# 428: HETEROTROPHIC MEDIUM H3P


## Main sol. 428

50	ml	**Solution A**	
855	ml	**Solution B**	
20	ml	**Solution C**	
30	ml	**Solution D**	
50	ml	**Solution E**	
5	ml	**Standard vitamin solution**	

1. H3P is a heterotrophic medium for growth, purity checking and isolation of a broad spectrum of aerobic bacteria (Ref. 3367).
2. Solutions A, B, C and D are autoclaved separately for 15 min at 121°C, cooled down to 50°C and then mixed aseptically with filter-sterilized solution E (at 50°C) and 5.0 ml of standard vitamin solution. The final pH of this medium should be 6.8 without adjustment.


## Solution A

2.3	g	KH2PO4	
2.9	g	Na2HPO4 x 2 H2O	
50	ml	Distilled water	


## Solution B

1	g	NH4Cl	
0.5	g	MgSO4 x 7 H2O	
0.01	g	CaCl2 x 2 H2O	
5	mg	MnCl2 x 4 H2O	
5	mg	NaVO3 x H2O	
5	ml	**Trace element solution SL-6**	
850	ml	Distilled water	
20	g	Agar, if required	


## Solution C

0.05	g	Ferric ammonium citrate	
20	ml	Distilled water	


## Solution D

1	g	Yeast extract	
1	g	Na-acetate	
1	g	Disodium succinate	
1	g	DL-Malate	
30	ml	Distilled water	

1. pH 7.0


## Solution E

1	g	Na-lactate	
1	g	Na-pyruvate	
1	g	D-Mannitol	
2	g	D-Glucose	
50	ml	Distilled water	

1. pH 7.0
2. filter-sterilized


## Standard vitamin solution

10	mg	Riboflavin	
50	mg	Thiamine-HCl x 2 H2O	
50	mg	Nicotinic acid	
50	mg	Pyridoxine hydrochloride	
50	mg	Calcium pantothenate	
0.1	mg	Biotin	
0.2	mg	Folic acid	
1	mg	Vitamin B12	
100	ml	Distilled water	

1. Filter sterilize (0.2 µm pore-size) and store at 4°C.


## Trace element solution SL-6

0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.3	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.01	g	CuCl2 x 2 H2O	
0.02	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water