339. ACETOBACTERIUM MEDIUM

NH₄Cl 1.0 g

KH₂PO₄ 0.33 g

K₂HPO₄ 0.45 g

MgSO₄ × 7 H₂O 0.1 g

Trace element solution (see below) 20.0 ml

Yeast extract 2.0 g

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

NaHCO₃ 10.0 g

D-Fructose 10.0 g

Vitamin solution (see below) 10.0 ml

L-Cysteine-HCl × H₂O 0.5 g

Na₂S × 9 H₂O 0.5 g

Distilled water 1000.0 ml

Trace element solution:

Nitrilotriacetic acid 1.5 g

MgSO₄ × 7 H₂O 3.0 g

MnSO₄ × H₂O 0.5 g

NaCl 1.0 g

FeSO₄ × 7 H₂O 0.1 g

CoSO₄ × 7 H₂O 0.18 g

CaCl₂ × 2 H₂O 0.1 g

ZnSO₄ × 7 H₂O 0.18 g

CuSO₄ × 5 H₂O 0.01 g

KAl(SO₄)₂ × 12 H₂O 0.02 g

H₃BO₃ 0.01 g

Na₂MoO₄ × 2 H₂O 0.01 g

NiCl₂ × 6 H₂O 0.03 g

Na₂SeO₃ × 5 H₂O 0.3 mg

Na₂WO₄ × 2 H₂O 0.4 mg

Distilled water 1000.0 ml

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.

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, fructose, vitamins, cysteine and sulfide, bring to the boil and cool to room temperature under 80% N₂ and 20% CO₂ gas mixture. Add bicarbonate (solid) and equilibrate the medium with the gas until a pH of around 7.4 is reached. Then distribute under the same gas atmosphere in anoxic Hungate-type tubes or serum vials and autoclave. Before use adjust the pH to 8.0 - 8.2 by adding a sterile anoxic stock solution of sodium carbonate (5% w/v) prepared under 80% N₂ and 20% CO₂ gas mixture (0.25 ml per 10.0 ml medium) and add fructose, vitamins (sterilized by filtration), cysteine and sulfide from anoxic sterile stock solutions prepared under 100% N₂.

Updated 11.08.2021