# 322: METHANOSPHAERA MEDIUM


## Main sol. 322

100	ml	**Clarified rumen fluid**	
2	g	Trypticase peptone (BD BBL)	
2	g	Yeast extract	
0.5	g	Na-acetate	
0.5	g	Na-formate	
10	ml	**Modified Wolin's mineral solution**	
1.9	ml	Na2SeO4 (0.1% w/v)	
0.7	ml	NiCl2 x 6 H2O (0.1% w/v)	
3	ml	**FeSO4 x 7 H2O solution (0.1% w/v)**	
0.6	g	K2HPO4	
2.8	g	KH2PO4	
0.3	g	(NH4)2SO4	
1	g	NH4Cl	
0.6	g	NaCl	
0.15	g	MgSO4 x 7 H2O	
0.08	g	CaCl2 x 2 H2O	
0.5	ml	Sodium resazurin (0.1% w/v)	
4	g	NaHCO3	
10	ml	Methanol (50% v/v)	
2	ml	**Wolin's vitamin solution (10x)**	
0.5	g	DL-Dithiothreitol	
900	ml	Distilled water	

1. Dissolve ingredients except bicarbonate, methanol, and dithiothreitol (DTT), then sparge medium with 80% H2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add and dissolve bicarbonate, adjust pH of medium to 6.8 - 7.0, then 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. Add methanol (50% v/v solution), vitamins, and DTT from sterile anoxic stock solutions prepared under 100% N2 gas. Vitamins and DTT should be sterilized by filtration. Prior to use check pH of complete medium and adjust to 6.7 - 6.9, if necessary.
2. After inoculation add sterile 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.


## 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.


## Modified Wolin's mineral solution

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.18	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.18	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.02	g	AlK(SO4)2 x 12 H2O	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.3	mg	Na2SeO3 x 5 H2O	
0.4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.


## 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.