DSMZ 377 . PYROCOCCUS MEDIUM

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Equipment needed: Autoclave Gassing station Hungate tubes

Final gas composition: 100 % N2 read more

Final pH: 6.5

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ml
Compound Amount Unit
NaCl 13.85 g
MgSO4 x 7 H2O 3.50 g
MgCl2 x 6 H2O 2.75 g
KCl 0.33 g
NaBr 0.05 g
H3BO3 15.00 mg
SrCl2 x 6 H2O
(0.1% w/v)
7.00 ml
(NH4)2SO4 10.00 mg
Citric acid
(0.1% w/v)
5.00 ml
KI
(0.01% w/v)
0.50 ml
CaCl2 x 2 H2O 0.75 g
KH2PO4 0.50 g
NiCl2 x 6 H2O
(0.1% w/v)
2.00 ml
Modified Wolin's mineral solution 10.00 ml
Sodium resazurin
(0.1% w/v)
0.50 ml
Sulfur
(powdered)
30.00 g
Peptone
(BD Bacto)
5.00 g
Yeast extract
(OXOID)
1.00 g
Neutralized sulfide solution 3% (w/v) 15.00 ml
Distilled water 960.00 ml
1 Dissolve ingredients except for sulfur, peptone, yeast extract, 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 the same gas atmosphere into anoxic Hungate-type tubes or serum vials that already contain the appropriate amount of sulfur. Sterilize the medium by autoclaving at 110°C for 20 min. After sterilization, add peptone and yeast extract from sterile stock solutions prepared under 100% N2 gas atmosphere and sulfide from a neutralized stock solution.

Modified Wolin's mineral solution #

1000
Compound Amount Unit
Nitrilotriacetic acid 1.50 g
MgSO4 x 7 H2O 3.00 g
MnSO4 x H2O 0.50 g
NaCl 1.00 g
FeSO4 x 7 H2O 0.10 g
CoSO4 x 7 H2O 0.18 g
CaCl2 x 2 H2O 0.10 g
ZnSO4 x 7 H2O 0.18 g
CuSO4 x 5 H2O 0.01 g
AlK(SO4)2 x 12 H2O 0.02 g
H3BO3 0.01 g
Na2MoO4 x 2 H2O 0.01 g
NiCl2 x 6 H2O 0.03 g
Na2SeO3 x 5 H2O 0.30 mg
Na2WO4 x 2 H2O 0.40 mg
Distilled water 1000.00 ml
1 First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.

Neutralized sulfide solution 3% (w/v) #

100
Compound Amount Unit
Na2S x 9 H2O 3 g
Distilled water 100 ml
1 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 M H2SO4 drop-wise with a syringe without opening the bottle.