# 666a: CELLULOSILYTICUM MEDIUM


## Main sol. 666a

400	ml	**Clarified rumen fluid**	
0.23	g	K2HPO4	
0.23	g	KH2PO4	
0.45	g	NaCl	
0.45	g	(NH4)2SO4	
0.06	g	CaCl2 x 2 H2O	
0.09	g	MgSO4 x 7 H2O	
1	g	Agar	
5	mg	Indigocarmine	
6.4	g	NaHCO3	
2.5	g	Cellobiose	
5	g	Yeast extract	
0.3	g	L-Cysteine HCl x H2O	
0.3	g	DL-Dithiothreitol (DTT)	
600	ml	Distilled water	

1. Dissolve ingredients (except bicarbonate, cellobiose, yeast extract, cysteine and DTT), bring medium to the boil, then cool to room temperature under 100% CO2 gas atmosphere. Add the bicarbonate and equilibrate the medium with the CO2 gas to pH 6.8. Distribute under 100% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Thereafter, add cellobiose, yeast extract, cysteine and DTT from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Cellobiose has to be sterilzed by filtration. Adjust pH of complete medium to 6.7 - 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.