117. METHANOSARCINA MEDIUM

Solution 1:

NaCl 0.9 g

MgCl₂ × 6 H₂O 0.2 g

CaCl₂ × 2 H₂O 0.1 g

NH₄Cl 1.0 g

Yeast extract 2.0 g

Resazurin 10.0 mg

Methanol 10% 10.0 ml

Trace element solution (see below) 10.0 ml

Vitamin solution (see below) 5.0 ml

Distilled water 965.0 ml

Solution 2 (reducing agents):

L-Cysteine-HCl 0.5 g

Na₂S × 9 H₂O 0. 5 g

Distilled water 10.0 ml

Buffer solutions:

a) K₂HPO₄ 29.0 g

Distilled water 100.0 ml

b) KH₂PO₄ 15.0 g

Distilled water 100.0 ml

Trace element solution:

Nitrilotriacetic acid 12.8 g

FeSO₄ × 7 H₂O 0.1 g

MnCl₂ × 6 H₂O 0.1 g

CoCl₂ × 2 H₂O 0.17 g

CaCl₂ × 2 H₂O 0.1 g

ZnCl₂ 0.1 g

CuCl₂ × 2 H₂O 0.02 g

H₃BO₃ 0.01 g

Na₂MoO₄ × 2 H₂O 0.01 g

NaCl 1.0 g

Na₂SeO₄ 0.017 g

Distilled water 1000.0 ml

Vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine 10.0 mg

Riboflavin 5.0 mg

Pantotenoic acid 5.0 mg

p-Aminobenzoic acid 5.0 mg

Thiamine-HCl 5.0 mg

Nicotinic acid 5.0 mg

Vitamin B₁₂ 0.1 mg

Lipoic acid 5.0 mg

Distilled water 1000.0 ml

pH 7.2-7.4

Prepare medium in anaerobic conditions, blowing through with N₂ without O₂ up to sterilization. Solutions of reducing agents (10.0 ml) and of buffer (per 1 ml) add to base medium after separate sterilization. Base medium, trace elements, buffer solutions and reducing agents autoclave at 121°C for 15 min. Vitamin solution is filter sterilized.

 

Updated 1.02.2024