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Main sol. 318

Internal Solution-ID: S588

Equipment needed: Filter Hungate tubes Autoclave Gassing station
Compound Amount Unit Conc. [g/L] Conc. [mM]
KH2PO4 0.30 g 0.295 2.17
NaCl 0.60 g 0.591 10.105
MgCl2 x 6 H2O 0.10 g 0.098 0.484
CaCl2 x 2 H2O 0.08 g 0.079 0.536
Trace element solution 10.00 ml - -
NH4Cl 1.00 g 0.984 18.401
Yeast extract
(OXOID)
0.50 g 0.492 -
Trypticase peptone
(BD BBL)
0.50 g 0.492 -
Sodium resazurin
(0.1% w/v)
0.50 ml 4.9e-4 0.002
KHCO3 2.00 g 1.969 19.662
Methanol 5.00 ml 3.898 121.642
Wolin's vitamin solution (10x) 1.00 ml - -
L-Cysteine HCl x H2O 0.30 g 0.295 1.681
Na2S x 9 H2O 0.30 g 0.295 1.229
Distilled water 1000.00 ml - -
1 Dissolve ingredients except bicarbonate, vitamins, methanol, cysteine and sulfide, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add vitamins (sterilized by filtration), methanol, cysteine 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 6.8, if necessary.
318 METHANOSARCINA THERMOPHILA (BCYT) MEDIUM