# J687: MB MEDIUM WITH METHANOL


## Main sol. J687


## Solution A

10	g	NaCl	
2	g	Yeast extract	
2	g	Trypticase peptone	
1	g	NH4Cl	
1	g	MgCl2 x 6 H2O	
0.5	g	KCl	
0.4	g	CaCl2 x 2 H2O	
0.4	g	K2HPO4	
0.5	mg	Resazurin	
10	ml	**Trace minerals**	
1	ml	**Selenite-tungstate solution**	
4	g	NaHCO3	
920	ml	Distilled water	

1. Mix components except NaHCO3, bring to a boil for several seconds and cool down under a stream of N2-CO2 (80:20). Add NaHCO3, mix thoroughly and distribute the medium into culture vessels under the same gas mixture. Seal with butyl rubber stoppers and autoclave. Finally, add per liter the following sterile solutions (autoclaved or *filter-sterilized): 

50	ml	Methanol solution (see below) (*)	
10	ml	Trace vitamins (see Medium No. 197) (*)	
5	ml	L-Cysteine HCl x H2O (5%)	
5	ml	Na2S x 9 H2O (5%)	


## Methanol solution

2.28	ml	Methanol	
50	ml	Distilled water	


## Trace minerals

1.5	g	Nitrilotriacetic acid	
3	g	MgSO4 x 7 H2O	
0.5	g	MnSO4 x n H2O	
1	g	NaCl	
0.1	g	FeSO4 x 7 H2O	
0.1	g	CoSO4 x 7 H2O	
0.1	g	CaCl2 x 2 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.01	g	CuSO4 x 5 H2O	
0.01	g	AlK(SO4)2	
0.01	g	H3BO3	
0.01	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH solution. Then proceed to add minerals. Adjust final pH to 7.0.


## Selenite-tungstate solution

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