434. TENIUFILUM THALASSICUM MEDIUM (TTM)

KH₂PO4 0.33 g

NH₄Cl 0.33 g

KCl 0.33 g

NaCl 10.0 g

MgCl₂ × 6 H₂O 0.33 g

CaCl₂ × 6 H₂O 0.033 g

NaHCO₃ 2.0 g

Yeast extract 0.05 g

Tryptone 2.0 g

Fructose 2.0 g

Resazurin 1.0 mg

Trace element solution (see below) 10.0 ml

Vitamin solution (see below) 10.0 ml

Na₂S × 9 H₂O 0.50 g

Distilled water 1000.0 ml

Trace element solution:

Nitrilotriacetic acid 1.50 g

MgSO₄ × 7 H₂O 3.0 g

MnSO₄ × H₂O 0.50 g

NaCl 1.0 g

FeSO₄ × 7 H₂O 0.10 g

CoSO₄ × 7 H₂O 0.18 g

CaCl₂ × 2 H₂O 0.10 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 0.30 mg

Na₂WO₄ × 2 H₂O 0.40 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.

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 carbonate, and sulfide), adjust pH to 8.0, boil for 10 min. and sparge medium with 100% N₂ gas for 30 min to make it anoxic. Distribute under N₂ into Hungate tubes (10 ml) and autoclave. Add sulfide from a sterile anoxic stock solution prepared under 100% N₂ gas and carbonate from a sterile stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Filter sterilize vitamin solution and aseptically add 100 μl per Hungate tube. Adjust pH of the complete medium to 8.0, if necessary.