314. NATRANAEROBACULUM MEDIUM

KH2PO4 0.2 g

MgCl2 6 H2O 0.1 g

NH4Cl 1.0 g

KCl 0.2 g

NaCl 16.0 g

Trace element solution SL-11 (see below) 1.0 ml

Yeast extract 0.2 g

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

Na2CO3 68.0 g

NaHCO3 38.0 g

Ethanol 5.0 ml

Vitamin solution (see below) 10.0 ml

Na2S 9 H2O 1.0 g

Distilled water 1000.0 ml

Trace element solution SL-11:

Na2-EDTA 2 H2O 5.2 g

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 1000.0 ml

Dissolve EDTA in 800.0 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000.0 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 ingredients except carbonate, bicarbonate, ethanol, vitamins and sulfide. Sparge medium with 100% N2 gas for 3045 min to make it anoxic. Add and dissolve carbonates and sulfide while gassing the head space only. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Before use add ethanol and vitamins from sterile anoxic stock solution prepared under 100% N2 gas. Vitamins are sterilized by filtration. Adjust pH of the complete medium to 9.510.0, if necessary.

Updated 11.08.2021