421. THIOHALOMONAS DENITRIFICANS MEDIUM (TMDM)

NaCl 120.0 g

K₂HPO₄ 1.50 g

NH₄Cl 0.50 g

KNO₃ 1.0 g

Trace element solution (see below) 1.0 ml

CaCl₂ × 2 H₂O 0.05 g

MgSO₄ × 7 H₂O 0.50 g

Na₂S₂O₃ × 5 H₂O 5.0 g

NaHCO₃ 5.0 g

Seven vitamins solution (see below) 1.0 ml

Trace element solution (Pfennig & Lippert,1966)

EDTA 5.0 g

FeSO₄ × 7 H₂O 2.2 g

ZnSO₄ × 7 H₂O 0.1 g

MnCl₂ × 4 H₂O 0.03 g

H₃BO₃ 0.03 g

CoCl₂ × 6 H₂O 0.2 g

CuCl₂ × 2 H₂O 0.03 g

NiCl₂ × 6 H₂O 0.03 g

Na₂MoO₄ × 2 H₂O 0.03 g

Distilled water 1000.0 ml

pH 3.0-4.0

Seven vitamins solution:

Vitamin B₁₂ 100.0 mg

p-Aminobenzoic acid 80.0 mg

D(+)-Biotin 20.0 mg

Nicotinic acid 200.0 mg

Calcium pantothenate 100.0 mg

Pyridoxine HCl 300.0 mg

Thiamine-HCl × 2 H₂O 200.0 mg

Distilled water 1000.0 ml

Dissolve sodium chloride, potassium hydrogenphosphate, ammonium chloride and potassium nitrate, then sparge solution with 80% N₂ and 20% CO₂ gas mixture for at least 30 – 45 min to remove dissolved oxygen. Dispense solution under same gas atmosphere in vials suitable for anaerobic cultures (e.g. Balch-type tubes) to 50% volume, close vials with butyl rubber septa and autoclave. Add trace elements, calcium chloride, magnesium sulfate, thiosulfate, and vitamins from sterile anoxic stock solutions prepared under 100% N₂ gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Vitamins should be sterilized by filtration. Adjust pH of the complete medium to 7.5 - 7.8 using a sterile anoxic stock solution of sodium carbonate (5% w/v) prepared under 80% N₂ and 20% CO₂ gas atmosphere.