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DSMZ

DSMZ 1501 . METHYLOCALDUM MARINUM MEDIUM

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Equipment needed:
Final pH: n.d.
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ml
Compound Amount Unit
Solution 1 (10x NMS salts) 100.0 ml
NaCl
(2 %) 		900.0 ml
Solution 2 (Fe EDTA) 0.1 ml
Solution 3 (Sodium molybdate) 1.0 ml
Trace elements 1.0 ml
Agar , for solid medium 1.5 %
Phosphate buffer 10.0 ml
1 Prepare the growth medium as follows:
2 Dilute 100 ml solution 1 to 1 litre with 2% NaCl and then add 1 ml of solution 3, 1 ml of the trace elements, and 0.1 ml of solution 2. If solid media is required add 1.5% agar. Dispense the medium into the growth vessels. If using sealed vessels it is appropriate to add 25% methane to the gas phase and autoclave at 121C (15psi) for 15 minutes. Autoclave the phosphate buffer separately. When the growth medium is cool 10 ml/l of the phosphate buffer is added - if too warm the phosphate will precipitate. Liquid cultures should be grown with shaking.

Trace elements #

100
Compound Amount Unit
Na2-EDTA 25.0 mg
CuSO4 x 5 H2O 20.0 mg
FeSO4 x 7 H2O 50.0 mg
ZnSO4 x 7 H2O 40.0 mg
H3BO3 1.5 mg
CoCl2 x 6 H2O 5.0 mg
MnCl2 x 4 H2O 2.0 mg
NiCl2 x 6 H2O 1.0 mg
Distilled water 100.0 ml
1 May be stored at 4°C in the dark

Phosphate buffer #

1000
Compound Amount Unit
Na2HPO4 x 12 H2O 71.6 g
KH2PO4 26.0 g
Distilled water 1000.0 ml
1 Dissolve in about 800 ml of water, adjust the pH to 6.8 and make up to 1 litre.

Solution 1 (10x NMS salts) #

1000
Compound Amount Unit
KNO3 10 g
MgSO4 x 6 H2O 10 g
CaCl2 x 2 H2O 2 g
Distilled water 1000 ml
1 Dissolve the ingredients listed above (in that order) in about 700 ml of 2% NaCl, and then make up to 1 litre with 2% NaCl.

Solution 2 (Fe EDTA) #

100
Compound Amount Unit
Fe(III)-EDTA 3.8 g
Distilled water 100.0 ml

Solution 3 (Sodium molybdate) #

1000
Compound Amount Unit
Na2MoO4 x 2 H2O 0.26 g
Distilled water 1000.00 ml