# 734a: METHANOBREVIBACTER CUTICULARIS MEDIUM


## Main sol. 734a

20	ml	**Clarified rumen fluid**	
1	g	NaCl	
0.5	g	KCl	
0.4	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.3	g	NH4Cl	
0.2	g	KH2PO4	
0.15	g	Na2SO4	
0.5	g	Casamino acids (BD Bacto)	
0.5	g	Yeast extract (OXOID)	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
2	g	Na2CO3	
2.1	g	MOPS (SIGMA)	
1	ml	**Seven vitamins solution**	
0.16	g	DL-Dithiothreitol (DTT)	
980	ml	Distilled water	

1. Dissolve ingredients (except carbonate, MOPS buffer, vitamins and DTT), bring medium to the boil, then cool to room temperature under 80% H2 and 20% CO2 gas mixture. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add MOPS buffer adjusted to pH 7.7, vitamins and DTT from anoxic stock solutions prepared under 100% N2 gas and sterilized by filtration and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH of complete medium to 7.7, if necessary.


## 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.


## 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.


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Seven vitamins solution

100	mg	Vitamin B12	
80	mg	p-Aminobenzoic acid	
20	mg	D-(+)-biotin	
200	mg	Nicotinic acid	
100	mg	Calcium pantothenate	
300	mg	Pyridoxine hydrochloride	
200	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water