# 1310: BUTYRIVIBRIO M2 MEDIUM


## Main sol. 1310

10	g	Casitone (BD Bacto)	
2.5	g	Yeast extract (BD Bacto)	
2	g	D-Glucose	
2	g	Cellobiose	
2	g	Maltose	
5	g	Na-DL-lactate	
200	ml	**Clarified rumen fluid**	
0.05	g	K2HPO4	
0.05	g	KH2PO4	
0.1	g	(NH4)2SO4	
0.1	g	NaCl	
0.01	g	CaCl2 x 2 H2O	
0.01	g	MgSO4 x 7 H2O	
0.5	ml	Sodium resazurin (0.1% w/v)	
4	g	Na2CO3	
0.3	g	L-Cysteine HCl x H2O	
0.3	g	Na2S x 9 H2O	
800	ml	Distilled water	

1. Dissolve ingredients (except carbonate, cysteine and sulfide), boil medium for 1 min, then cool to room temperature under 100% CO2 gas atmosphere. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Add cysteine and sulfide form sterile anoxic stock solutions prepared under 100% N2 gas. Adjust pH of the complete medium to 6.5 - 6.8, 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.