330. CLOSTRIDIUM CELLULOLYTICUM MEDIUM

(NH₄)₂SO₄ 1.3 g

KH₂PO₄ 1.5 g

K₂HPO₄ × 3 H₂O 2.9 g

FeSO₄ × 7 H₂O solution (0.1% w/v in 0.1 N H₂SO₄) 1.25 ml

Trace element solution SL-10 (see below) 1.0 ml

Yeast extract 2.0 g

Na-resazurin solution (0.1% w/v) 0.5 ml

MgCl₂ × 6 H₂O 0.2 g

CaCl₂ × 2 H₂O 75.0 mg

Cellobiose 6.0 g

L-Cysteine-HCl × H₂O 0.5 g

Na₂CO₃ 2.5 g

Distilled water 1000.0 ml

Trace element solution SL-10:

HCl (25%; 7.7 M) 10.0 ml

FeCl₂ × 4 H₂O 1.5 g

ZnCl₂ 70.0 mg

MnCl₂ × 4 H₂O 100.0 mg

H₃BO₃ 6.0 mg

CoCl₂ × 6 H₂O 190.0 mg

CuCl₂ × 2 H₂O 2.0 mg

NiCl₂ × 6 H₂O 24.0 mg

Na₂MoO₄ × 2 H₂O 36.0 mg

Distilled water 990.0 ml

Dissolve ingredients except magnesium chloride, calcium chloride, cellobiose, cysteine and carbonate, then sparge medium with 80% N₂ and 20% CO₂ gas mixture for 30 – 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes and autoclave. After autoclaving add magnesium chloride, calcium chloride and cellobiose from anoxic stock solutions prepared under 100% N₂ gas. Cellobiose has to be sterilized by filtration. Prior to inoculation add cysteine from a sterile anoxic stock solution prepared under 100% N₂ gas and adjust pH to 7.2 by adding a sterile anoxic stock solution of Na₂CO₃ (5% w/v) prepared under 80% N₂ and 20% CO₂ gas atmosphere.

 

Updated 07.07.2021