# 523: CLOSTRIDIUM CHARTATABIDUM MEDIUM


## Main sol. 523

38	ml	**Mineral solution**	
200	ml	**Clarified rumen fluid**	
0.3	g	K2HPO4	
2	g	Trypticase peptone (BD BBL)	
0.5	g	Yeast extract (OXOID)	
2	ml	**Haemin solution** (0.05% w/v)	
0.5	ml	Sodium resazurin (0.1% w/v)	
4	g	Na2CO3	
5	g	Cellobiose	
0.25	g	L-Cysteine HCl x H2O	
0.25	g	Na2S x 9 H2O	
760	ml	Distilled water	

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


## Mineral solution

6	g	KH2PO4	
12	g	NaCl	
6	g	(NH4)2SO4	
1.6	g	CaCl2 x 2 H2O	
2.5	g	MgSO4 x 7 H2O	
1000	ml	Distilled water	


## 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.


## Haemin solution

50	mg	Haemin	
1	ml	NaOH (1 N)	
100	ml	Distilled water	

1. Dissolve 50 mg haemin in 1 ml 1 N NaOH; make up to 100 ml with distilled water and filter sterilize. Store refrigerated.