# 833: DEHALOSPIRILLUM MEDIUM


## Main sol. 833

892	ml	**Solution A**	
10	ml	**Solution B**	
2	ml	**Solution C**	
35	ml	**Solution D**	
20	ml	**Solution E**	
40	ml	**Solution F**	
3	ml	**Solution G**	
1	ml	**Solution H**	

1. Sparge solution A with 100% N2 gas for 30 - 45 min to make it anoxic, then distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions B and H are autoclaved separately under 100% N2 gas. Solutions C, E, F and G are prepared under 100% N2 gas and sterilized by filtration. Solution D is autoclaved under 80% N2 and 20% CO2 gas atmosphere. 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 complete medium to 7.3 - 7.6, if necessary.
2. Note: Before inoculation, the medium must be completely reduced, i.e. resazurin must be colorless. The addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) can help to reduce the medium.


## Solution A

0.7	g	Na2SO4	
0.2	g	KH2PO4	
0.25	g	NH4Cl	
0.25	g	NaCl	
0.4	g	MgCl2 x 6 H2O	
0.5	g	KCl	
0.15	g	CaCl2 x 2 H2O	
2	g	Yeast extract	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
0.5	ml	Sodium resazurin (0.1% w/v)	
890	ml	Distilled water	


## Solution B

10	ml	Potassium phosphate buffer (0.1 M, pH 7.5)	


## Solution C

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


## Solution D

1.75	g	Na2CO3	
35	ml	Distilled water	


## Solution E

4.5	g	Na-pyruvate	
20	ml	Distilled water	


## Solution F

6.4	g	Na2-fumarate	
40	ml	Distilled water	


## Solution G

30	mg	FeSO4 x 7 H2O	
3	ml	H2SO4 (0.1 N)	


## Solution H

50	mg	L-Cysteine HCl x H2O	
1	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	


## 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