422. THIOHALORHABDUS DENITRIFICANS MEDIUM (TRDM)

NaCl 180.0 g

K₂HPO₄ 1.50 g

NH₄Cl 0.50 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 and ammonium chloride, then sparge solution with 80% N₂ and 20% CO₂ gas mixture for at least 30 – 45 min to remove dissolved oxygen and to saturate the solution with CO₂. Dispense solution in vials suitable for anaerobic cultures (e.g. Balch-type tubes) to 30% volume under 80% N₂ and 20% CO₂ gas atmosphere. Close vials with butyl rubber septa and autoclave. Add trace elements, calcium chloride, magnesium sulfate, thiosulfate, bicarbonate and vitamins from sterile stock solutions and adjust pH of the medium to 7.5 - 7.8 using a sterile stock solution of sodium carbonate (5% w/v). After inoculation, add sterile air (with hypodermic needle through the rubber closure) to a concentration of ca. 4% (v/v) O₂ (e.g., add 7 ml of air to a Balch-type tube with a total volume of 27 ml).