg

Pyridoxine-HCl 300.0 mg

Thiamine-HCl × 2 H₂O 200.0 mg

Distilled water 1000.0 ml

Dissolve ingredients except bicarbonate, D-glucose, yeast extract, vitamins and sulfide, then sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 -45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave.

Add D-glucose, yeast extract, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N₂ gas atmosphere and bicarbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Stock solutions of D-glucose and vitamins are sterilized by filtration. After completing the medium adjust pH to 6.8-7.0 with a sterile anoxic stock solution of Na₂CO₃ (5% w/v), if necessary.

 

Updated 11.07.2021