# 120b: METHANOMICROCOCCUS MEDIUM


## Main sol. 120b

0.35	g	K2HPO4	
0.23	g	KH2PO4	
0.5	g	NH4Cl	
0.5	g	MgSO4 x 7 H2O	
0.25	g	CaCl2 x 2 H2O	
2.25	g	NaCl	
2	ml	**FeSO4 x 7 H2O solution (0.1% w/v)**	
1	ml	**Trace element solution SL-10**	
2	g	Yeast extract (OXOID)	
2	g	Casitone (BD BBL)	
2.5	g	Na-acetate	
0.5	ml	Sodium resazurin (0.1% w/v)	
2.5	g	NaHCO3	
0.14	g	2-Mercaptoethanesulfonate (coenzyme M)	
20	ml	Methanol (50% v/v)	
1	ml	**Wolin's vitamin solution (10x)**	
0.36	g	L-Cysteine HCl x H2O	
0.36	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except bicarbonate, coenzyme M, vitamins, methanol, cysteine and sulfide) and sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Then add and dissolve bicarbonate, adjust pH to 6.5 and dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclave. Methanol (50% v/v stock solution) and the reducing agents are each autoclaved separately under 100% N2 gas atmosphere as concentrated solutions in tightly closed tubes. Vitamins and coenzyme M are prepared under 100% N2 gas atmosphere and sterilized by filtration. Appropriate volumes of the stock solutions are injected into the sterile medium with hypodermic syringes. Adjust pH of the complete medium to 7.0 - 7.2, if necessary.
2. After inoculation, pressurize culture vessels with sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.


## Trace element solution SL-10

10	ml	HCl (25%)	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
990	ml	Distilled water	

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water	


## FeSO4 x 7 H2O solution (0.1% w/v)

1	g	FeSO4 x 7 H2O	
1000	ml	H2SO4 (0.1 N)	

1. The ferrous sulfate solution is not stable and should be freshly prepared.