425. PROTEINIVORAX MEDIUM (PHM)

KH₂PO₄ 0.30 g

MgSO₄ × 7 H₂O 0.12 g

CaCl₂ × 2 H₂O 0.03 g

NaCl 20.0 g

Tryptone (BD Bacto) 3.0 g

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

Na₂CO₃ 60.0 g

NaHCO₃ 50.0 g

Na-Thioglycolate 1.0 g

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

Dissolve ingredients (except carbonates and thioglycolate), then sparge medium with 100% N₂ gas for 30 - 45 min to make it anoxic. Add carbonate and hydrogencarbonate, adjust pH to 8.5 – 9.0, dispense medium under 100% N₂ gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add thioglycolate from an anoxic stock solution prepared under 100% N₂ gas and sterilized by filtration.