419. THIOHALOSPIRA HALOPHILA MEDIUM (T HM)

NaCl 233.0 g

K₂HPO₄ 1.50 g

NH₄Cl 0.50 g

Trace element solution (see below) 1.0 ml

CaCl₂ × 2 H₂O 0.05 g

MgCl₂ × 6 H₂O 0.4 g

Na₂S₂O₃ × 5 H₂O 5.0 g

NaHCO₃ 2.50 g

Seven vitamins solution (see below) 1.0 ml

Distilled water 1000.0 ml

Trace elements 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 × 6H₂O 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

Ca-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 under air atmosphere in Erlenmeyer flasks to 10% of volume, close with screw caps and autoclave. Add trace elements, calcium chloride, magnesium chloride, thiosulfate, bicarbonate and vitamins from sterile stock solutions and adjust pH of the medium to 7.0-7.2, if necessary. Incubate with shaking.