317. ORNATILINEA MEDIUM

KH₂PO₄ 0.14 g

MgCl₂ × 6 H₂O 0.2 g

CaCl₂ × 2 H₂O 0.15 g

NH₄Cl 0.54 g

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

Selenite-tungstate solution (see below) 1.0 ml

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

NaHCO₃ 2.5 g

Yeast extract 2.3 g

Vitamins solution (see below) 10.0 ml

D-Glucose 2.2 g

L-Cysteine-HCl × H₂O 0.25 g

Na₂S × 9 H₂O 0.25 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.

Selenite-tungstate solution:

NaOH 0.5 g

Na₂SeO₃ × 5 H₂O 3 mg

Na₂WO₄ × 2 H₂O 4 mg

Distilled water 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 bicarbonate, yeast extract, vitamins, glucose and reducing agents), then sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30–45 min to make it anoxic. Add solid bicarbonate and adjust pH to 7.0. Dispense medium under 80% N₂ and 20% CO₂ gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add yeast extract, vitamins (sterilized by filtration), glucose, sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N₂ gas. Adjust pH of complete medium to 7.0, if necessary.

 

Updated 1.02.2024