# 1778a: ANB aerobic


## Main sol. 1778a

15	g/l	Na2CO3	
20	g/l	NaHCO3	
3	g/l	NaCl	
1	g/l	K2HPO4	
1	ml	**Trace element solution**	

1. mix compounds.
2. check pH (should be 10)
3. Fill 100 mL each into 200 mL serum-flasks.
4. Autoclave at 121 °C for 20 min.
5. After autoclaving, add the following sterile solutions:

1	ml/l	MgSO4 (1M in H2O)	
2	ml/l	NH4Cl (2M in H2O)	
1	ml/l	**Vitamine mixture**	
10	ml/l	Melezitose (10% in H2O)	
2	ml/l	Yeast extract (1% solution in water)	

6. Mix well and aliquot into the desired volumes and flasks or tubes (e.g., 20 ml in 100 ml flasks).


## Trace element solution

5	g	EDTA	
2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4	
0.03	g	MnCl2	
0.3	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.01	g	CuCl2 x 5 H2O	
0.02	g	NiCl2 x 6 H2O	
0.02	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. Adjust pH to 3.0-4.0 with HCl.
2. Autoclave at 121 °C for 15 min.
3. Note: autoclave in closed bottles


## Vitamine mixture

100	ml	**Acidic thiamine / Ca pantothenate solution**	
100	ml	**Basic vitamin solution**	
100	ml	**Neutral folic acid / riboflavin solution**	
100	ml	**Neutral B12 solution 0.005 g/L**	

1. Sterilize by filtration.
2. Mix the four solutions in the ratio 1:1:1:1.


## Acidic thiamine / Ca pantothenate solution

5	mg	Thiamine	
5	mg	Calcium pantothenate	
100	ml	Distilled water (acidified to pH 3.0 with HCl)	


## Basic vitamin solution

2	mg	Biotin	
5	mg	PABA (p-aminobenzoic acid)	
5	mg	Nicotinic acid	
10	mg	Pyridoxine	
100	ml	NaOH (0.1N)	


## Neutral folic acid / riboflavin solution

2	mg	Folic acid	
5	mg	Riboflavin	
100	ml	Distilled water	


## Neutral B12 solution 0.005 g/L

0.5	mg	B12	
100	ml	Distilled water