# J827: DEHALOCOCCOIDES MEDIUM


## Main sol. J827

1	g	NaCl	
0.41	g	MgCl2 x 6 H2O	
0.27	g	NH4Cl	
0.52	g	KCl	
0.015	g	CaCl2 x 2 H2O	
0.2	g	KH2PO4	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
1	ml	**Selenite-tungstate solution**	
0.41	g	Sodium acetate	
0.25	mg	Resazurin	
1000	ml	Distilled water	

1. Mix components thoroughly, bring to a boil and cool down under a N2 atmosphere. Add the following solutions from anaerobic stocks, then dispense the medium into culture vessels leaving 40 to 50% volume of a gas phase (e.g., 30 ml of the medium in a 50 ml serum bottle) and autoclave under a N2 atmosphere: 

1	ml	L-Cysteine HCl x H2O (5%)	
1	ml	Na2S x 9 H2O (5%)	

2. After cooling, replace the gas phase with a N2-CO2-H2 (8:1:1, v/v) gas mixture. Add per liter the following filter-sterilized solutions from anaerobic stocks: 

10	ml	NaHCO3 (8%)	
20	ml	MOPS (pH 7.4, 1.0 M)	
20	ml	**25 mM Ti(III) NTA solution** (25 mM)	
1	ml	**Vitamin solution**	

3. To complete the medium, add per liter the following filter-sterilized chlorosolvent: 

0.7	ml	Trichloroethylene (10 mg/ml in methanol)	

4. Check final pH to be about 7.2.


## Vitamin solution

10	mg	Biotin	
2	mg	Folic acid	
250	mg	Pyridoxine hydrochloride	
20	mg	Thiamine HCl	
5	mg	Riboflavin	
100	mg	Nicotinic acid	
10	mg	DL-Calcium pantothenate	
25	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water	


## 25 mM Ti(III) NTA solution

9.6	g	Nitrilotriacetic acid	
300	ml	Water (anaerobic )	

1. To prepare anaerobic water, boil 1.0 L distilled water and cool down under a stream of oxygen-free N2 gas. Mix components under N2 gas. Adjust pH to 9.0 with NaOH. Slowly add 9.6 ml of 20% TiCl3 to the chelator solution. Keep the pH above 2.0 by the addition of anaerobic saturated Na2CO3 solution. Adjust pH to 7.0 with Na2CO3 solution, adjust pH to 7.0 and bring the final volume to 500 ml with the anaerobic water. Filter-sterilize and keep the solution under a N2 gas atmosphere.


## FeCl2 solution

10	ml	HCl (7.7 M, 25%)	
1.5	g	FeCl2 x 4 H2O	
990	ml	Distilled water	


## Trace element solution

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	
1000	ml	Distilled water	


## Selenite-tungstate solution

0.4	g	NaOH	
6	mg	Na2SeO3 x 5 H2O	
8	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water