326. METHANOCALCULUS NATRONOPHILUS MEDIUM

KH₂PO₄ 0.2 g

MgCl₂ × 6 H₂O 0.1 g

NH₄Cl 1.0 g

KCl 0.2 g

NaCl 60.0 g

Trace element solution SL-11 (see below) 1.0 ml

Na-acetate × 3 H₂O 0.2 g

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

Na₂CO₃ 68.0 g

NaHCO₃ 38.0 g

Vitamin solution (see below) 10.0 ml

Na₂S × 9 H₂O 1.0 g

Distilled water 1000.0 ml

Trace element solution SL-11:

Na₂-EDTA × 2 H₂O 5.2 g

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 1000.0 ml

Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000.0 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 carbonate, bicarbonate, vitamins and sulfide, then sparge medium with 80% H₂ and 20% CO₂ gas mixture for 30–45 min to make it anoxic. Add and dissolve carbonates and sulfide while gassing the head space only. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Before use add vitamins from an anoxic stock solution sterilized by filtration. Adjust pH of the complete medium to 9.0.

 

Updated 1.02.2024