DSMZ 924b . METHANOSARCINA MB MEDIUM (1 g/l NaCl)

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* Medium for Methanosarcina mazei DSM 27785

Omit formate and acetate and supplement medium with 4.00 ml/l methanol added from a sterile anoxic stock solution.


Growth conditions: 2-3 days anaerobic 37 °C
Equipment needed: Autoclave Filter Gassing station Hungate tubes

Final gas composition: 80 % N2 20 % CO2 read more

Final pH: 7.2

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ml
Compound Amount Unit
NaCl 1.00 g
MgCl2 x 6 H2O 1.00 g
KCl 0.50 g
CaCl2 x 2 H2O 0.40 g
K2HPO4 0.40 g
NH4Cl 1.00 g
Modified Wolin's mineral solution 10.00 ml
Yeast extract
(OXOID)
2.00 g
Trypticase peptone
(BD BBL)
2.00 g
Sodium resazurin
(0.1% w/v)
0.50 ml
NaHCO3 4.00 g
Na-formate* 6.80 g
Na-acetate* 1.60 g
Wolin's vitamin solution (10x) 1.00 ml
L-Cysteine HCl x H2O 0.25 g
Na2S x 9 H2O 0.25 g
Methanol 4.00 ml
Distilled water 1000.00 ml
1 Dissolve ingredients (except bicarbonate, formate, acetate vitamins, and reducing agents), then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add solid bicarbonate, adjust pH to 6.8 - 7.0, dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add formate, acetate, vitamins (sterilized by filtration), cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The pH of the complete medium should be 7.2.

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.