# J574: DB CHARACTERIZATION MEDIUM NO. 2


## Main sol. J574

833	ml	**MDS salt water**	
1	ml	**FeCl2 solution**	
1	ml	**Trace element solution**	
0.25	g	Peptone (Oxoid)	
0.05	g	Yeast extract (BD-Difco)	

1. Add components and bring volume to 980 ml with distilled water. For preparation of solid medium, add 20 g/L agar (if necessary, use washed agar as described below). Autoclave, and after cooling, add the following solutions (filter-sterilized or *autoclaved) to the medium: 

5	ml	NH4Cl (1 M)	
2	ml	**Potassium phosphate buffer** (*)	
3	ml	**Vitamin solution**	
10	ml	Sodium pyruvate (1 M)	

2. Readjust pH to 7.0 - 7.5 with a sterilized 10% Na2CO3 solution, if necessary.


## Washed agar

1. Stir agar in distilled water and stand still for 30 min. Then, remove supernatant by decantation. Repeat the washing at least twice.


## MDS salt water

240	g	NaCl	
30	g	MgCl2 x 6 H2O	
35	g	MgSO4 x 7 H2O	
7	g	KCl	
5	ml	CaCl2 (1 M)	

1. Add components to distilled water and bring volume to 1.0 L. Adjust pH to 7.5 with 1 M Tris base.


## Potassium phosphate buffer

83.4	ml	K2HPO4 (1 M)	
16.6	ml	KH2PO4 (1 M)	

1. Combine the two solutions and check that pH is close to 7.5. Add an equal volume of distilled water.


## Vitamin solution

3	mg	Biotin	
4	mg	Folic acid	
50	mg	Pyridoxine hydrochloride	
33	mg	Thiamine HCl	
10	mg	Riboflavin	
33	mg	Nicotinic acid	
17	mg	DL-Calcium pantothenate	
17	mg	Vitamin B12	
13	mg	p-Aminobenzoic acid	
10	mg	Lipoic acid	
1000	ml	Distilled water	


## 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