432. PYROBACULUM FERRIREDUCENS MEDIUM (PFM)

NH₄Cl 0.33 g

KCl 0.33 g

KH₂PO₄ 0.33 g

MgCl₂ × 6 H₂O 0.33 g

CaCl₂ × 2 H₂O 0.33 g

Trace element solution SL-10 (see below) 1.0 ml

Selenite-tungstate solution (see below) 1.0 ml

Yeast extract 1.0 g

KNO₃ 1.0 g

NaHCO₃ 2.0 g

Wolin's vitamin solution (see below) 10.0 ml

Distilled water 1000.0 ml

Trace element solution SL-10

HCl (25%; 7.7 M) 10.0 ml

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 990.0 ml

Selenite-tungstate solution:

NaOH 0.5 g

Na₂SeO₃ × 5 H₂O 3.0 mg

Na₂WO₄ × 2 H₂O 4.0 mg

Distilled water 1000.0 ml

Wolin’s vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine-HCl 10.0 mg

Thiamine-HCl 5.0 mg

Riboflavin 5.0 mg

Nicotinic acid 5.0 mg

Ca-D-pantothenate 5.0 mg

Vitamin B₁₂ 0.10 mg

p-Aminobenzoic acid 5.0 mg

(±)-α-Lipoic acid 5.0 mg

Distilled water 1000.0 ml

Dissolve ingredients (except yeast extract, nitrate, bicarbonate and vitamins) and sparge medium with 100% CO₂ gas for at least 30 – 45 min to make it anoxic, then dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add yeast extract, nitrate 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 atmosphere. Vitamins should be sterilized by filtration. The pH of the complete medium should be adjusted to 6.5 – 6.8, if necessary.