# 119a: METHANOBREVIBACTER MEDIUM


## Main sol. 119a

0.5	g	KH2PO4	
0.4	g	MgSO4 x 7 H2O	
0.4	g	NaCl	
0.4	g	NH4Cl	
0.05	g	CaCl2 x 2 H2O	
1	ml	**Trace element solution SL-10**	
2	ml	**FeSO4 x 7 H2O solution (0.1% w/v)**	
1	g	Yeast extract (OXOID)	
1	g	Na-acetate	
2	g	Na-formate	
200	ml	**Clarified rumen fluid**	
20	ml	**Fatty acid mixture**	
0.5	ml	Sodium resazurin (0.1% w/v)	
4	g	NaHCO3	
0.5	g	L-Cysteine HCl x H2O	
0.5	g	Na2S x 9 H2O	
780	ml	Distilled water	

1. Dissolve ingredients except bicarbonate, cysteine and sulfide. Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, then dispense medium under 80% H2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes to 30% of their volume and autoclave. Add cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. Prior to use check pH of complete medium and adjust to 6.8 - 7.0, if necessary.
2. After inoculation add 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.


## Clarified rumen fluid

1. Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and then stored frozen at -20°C.


## Fatty acid mixture

23	ml	Isobutyric acid	
27	ml	DL-2-Methylbutyric acid	
27	ml	Valeric acid	
27	ml	Isovaleric acid	
896	ml	Distilled water	

1. Adjust pH to 7.5 with concentrated NaOH.


## 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.