# 739: DESULFONEMA ISHIMOTOI MEDIUM


## Main sol. 739

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

1. Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic and reach a pH of around 6, then distribute under the same gas atmosphere into anoxic Hungate-type tubes and autoclave. Solutions B and C are prepared under 100% N2 gas atmosphere and sterilized by filtration. Solutions D and F are autoclaved separately under 100% N2 gas. Solution E is prepared under 80% N2 and 20% CO2 gas gas atmosphere. To complete the medium appropriate amounts of solutions B to F are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be at 7.0. After combining solutions the medium should equilibrate overnight and a white precipitate should be apparent.
2. 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% (v/v) inoculum.


## Solution A

25	g	NaCl	
5.6	g	MgCl2 x 6 H2O	
6.8	g	MgSO4 x 7 H2O	
1.4	g	CaCl2 x 2 H2O	
0.72	g	KCl	
0.14	g	KH2PO4	
0.25	g	NH4Cl	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
950	ml	Distilled water	


## Solution B

2.5	g	Na-acetate x 3 H2O	
0.1	g	Disodium succinate	
0.19	ml	Isobutyric acid	
10	ml	Distilled water	

1. Adjust to pH 7.0 with 1 N NaOH.


## Solution C

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


## Solution D

0.48	g	AlK(SO4)2 x 12 H2O	
10	ml	Distilled water	


## Solution E

1	g	Na2CO3	
20	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.


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## 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