313. PYROCOCCUS MEDIUM

NaCl 13.85 g

MgSO₄ × 7 H₂O 3.5 g

MgCl₂ × 6 H₂O 2.75 g

KCl 0.33 g

NaBr 0.05 g

H₃BO₃ 15.00 g

SrCl₂ × 6 H₂O solution (0.1% w/v) 7.0 ml

NH₄SO₄ 10.0 mg

Citric acid solution (0.1% w/v) 5.0 ml

KI solution (0.01% w/v) 0.5 ml

CaCl₂ × 2 H₂O 0.75 g

KH₂PO₄ 0.5 g

NiCl₂ × 6 H₂O solution (0.1% w/v) 2.0 ml

Trace element solution (see below) 10.0 ml

Peptone (BD Bacto) 5.0 g

Yeast extract (OXOID) 1.0 g

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

Sulfur, powdered 30.0 g

Neutralized sulfide solution 16.7 ml

Distilled water 1000.0 ml

Neutralized sulfide solution:

Na₂S × 9 H₂O 3.0 g

Distilled water 100.0 ml

The sulfide solution is prepared in a 250 ml screw-capped bottle with a butyl rubber septum and a magnetic stirrer. The solution is bubbled with nitrogen gas, closed and autoclaved for 15 min. at 121°C. After cooling to room temperature the pH is adjusted to about 7.0 by adding of sterile 2 N H₂SO₄ drop-wise with a syringe without opening the bottle. Appearance of a yellow color indicates the drop of pH to about 8. The solution should be stirred continuously to avoid precipitation of elemental sulfur. The final solution should be clear and is yellow in color.

Trace element solution:

Nitrilotriacetic acid 1.5 g

MgSO₄ × 7 H₂O 3.0 g

MnSO₄ × H₂O 0.5 g

NaCl 1.0 g

FeSO₄ × 7 H₂O 0.1 g

CoSO₄ × 7 H₂O 0.18 g

CaCl₂ × 2 H₂O 0.1 g

ZnSO₄ × 7 H₂O 0.18 g

CuSO₄ × 5 H₂O 0.01 g

KAl(SO₄)₂ × 12 H₂O 0.02 g

H₃BO₃ 0.01 g

Na₂MoO₄ × 2 H₂O 0.01 g

NiCl₂ × 6 H₂O 0.03 g

Na₂SeO₃ × 5 H₂O 0.3 mg

Na₂WO₄ × 2 H₂O 0.4 mg

Distilled water 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 sulfur and sulfide, adjust the pH to 6.5, and sparge medium with 100% N2 gas for 30–45 min to make it anoxic. Distribute medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials that contain already the appropriate amount of sulfur. Sterilize medium by heating cultivation vessels in a boiling water bath for 1-2 hours on each of 3 successive days. Add neutralized sulfide stock solution.