312. THERMOTOGA NEAPOLITANA MEDIUM

Starch, soluble 5.0 g

KH₂PO₄ 0.5 g

Trace element solution (see below) 15.0 ml

NiCl₂ × 6 H₂O solution (0.1% w/v) 2.0 ml

Sea water (see below) 250.0 ml

Yeast extract 2.0 g

Na-resazurin solution (0.1% w/v) 0.5 ml

L-Cysteine-HCl × H₂O 0.5 g

Na₂S × 9 H₂O 0.5 g

Distilled water 750.0 ml

Trace element solution:

Nitrilotriacetic acid 1.5 g

MgSO₄ × 7 H₂O 3.0 g

MnSO₄ × H₂O 0.5 g

NaCl 1.0 g

FeSO₄ × 7 H₂O 0.1 g

CoSO₄ × 7 H₂O 0.18 g

CaCl₂ × 2 H₂O 0.1 g

ZnSO₄ × 7 H₂O 0.18 g

CuSO₄ × 5 H₂O 0.01 g

KAl(SO₄)₂ × 12 H₂O 0.02 g

H₃BO₃ 0.01 g

Na₂MoO₄ × 2 H₂O 0.01 g

NiCl₂ × 6 H₂O 0.03 g

Na₂SeO₃ × 5 H₂O 0.3 mg

Na₂WO₄ × 2 H₂O 0.4 mg

Distilled water 1000.0 ml

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.

Artificial sea water:

NaCl 27.7 g

MgSO₄ × 7 H₂O 7.0 g

MgCl₂ × 6 H₂O 5.5 g

KCl 0.65 g

NaBr 0.1 g

H₃BO₃ 30.0 mg

SrCl₂ × 6 H₂O 15.0 mg

Citric acid 10.0 mg

KI 0.05 mg

CaCl₂ × 2 H₂O 2.25 g

Distilled water 1000.0 ml

Dissolve ingredients (except sulfide and cysteine) and adjust pH to 6.5. Boil medium for 1 min, then cool to room temperature under 100% N₂ gas atmosphere. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of volume and autoclave. Add sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N₂ gas. Adjust pH of complete medium to 6.5, if necessary.

 

Updated 1.02.2024