# 465c: MINERAL MEDIUM WITH DICHLOROMETHANE


## Main sol. 465c

1000	ml	**Main sol. 465**	

1. Prepare medium 465 (addition of 50 mg/l bromothymol blue as pH-indicator is recommended) and autoclave. Grow the cells in medium 465 with methanol supplied via incubation atmosphere (up to 10 ml methanol per liter medium). Incubate these adapted cells in medium 465 and consecutively feed small amounts of dichloromethane (toxic! 2 mmol per liter or less) via incubation atmosphere. Readjust pH of the culture with sterile 1 M NaOH if necessary.


## Main sol. 465

3.5	g	Na2HPO4 x 2 H2O	
1	g	KH2PO4	
0.5	g	(NH4)2SO4	
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.