# 159: TREPONEMA MEDIUM


## Main sol. 159

300	ml	**Clarified rumen fluid**	
0.12	g	CaCl2 x 2 H2O	
0.18	g	MgSO4 x 7 H2O	
0.45	g	KH2PO4	
0.45	g	K2HPO4	
0.9	g	NaCl	
0.9	g	(NH4)2SO4	
0.5	ml	Sodium resazurin (0.1% w/v)	
1	g	L-Cysteine HCl x H2O	
1.5	g	Na2CO3	
2	g	D-Glucose	
700	ml	Distilled water	

1. Dissolve ingredients (except cysteine, carbonate and glucose) and sparge medium with 100% CO2 gas for 30 - 45 min to make it anoxic. Add cysteine and adjust pH with KOH to 6.5. Distribute medium under 100% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Therafter, add glucose from an anoxic stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere. Final pH of the medium should be 6.7 - 7.0.


## 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.