# 465d: MINERAL MEDIUM WITH BENZYLCYANIDE AS NITROGEN SOURCE


## Main sol. 465d

1000	ml	**Main sol. 465 (modified)**	
1.8	g/l	Glucose	
120	mg/l	Benzylcyanide	

1. Prepare and autoclave medium 465 without ammonium sulfate. Add 120 mg/l benzylcyanide (not sterilized) to the incubation atmosphere or directly to the medium.


## Main sol. 465 (modified)

3.5	g	Na2HPO4 x 2 H2O	
1	g	KH2PO4	
0.1	g	MgCl2 x 6 H2O	
0.05	g	Ca(NO3)2 x 4 H2O	
1	ml	**Trace element solution SL-4**	
1000	ml	Distilled water	

1. pH 7.25
2. Rehydrate and cultivate lyophilized cells in the complex medium recommended for the specific strain (e.g. medium 1, 220, 535 or 830). After this reactivation, cultivate on mineral medium with the appropriate carbon source.


## Trace element solution SL-4

0.5	g	Na2-EDTA	
0.2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.3	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.01	g	CuCl2 x 2 H2O	
0.02	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. First dissolve EDTA in distilled water and adjust pH to 7.0 using 2 N NaOH; then add other compounds.