# 201: DESULFONEMA LIMICOLA MEDIUM


## Main sol. 201

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

1. Solution A is sparged with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to reach a pH of around 6, then distributed under same gas atmosphere into anoxic Hungate-type tubes and autoclaved.
2. Solutions B, D, E, G, and H are autoclaved separately under 100% N2 gas. Solution C is autoclaved under 80% N2 and 20% CO2 gas atmosphere. Solution F is prepared under 100% N2 gas and sterilized by filtration. To complete the medium appropriate amounts of solutions B to H are added to the sterile solution A in the sequence as indicated. Adjust pH of the complete medium to 7.6, if necessary.
3. Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution freshly prepared under N2 and filter-sterilized) just before inoculation may stimulate growth at the beginning. For transfers use 5 - 10% inoculum.
4. Cultures of Desulfonema spp. show usually improved growth in media containing an artificial light sediment of aluminum phosphate. Alternatively, a viscous medium prepared with 1 - 2 g washed agar per liter can be used (Widdel et al., Arch. Microbiol. 134:286-294 (1983)): Agar (Difco) is washed three times for one hour with distilled water at room temperature. Finally, the agar is suspended (c. 20.0 g/l) in distilled water and autoclaved. 50 ml of the hot agar is added to the hot sterile medium part A (when preparing part A calculated for 1 liter, subtract the volume of the agar solution). After cooling to room temperature complete the medium by adding part B to I. For mass cultures the aluminum phosphate as artificial sediment can be used.


## Solution A

3	g	Na2SO4	
15	g	NaCl	
2.2	g	MgCl2 x 6 H2O	
0.15	g	CaCl2 x 2 H2O	
0.5	g	KCl	
0.2	g	KH2PO4	
0.3	g	NH4Cl	
0.5	ml	Sodium resazurin (0.1% w/v)	
930	ml	Distilled water	


## Solution B

1	ml	**Trace element solution SL-10**	


## Solution C

1.5	g	Na2CO3	
30	ml	Distilled water	


## Solution D

2.5	g	Na-acetate x 3 H2O	
10	ml	Distilled water	


## Solution E

0.1	g	Disodium succinate	
1	ml	Distilled water	


## Solution F

1	ml	**Wolin's vitamin solution (10x)**	


## Solution G

20	ml	**Clarified rumen fluid**, clarified	


## Solution H

0.4	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.


## 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