363. ANOXYBACILLUS MEDIUM

KH₂PO₄ 0.2 g

MgCl₂ × 6 H₂O 0.1 g

KCl 0.2 g

NH₄Cl 1.0 g

NaCl 5.0 g

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

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

Na₂CO₃ 2.76 g

NaHCO₃ 10.0 g

Yeast extract 0.5 g

D-Glucose 5.0 g

Vitamin solution (see below) 10.0 ml

Na₂S × 9 H₂O 0.5 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

Vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine-HCl 10.0 mg

Thiamine-HCl × 2 H₂O 5.0 mg

Riboflavin 5.0 mg

Nicotinic acid 5.0 mg

D-Ca-pantothenate 5.0 mg

Vitamin B₁₂ 0.1 mg

p-Aminobenzoic acid 5.0 mg

Lipoic acid 5.0 mg

Distilled water 1000.0 ml

Dissolve all ingredients except carbonates, yeast extract, glucose, vitamins and sulfide, then sparge medium with 100% N₂ gas for 30 – 45 min to make it anoxic. Add carbonate and hydrogencarbonate, adjust pH to 9.0 - 9.5, dispense medium under 100% N₂ gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add yeast extract, glucose, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N₂ gas. Vitamins should be sterilized by filtration. Adjust pH of complete medium to 9.5 – 9.7.

 

Updated 11.07.2021