# 1569: ORENIA CHITINITROPHA


## Main sol. 1569

120	g	NaCl	
2.5	g	K2HPO4	
0.5	g	(NH4)2SO4	
1000	ml	Distilled water	

1. Adjust pH to 7.0 with KH2PO4 or K2HPO4. Make anoxic under nitrogen stream and autoclave at 121°C.
2. After autoclaving add sterile, anoxic solutions of:

1.5	g/l	Chitin (amorphous, from 5 % solution), adjust in range from 1 - 2 if required	
5	mM	MgCl2 x 6 H2O	
20	mg/l	Yeast extract	
20	mM	NaHCO3	
1	mM	Na2S	
1	ml/l	**Acidic trace metals (Pfennig & Lippert, 1965)**	
1	ml/l	**Vitamine mixture (Pfennig & Lippert, 1965)**	


## Vitamine mixture (Pfennig & Lippert, 1965)

50	ml	**Basic solution (100 ml 0.1 N NaOH)**	
50	ml	**Neutral solution (100 ml Aqua dem.)**	
50	ml	**Acidic solution (HCl, pH 3; 100 ml)**	

1. Sterilize by filtration and keep at 4°C. Before addition to the medium mix 1:1:1 (vol:vol:vol).


## Acidic solution (HCl, pH 3; 100 ml)

5	mg	Thiamin	
5	mg	Calcium pantothenate	
0.5	mg	B12	
100	ml	Distilled water	


## Basic solution (100 ml 0.1 N NaOH)

2	mg	Biotin	
5	mg	PABA	
5	mg	Nicotinic acid	
10	mg	Pyridoxine	
100	ml	Distilled water	


## Neutral solution (100 ml Aqua dem.)

2	mg	Folic acid	
5	mg	Riboflavin	
100	ml	Distilled water	


## Acidic trace metals (Pfennig & Lippert, 1965)

5	g/l	EDTA	
2	g/l	FeSO4 x 7 H2O	
100	mg/l	ZnSO4 x 7 H2O	
30	mg/l	MnCl2	
300	mg/l	H3BO3	
200	mg/l	CoCl2 x 6 H2O	
10	mg/l	CuCl2	
20	mg/l	NiCl2 x 2 H2O	
20	mg/l	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. adjust pH to 3-4 with HCl; autoclave in closed bottles, 120°C, 20 min.