# 275: TREPONEMA SUCCINIFACIENS MEDIUM


## Main sol. 275

920	ml	**Solution A**	
50	ml	**Solution B**	
30	ml	**Solution C**	

1. Dissolve ingredients of solution A and sparge with 80% N2 and 20% CO2 gas atmosphere to make it anoxic. Then distribute medium under the same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions B is prepared under 100% N2 gas atmosphere and autoclaved. Solution C is prepared under 80% N2 and 20% CO2 gas atmosphere and autoclaved. To complete the medium appropriate amounts of solutions B and C are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be at 7.4.


## Solution A

0.1	g	CaCl2 x 2 H2O	
0.1	g	MgSO4 x 7 H2O	
0.5	g	KH2PO4	
0.5	g	K2HPO4	
1	g	NaCl	
300	ml	**Clarified rumen fluid**	
0.5	g	Yeast extract (OXOID)	
0.5	g	Peptone (BD BACTO)	
0.5	g	(NH4)2SO4	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.5	g	L-Cysteine HCl x H2O	
620	ml	Distilled water	


## Solution B

10	g	Glucose	
50	ml	Distilled water	


## Solution C

1.5	g	Na2CO3	
30	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.