387. NATRANAEROBIUS MEDIUM

KH₂PO₄ 0.2 g

MgCl₂ × 6 H₂O 0.1 g

KCL 0.2 g

NH₄Cl 0.5 g

NaCl 100 g

Yeast extract (Difco) 6.0 g

Tryptone (Difco) 6.0 g

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

Vitamin solution (see below) 10 mL

Na₂CO₃ 68.0 g

NaHCO₃ 38.0 g

L-Cysteine-HCl × H₂O 0.7 g

Glucose 5.0 g

Distilled water to 1000.0 mL

pH 10.5 (measured at 60^oC)

 

Dissolve ingredients (except carbonates, cysteine, vitamins and sucrose), then sparge medium with 100% N₂ gas for 30 – 45 min to make it anoxic. Add carbonate, bicarbonate and cysteine and adjust pH to 10.5. Dispense medium under 100% N₂ gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add vitamins and glucose from sterile anoxic stock solutions prepared under 100% N₂ gas and sterilized by filtration.

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 (B₂) 0.1 mg

Riboflavin (B₁) 5.0 mg

Pantotenoic acid 5.0 mg

p-Aminobenzoic acid 5.0 mg

Thiamine-HCl 5.0 mg

Nicotinic acid 5.0 mg

Cyanocobalamin (B₁₂) 0.1 mg

Lipoic acid 5.0 mg

Distilled water 1000.0 ml

pH 7.2-7.4.

 

Updated 15.07.2021