# 337: SYNTROPHOCOCCUS SUCROMUTANS MEDIUM


## Main sol. 337

921	ml	**Solution A**	
50	ml	**Solution B**	
1	ml	**Solution C**	
25	ml	**Solution D**	
10	ml	**Solution E**	
1	ml	**Solution F**	

1. Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Adjust pH to 6.4 and dispense 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 atmosphere and sterilized by filtration. Solutions D, E and F are autoclaved separately under 100% N2 gas. To complete the medium add appropriate amounts of solutions B - F to the sterile solution A in the sequence as indicated. Adjust pH of the complete medium to 6.4 - 6.8, if necessary.
2. Note: Rumen fluid may be replaced by supplementing the medium with 200 µg/ml of crude egg yolk phosphatidylcholine (SIGMA, type IX-E).


## Solution A

50	ml	**Mineral solution**	
1	ml	**Trace element solution SL-10**	
300	ml	**Clarified rumen fluid**	
5	g	Trypticase peptone (BD Bacto/BD BBL)	
0.6	g	Na-formate	
0.5	ml	Sodium resazurin (0.1% w/v)	
570	ml	Distilled water	


## Solution B

2.5	g	NaHCO3	
50	ml	Distilled water	


## Solution C

1	ml	**Seven vitamins solution**	


## Solution D

5	g	Lactose	
25	ml	Distilled water	


## Solution E

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


## Solution F

78	mg	Na2S x 9 H2O	
1	ml	Distilled water	


## Mineral solution

10	g	KH2PO4	
6.6	g	MgCl2 x 6 H2O	
8	g	NaCl	
8	g	NH4Cl	
1	g	CaCl2 x 2 H2O	
1000	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