# J995: M30


## Main sol. J995


## Solution 1

20	ml	**Modified Hutner's basal salts**	
700	ml	Sea water (aged)	
50	ml	Tris-HCl buffer (pH 7.5, 0.1 M)	
260	ml	Distilled water	

1. For solid medium, add 5.0 g/L gelrite (Gellan Gum).


## Solution 2

2	g	N-Acetyl-D-glucosamine (Sigma)	
0.01	g	Na2HPO4 x 2 H2O	
4	ml	Ampicillin (50 mg/ml)	
10	ml	**Vitamin solution No. 6**	
40	ml	Distilled water	

1. Add 0.2 ml filter-sterilized Solution 2 to Solution 1.


## Vitamin solution No. 6

4	mg	Biotin	
20	mg	Pyridoxine hydrochloride	
10	mg	Thiamine-HCl x 2 H2O	
10	mg	Calcium pantothenate	
10	mg	p-Aminobenzoic acid	
4	mg	Folic acid	
10	mg	Riboflavin	
10	mg	Nicotinamide	
0.2	mg	Vitamin B12	
1000	ml	Distilled water	

1. Filter-sterilize.


## Modified Hutner's basal 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 nitrilotriacetic acid and adjust pH to 7.0 with KOH (about 7.3 g). Then proceed to add salts. Readjust pH to 6.8 with NaOH or H2SO4.