444. ISACHENKONIA MEDIUM

NH₄Cl 0.5 g

NaCl 20.0 g

CaCl₂ × 6 H₂O 0.033 g

KH₂PO₄ 0.2 g

MgCl₂ × 6 H₂O 0.1 g

Na₂CO₃ 50.0 g

NaHCO₃ 60.0 g

Glucose 3.0 g

Yeast extract 0.05 g

Trace element solution (see below) 1 ml

Vitamin solution (see below) 10 ml

Na₂S × 9 H₂O 3.0 g

pH 9.2

Trace element solution:

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

HCl (concentrated) 5.0 ml

CoCl₂ × H₂O 238.0 mg

(NH₄)₂Ni(SO₄)₂ × 6 H₂O 395.0 mg

Na₂MoO₄ × 2 H₂O 24.0 mg

Na₂WO₄ × 2 H₂O 33.0 mg

ZnSO₄ × 7 H₂O 144.0 mg

CuCl₂ × 2 H₂O 2.0 mg

Na₂SeO₄ 94.0 mg

H₃BO₃ 6.0 mg

MnCl₂ × 4 H₂O 99.0 mg

Distilled water 995.0 ml

Mohr's salt is dissolved firstly in concentrated HCl. Then it is 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 100 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

The medium is prepared anaerobically. NaHCO₃, Na₂CO₃ and vitamins, Na₂S × 9 H₂O are added after boiling and cooling of the medium under 100% CO₂ in gas phase. Adjust the pH between 9.0 and 9.2 with sterile NaOH after autoclaving. Yeast extract must be added to the final solution before inoculation.