# J994: MBM MEDIUM 2


## Main sol. J994

80	g	NaCl	
6	g	MgSO4 x 7 H2O	
1.5	g	KCl	
1	g	NH4Cl	
0.4	g	CaCl2 x 2 H2O	
0.4	g	K2HPO4	
10	ml	**Trace minerals**	
1.6	g	Sodium acetate x 3 H2O	
1	mg	Resazurin	
900	ml	Distilled water	

1. Mix components thoroughly. Bring to a boil for a few seconds, cool down under a N2-CO2 (4:1, v/v) gas mixture, seal with butyl rubber stoppers and autoclave. After cooling, add the following solutions (autoclaved or *filter-sterilized): 

40	ml	Disodium fumarate (1 M)	
50	ml	NaHCO3 (*, 8%)	
10	ml	**Trace vitamins** (*)	

2. Aseptically distribute the medium into culture vessels (e.g., 5 ml of the medium in the Hungate tubes) under a N2-CO2 (4:1, v/v) gas mixture and seal with butyl rubber stoppers. Add per liter the following solutions from anaerobic stocks (autoclaved and stored under a N2 gas atmosphere): 

6	ml	L-Cysteine HCl x H2O (5%)	
6	ml	Na2S x 9 H2O (5%)	

3. Readjust pH to 7.5, if necessary.


## 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.


## Trace vitamins

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine HCl	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	Calcium pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
5	mg	Lipoic acid	
1000	ml	Distilled water