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

FeCl3 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 24/05/2020