245. THERMINCOLA MEDIUM

NH₄Cl 1.0 g

MgCl₂ × 6 H₂O 0.33 g

CaCl₂ × 2 H₂O 0.1 g

KCl 0.33 g

KH₂PO₄ 0.5 g

Resazurin 0.5 mg

Wolfe's mineral elixir (see below) 1.0 ml

Vitamin solution (see below) 20.0 ml

NaHCO₃ 0.5 g

Na₂CO₃ 0.5 g

Na-acetate × 3 H₂O 0.2 g

Yeast extract 0.2 g

Na₂S × 9 H₂O 1.0 g

Distilled water 1000.0 ml

Wolfe's mineral elixir:

MgSO₄ × 7 H₂O 30.0 g

MnSO₄ × H₂O 5.0 g

NaCl 10.0 g

FeSO₄ × 7 H₂O 1.0 g

CoCl₂ × 6 H₂O 1.8 g

CaCl₂ × 2 H₂O 1.0 g

ZnSO₄ × 7 H₂O 1.8 g

CuSO₄ × 5 H₂O 0.1 g

KAl(SO₄)₂ × 12 H₂O 0.18 g

H₃BO₃ 0.1 g

Na₂MoO₄ × 2 H₂O 0.1 g

(NH₄)₂Ni(SO₄)₂ × 6 H₂O 2.8 g

Na₂WO₄ × 2 H₂O 0.1 g

Na₂SeO₄ 0.1 g

Distilled water 1000.0 ml

First adjust pH to 1.0 with diluted H₂SO₄, then add and dissolve the salts.

Vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine (B₂) 10.0 mg

Riboflavin (B₁) 5.0 mg

Pantotenoic acid 5.0 mg

p-Aminobenzoic acid 5.0 mg

Thiamine-HCl 5.0 mg

Nicotinic acid 5.0 mg

Vitamin B₁₂ 0.1 mg

Lipoic acid 5.0 mg

Distilled water 1000.0 ml

Dissolve ingredients except carbonates, vitamin solution, yeast extract and sulfide, boil medium for 1 min, then cool to room temperature under N₂ gas atmosphere. Add carbonates and sulfide to the medium, dispense under CO gas atmosphere in culture vessels (e.g., 10.0 ml medium in 50 ml serum bottles) and autoclave at 121°C 15 min. Prior to inoculation add yeast extract from a sterile, anoxic stock solution prepared under N₂ and adjust pH of final medium to 8.0 with a sterile, anoxic solution of 1 N HCl (this solutions autoclave at 121°C 15 min). Add filter-sterilized vitamin solution.

 

Updated 1.02.2024