334. METHANOSPIRILLUM MEDIUM

KH₂PO₄ 0.23 g

K₂HPO₄ 0.23 g

MgSO₄ × 7 H₂O 0.09 g

NaCl 0.46 g

NH₄Cl 0.4 g

CaCl₂ × 2 H₂O 60.0 mg

(NH₄)₂SO₄ 0.23 g

Trace element solution SL-10 (see below) 10.0 ml

Vitamin solution (see below) 10.0 ml

Yeast extract 1.0 g

Na-acetate × 3 H₂O 1.0 g

Na-formate 2.0 g

Trypticase 0.5 g

Casamino acids 0.2 g

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

NaHCO₃ 4.0 g

L-Cysteine-HCl × H₂O 0.5 g

Na₂S × 9 H₂O 0.5 g

Distilled water 980.0 ml

Trace element solution SL-10:

HCl (25%; 7.7 M) 10.0 ml

FeCl₂ × 4 H₂O 1.5 g

ZnCl₂ 70.0 mg

MnCl₂ × 4 H₂O 100.0 mg

H₃BO₃ 6.0 mg

CoCl₂ × 6 H₂O 190.0 mg

CuCl₂ × 2 H₂O 2.0 mg

NiCl₂ × 6 H₂O 24.0 mg

Na₂MoO₄ × 2 H₂O 36.0 mg

Distilled water 990.0 ml

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, cysteine and sulfide. Sparge medium with 80% H₂ and 20% CO₂ gas mixture for 30–45 min to make it anoxic. Add and dissolve bicarbonate, then dispense medium under 80% H₂ and 20% CO₂ gas atmosphere into anoxic Hungate-type tubes and autoclave. Add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N₂ gas. Prior to use check pH of complete medium and adjust to 6.8-7.0, if necessary.

 

Updated 01.02.2024