# 1419: HYPHOMICROBIUM MEDIUM


## Main sol. 1419

50	ml	**Mineral Salts Solution A**	
1000	ml	**Mineral Salts Solution B**	

1. Autoclave solution A and B separately and combine after cooling.
2. Prepare the liquid medium. Before inoculation add from sterile stock solutions: 

10	ml	**Vitamin solution**	
2.5	µl	Vitamin B12 (0.1%)	

3. For solid media add 15.0 g agar to solution B


## Mineral Salts Solution A

1.36	g	KH2PO4	
2.68	g	Na2HPO4 x 2 H2O	
3.38	g	Methylamine hydrochloride	
50	ml	Distilled water	

1. Adjust pH to 7.5


## Mineral Salts Solution B

0.5	g	(NH4)2SO4	
0.2	g	MgSO4 x 7 H2O	
0.25	g	Bacto peptone	
2	ml	**Trace Element Solution**	
20	ml	**Hutner's salts**	
10	ml	**Vitamin solution**, add after autoclaving	
2.5	µl	Vitamin B12 (0.1%), add after autoclaving	
1000	ml	Distilled water	
15	g	Agar, for solid medium	

1. Adjust pH to 7.5


## Trace Element Solution

0.199	g	CaCl2 x 2 H2O	
0.1	g	FeSO4 x 7 H2O	
0.035	g	MnSO4 x H2O	
0.05	g	Na2MoO4 x 2 H2O	
100	ml	Distilled water	


## Hutner's salts

10	g	Nitrilotriacetic acid (NTA)	
29.7	g	MgSO4 x 7 H2O	
3.335	g	CaCl2 x 2 H2O	
9.25	mg	(NH4)6Mo7O24 x 4 H2O	
99	mg	FeSO4 x 7 H2O	
50	ml	**"Metals 44"**	
950	ml	Distilled water	

1. Dissolve the nitrilotriacetic acid.
2. Adjust pH to 7.0 with KOH (~7.3 g).
3. Dissolve other salts separately and combine.
4. Adjust pH to 6.8 with NaOH or H2SO4.


## "Metals 44"

250	mg	Na-EDTA	
1095	mg	ZnSO4 x 7 H2O	
500	mg	FeSO4 x 7 H2O	
154	mg	MnSO4 x H2O	
39.2	mg	CuSO4 x 5 H2O	
24.8	mg	Co(NO3)2 x 6 H2O	
17.7	mg	Na2B4O7 x 10 H2O	
1000	ml	Distilled water	

1. Dissolve the EDTA and add a few drops of concentrated H2SO4 to retard precipitation of the heavy metal ions.


## Vitamin solution

2	mg	Biotin	
2	mg	Folic acid	
10	mg	Pyridoxine hydrochloride	
5	mg	Thiamine-HCl x 2 H2O	
5	mg	Riboflavin	
5	mg	Nicotinic acid	
5	mg	D-Calcium pantothenate	
0.1	mg	Vitamin B12	
5	mg	p-Aminobenzoic acid	
1000	ml	Distilled water