# 407: SYNTROPHOMONAS SD2 MEDIUM


## Main sol. 407

930	ml	**Solution A**	
30	ml	**Solution B**	
1	ml	**Solution C**	
20	ml	**Solution D**	
10	ml	**Solution E**	
10	ml	**Solution F**	

1. Add and dissolve ingredients of solution A, adjust pH to 7.2, then sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Distribute medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solution C is prepared under 100% N2 gas and sterilized by filtration. Solutions D, E and F are autoclaved under 100% N2 gas. To complete the medium, appropriate amounts of solutions B to F are added to the sterile solution A. Adjust pH of complete medium to 7.2, if necessary.
2. Note: Use 25 - 50% inoculum. Growth can be followed by the clearing of stearate. Maintain cultures at 25°C.


## Solution A

0.5	g	KH2PO4	
0.33	g	MgCl2 x 6 H2O	
0.4	g	NaCl	
0.4	g	NH4Cl	
0.05	g	CaCl2 x 2 H2O	
1	ml	**Trace element solution SL-10**	
50	ml	**Clarified rumen fluid**	
1	g	Trypticase peptone (BD BBL)	
2.8	g	Na2SO4	
0.5	ml	Sodium resazurin (0.1% w/v)	
880	ml	Distilled water	


## Solution B

1.5	g	Na2CO3	
30	ml	Distilled water	


## Solution C

1	ml	**Seven vitamins solution**	


## Solution D

0.61	g	Na-stearate	
20	ml	Distilled water	

1. Dissolve stearate by heating in water bath.


## Solution E

0.3	g	L-Cysteine HCl x H2O	
10	ml	Distilled water	


## Solution F

0.3	g	Na2S x 9 H2O	
10	ml	Distilled water	


## 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.


## 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.


## Seven vitamins solution

100	mg	Vitamin B12	
80	mg	p-Aminobenzoic acid	
20	mg	D-(+)-biotin	
200	mg	Nicotinic acid	
100	mg	Calcium pantothenate	
300	mg	Pyridoxine hydrochloride	
200	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water