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 01.02.2024