331. THERMOCOCCUS STETTERI MEDIUM

NH4Cl 0.33 g

CaCl2 2 H2O 0.33 g

MgCl2 6 H2O 0.33 g

KCl 0.33 g

KH2PO4 0.33 g

Casitone 5.0 g

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

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

Sulfur, powder 10.0 g

NaCl 2.5 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 ingredients (except sulfur, vitamins and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30-45 min to make it anoxic. Adjust pH to 5.7, dispense under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials containing already the appropriate amount of sulfur and sterilize by heating for 2-3 hours in a boiling water bath. Add vitamins (sterilized by filtration) and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to use adjust pH of complete medium to 6.5 with sterile, anaerobic NaHCO3 solution.

 

Updated 08.07.2021