# 912: TREPONEMA R8 MEDIUM


## Main sol. 912

1	g	NaCl	
0.5	g	MgCl2 x 6 H2O	
0.2	g	KH2PO4	
0.25	g	K2HPO4	
0.3	g	NH4Cl	
0.3	g	KCl	
15	mg	CaCl2 x 2 H2O	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
2.1	g	MOPS (SIGMA)	
0.9	g	D-Glucose	
0.19	g	Yeast extract	
0.19	g	Peptone (BD Bacto)	
0.5	ml	Sodium resazurin (0.1% w/v)	
2.5	g	NaHCO3	
50	ml	**Clarified rumen fluid**	
1	ml	**Wolin's vitamin solution (10x)**	
0.4	g	L-Cysteine HCl x H2O	
0.4	g	Na2S x 9 H2O	
950	ml	Distilled water	

1. Dissolve ingredients (except bicarbonate, rumen fluid, vitamins, cysteine and sulfide) and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Add bicarbonate, adjust pH to 7.2, distribute medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add rumen fluid, vitamins, cysteine and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere. Vitamins should be sterilized by filtration. Check pH of complete medium and adjust to 7.2, if necessary.


## 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.


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 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.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water