333. MEDIUM FOR PSYCHROPHILIC METHANOSARCINA

K₂HPO₄ 0.35 g

KH₂PO₄ 0.23 g

NH₄Cl 0.5 g

MgSO₄ × 7 H₂O 0.5 g

CaCl₂ × 2 H₂O 0.25 g

NaCl 2.25 g

FeSO₄ × 7 H₂O solution (0.1% w/v in 0.1 H₂SO₄) 2.0 ml

Trace element solution SL-10 (see below) 1.0 ml

Yeast extract 2.0 g

Casitone 2.0 g

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

NaHCO₃ 0.85 g

Vitamin solution (see below) 10.0 ml

Methanol 10.0 ml

L-Cysteine-HCl × H₂O 0.3 g

Na₂S × 9 H₂O 0.3 g

Distilled water 1000.0 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, vitamins, methanol, cysteine and sulfide) and sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 – 45 min to make it anoxic. Then add and dissolve bicarbonate, adjust pH to 6.8 and dispense medium under 80% N₂ and 20% CO₂ gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Methanol (50% v/v stock solution) and the reducing agents are each autoclaved separately under 100% N₂ gas atmosphere as concentrated solutions in tightly closed tubes. Vitamins are prepared under 100% N₂ gas atmosphere and sterilized by filtration. Appropriate volumes of the solutions are injected into the sterile medium with hypodermic syringes. Adjust pH of the complete medium to 6.5 - 6.8, if necessary.

 

Updated 01.02.2024