# 320: CLOSTRIDIUM CELLULOVORANS MEDIUM


## Main sol. 320

1	g	K2HPO4 x 3 H2O	
1	g	NH4Cl	
0.5	g	KCl	
0.5	g	MgSO4 x 7 H2O	
0.5	g	Trypticase peptone (BD BBL)	
0.5	g	Yeast extract	
20	ml	**Clarified rumen fluid**	
20	ml	**Sludge fluid**, alternative (optional)	
1	ml	**Trace element solution SL-10**	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.15	g	L-Cysteine HCl x H2O	
1	g	Na2CO3	
5	g	Cellobiose	
10	g	Cellulose, MN 301 (optional)	
0.15	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except cysteine, carbonate, cellobiose and sulfide), bring medium to the boil, then cool to room temperature under 80% N2 and 20% CO2 gas mixture and add cysteine. Dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Add sulfide from a sterile anoxic stock solution prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Sterilize cellobiose separately by filtration under 100% N2 gas. Adjust pH of the complete medium to 7.0, if necessary.
2. Note: Some strains can be adapted to cellulose as substrate using 10.0 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.


## 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.