492. CA. VULCANISAETA MOUTNOVSKIA MEDIUM (CVMM)

NH₄Cl 0.33 g

KCl 0.33 g

MgCl₂·x 2 H₂O 0.33 g

CaCl₂·x 6 H₂O 0.33 g

KH₂PO₄ 0.33 g

Na₂S·x 9 H₂O 0.5 g

Yeast extract 1.0 g

Trace element solution (see below) 1 ml

Vitamin solution (see below) 1 ml

Sodium sulfide 0.5 g

Resazurin 1.0 mg

Na₂SO₄ 1.0 g

So 10.0 g

Distilled water 1000 ml

Trace element solution:

(NH₄)₂Fe(SO₄)₂ x 6 H₂O (Mohr's salt) 784.0 mg

HCl (concentrated) 5.0 ml

CoCl₂ x H₂O 238.0 mg

(NH₄)₂Ni(SO₄)₂ x 6 H₂O 395.0 mg

Na₂MoO₄ x 2 H₂O 24.0 mg

Na₂WO₄ x 2 H₂O 33.0 mg

ZnSO₄ x 7 H₂O 144.0 mg

CuCl₂ x 2 H₂O 2.0 mg

Na₂SeO₄ 94.0 mg

H₃BO₃ 6.0 mg

MnCl₂ x 4 H₂O 99.0 mg

Distilled water 995.0 ml

Mohr's salt is dissolved firstly in concentrated HCl, and mixed with distilled water and other salts are dissolved in the sequence indicated.

Vitamin solution:

Biotin 20.0 mg

Folic acid 20.0 mg

Pyridoxine 0.1 mg

Riboflavin 50.0 mg

Pantotenoic acid 50.0 mg

p-Aminobenzoic acid 50.0 mg

Thiamine-HCl 50.0 mg

Nicotinic acid 50.0 mg

Vitamin B₁₂ 1.0 mg

Lipoic acid 50.0 mg

Distilled water 1000.0 ml

To adjust the pH of the medium to 4.8 3N HCl was added. The medium was dispensed in 5-ml portions into 17-ml Hungate tubes and the headspace was filled with CO₂. Dissolve ingredients except bicarbonate, vitamins, thioglycolate and ascorbic acid, then sparge medium with 100% N₂ gas for 30 – 45 min to make it anoxic. Add vitamins from anoxic stock solutions prepared under 100% N₂ gas and sterilized by filtration and bicarbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas mixture.

Updated: 10.01.2024