Main sol. 318

Equipment needed: Filter Hungate tubes Autoclave Gassing station

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
KH₂PO₄	0.30	g	0.294	2.159
NaCl	0.60	g	0.588	10.056
MgCl₂ x 6 H₂O	0.10	g	0.098	0.482
CaCl₂ x 2 H₂O	0.08	g	0.078	0.533
Trace element solution	10.00	ml	-	-
NH₄Cl	1.00	g	0.979	18.311
Yeast extract (OXOID)	0.50	g	0.49	-
Trypticase peptone (BD BBL)	0.50	g	0.49	-
Sodium resazurin (0.1% w/v)	0.50	ml	4.9e-4	0.002
KHCO₃	2.00	g	1.959	19.566
Methanol (50% v/v)	10.00	ml	7.757	242.092
Wolin's vitamin solution (10x)	1.00	ml	-	-
L-Cysteine HCl x H₂O	0.30	g	0.294	1.673
Na₂S x 9 H₂O	0.30	g	0.294	1.223
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 to 30% of their volume and autoclave. Add vitamins (sterilized by filtration), methanol (50% v/v solution), 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