# J1004: FRESHWATER MEDIUM FOR SOIL AOA


## Main sol. J1004


## Basic FWM solution

1	g	NaCl	
0.5	g	KCl	
0.4	g	MgCl2 x 6 H2O	
0.2	g	KH2PO4	
0.1	g	CaCl2 x 2 H2O	
1000	ml	Distilled water	

1. Autoclave Basic FWM solution and aseptically add the following solutions (autoclaved or *filter-sterilized) in the given order: 

1	ml	**Modified trace element mixture**	
1	ml	**Vitamin solution** (*)	
1	ml	FeNa-EDTA (pH 7.0, *, 7.5 mM)	
2	ml	NaHCO3 (*, 1 M)	
10	ml	**HEPES solution**	
1	ml	NH4Cl (1 M)	
1	ml	Sodium pyruvate (*, 1 M)	

2. Use glass or polystyrene containers for cultivation. Cultivate in the dark without shaking.


## Modified trace element mixture

12.5	ml	HCl (25%)	
30	mg	H3BO3	
100	mg	MnCl2 x 4 H2O	
190	mg	CoCl2 x 6 H2O	
24	mg	NiCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
144	mg	ZnSO4 x 7 H2O	
36	mg	Na2MoO4 x 2 H2O	
987	ml	Distilled water	


## Vitamin solution

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	DL-Calcium pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
2	g	Choline chloride	
1000	ml	Distilled water	

1. Adjust pH to 7.0 with KOH.


## HEPES solution

12	g	NaOH	
119.2	g	HEPES (free acid)	

1. Dissolve NaOH in 300 ml distilled water. While stirring vigorously, slowly add HEPES until completely dissolved. Fill up to 450 ml with distilled water. If 1 ml of this solution is added to 100ml of the medium (containing all additive but HEPES), pH should be around 7.6 at 30C. Otherwise, adjust pH of the HEPES solution with 10 N NaOH or conc. HCl. Finally fill up to 500 ml with distilled water.