# J355: METHANOSPHAERA MEDIUM


## Main sol. J355

100	ml	Rumen fluid (see below),  clarified	
2	g	Yeast extract (BD-Difco)	
2	g	Trypticase peptone (BD-BBL)	
0.5	g	Sodium acetate	
0.5	g	Sodium formate	
10	ml	**Trace minerals**	
1	ml	**Trace vitamins**	
0.19	mg	Na2SeO4	
0.7	mg	NiCl2 x 6 H2O	
3	mg	FeSO4 x 7 H2O	
2.8	g	KH2PO4	
0.6	g	K2HPO4	
0.3	g	(NH4)2SO4	
1	g	NH4Cl	
0.6	g	NaCl	
0.15	g	MgSO4 x 7 H2O	
0.076	g	CaCl2 x 2 H2O	
5	ml	Methanol	
1	mg	Resazurin	
0.875	g	L-Cysteine HCl x H2O	
0.375	g	Na2S x 9 H2O	
900	ml	Distilled water	

1. Mix ingredients, except methanol, L-cysteine x HCl x H2O and Na2S x 9H2O, and adjust pH to 7.0. Bring to a boil for 5-10 sec, then, cool down under a stream of N2 and add methanol to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in a 100-ml serum bottle) under a stream of N2 and seal. After autoclaving, stand the medium overnight. Separately autoclave L-cysteine x HCl x H2O and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, replace the gas phase with H2-CO2 (80:20, v/v), and add the L-cysteine x HCl x H2O and Na2S x 9H2O solutions aseptically and anaerobically. Pressurize the inoculated bottles to 200 kPa H2-CO2 (80:20, v/v). To prepare the clarified rumen fluid, preheat rumen content at 120C for 15 min. Use the supernatant after centrifuging at 25,000 x g for 15 min.


## Trace minerals

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x n H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.1	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.01	g	AlK(SO4)2	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH solution. Then proceed to add minerals. Adjust final pH to 7.0.


## Trace vitamins

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water