# 520: RUMINOCLOSTRIDIUM CELLULOLYTICUM (CM3) MEDIUM


## Main sol. 520

1.3	g	(NH4)2SO4	
1.5	g	KH2PO4	
2.9	g	K2HPO4 x 3 H2O	
1.25	ml	FeSO4 x 7 H2O (0.1% w/v in 0.1 N H2SO4)	
1	ml	**Trace element solution SL-10**	
2	g	Yeast extract	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.2	g	MgCl2 x 6 H2O	
75	mg	CaCl2 x 2 H2O	
6	g	Cellobiose	
10	g	Cellulose, MN 301 (optional)	
1.5	g	Na2CO3	
0.5	g	L-Cysteine HCl x H2O	
1000	ml	Distilled water	

1. Dissolve ingredients except magnesium chloride, calcium chloride, cellobiose, cysteine and carbonate, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add magnesium chloride, calcium chloride and cellobiose from anoxic stock solutions prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2. Cellobiose has to be sterilized by filtration. Prior to inoculation add cysteine from a sterile anoxic stock solution prepared under 100% N2 gas and adjust pH to 7.2.
2. Note: Some strains can be adapted to cellulose as substrate using 10.00 g/l cellulose powder MN 301 (MACHEREY-NAGEL).


## 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.