# 140: RUMINICLOSTRIDIUM MEDIUM


## Main sol. 140

0.5	g	KH2PO4	
1	g	NaCl	
0.5	g	(NH4)2SO4	
0.1	g	MgSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.5	g	K2HPO4	
300	ml	**Clarified rumen fluid**	
300	ml	**Sludge fluid**, alternative (optional)	
0.5	ml	Sodium resazurin (0.1% w/v)	
2.5	g	Na2CO3	
5	g	Cellobiose	
0.25	g	L-Cysteine HCl x H2O	
0.25	g	Na2S x 9 H2O	
700	ml	Distilled water	

1. Dissolve ingredients (except carbonate, cellobiose and reducing agents), bring medium to the boil, then cool to room temperature under 100% CO2 gas atmosphere. Use either clarified rumen fluid  or sludge fluid as supplement. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add cellobiose, sulfide and cysteine from sterile anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Cellobiose has to be sterilized by filtration. Adjust pH of complete medium to 6.8, if necessary.


## Sludge fluid

4	g	Yeast extract	
1000	ml	Sludge	

1. Add 0.4% yeast extract to sludge from an anaerobic digester, and after gassing with nitrogen gas for a few minutes incubate it at 37°C for 24 hours. Then centrifuge the sludge at 13000 g and autoclave the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at 8-12°C in the dark.


## 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.