456. INFIRMIFILUM MEDIUM (IM)

KH₂PO₄ 0.165 g

NH₄Cl 0.165 g

KCl 0.165 g

MgCl₂ × 6H₂O 0.165 g

CaCl₂ × 6H₂O, 0.06 g

Trace element solution (see below) 1.0 ml

Vitamin solution (see below) 1.0 ml

Na-resazurin solution (0.1% w/v) 0.50 ml

Yeast extract 0.05 g

Na₂S × 9H₂O 0.3 g

D-Glucose 1.0 g

Trace element solution:

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 10.0 mg

Riboflavin 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

The medium is prepared anaerobically. Vitamins, Na₂S × 9H₂O are added after boiling and cooling of the medium under 100% CO₂ in gas phase. Adjust the pH between 6.0 and 6.5 with sterile HCl or NaOH after autoclaving. Yeast extract must be added to the final solution before inoculation.