# 585a: BCYE alpha agar


## CYE-ACES alpha AGAR

490	ml	**Solution A**	
490	ml	**Solution B**	
10	ml	**Solution C**	
10	ml	**Solution D**	

1. Prepare OXOID Legionella CYE-Agar base or solution A.
2. Cool down to 50 °C.
3. Add solution C (Cysteine-HCl) and solution D (ferric pyrophosphate).
4. Adjust pH to 6.9 with sterile 1.0 N KOH or HCl.
5. The pH of the medium is critical.
6. Finally, add solution B. Swirl medium in flask during dispensing to petri dishes or tubes in order to keep charcoal suspended.


## OXOID Legionella CYE-Agar base

1000	ml	OXOID Legionella CYE-Agar base (with Legionella BCYE supplement)	

1. Dehydrated media preparations are available from media distributors.
2. We use OXOID Legionella CYE-Agar base with Legionella BCYE supplement.
3. Note: Alternatively use CYE-ACES AGAR


## Solution A

10	g	ACES (N-2-acetamido-2-aminoethane-sulfonic acid)	
10	g	Yeast extract (Difco 0127)	
2	g	Activated charcoal (Sigma C 5510)	
1	g	Na2 alpha-ketoglutarate	
490	ml	Distilled water	

1. Dissolve ACES and bring to pH 6.9 by slowly adding 1 N KOH. Add the other components and dissolve by boiling. Autoclave at 121°C for 15 minutes. Cool to 50 - 55°C.


## Solution B

15	g	Agar	
490	ml	Distilled water	

1. Autoclave at 121°C and cool to 50 - 55°C.


## Solution C

0.4	g	L-Cysteine HCl x H2O	
10	ml	Distilled water	

1. Prepare a fresh solution and filter-sterilize it.


## Solution D

0.25	g	Fe4(PO4)2	
10	ml	Distilled water	

1. Prepare a fresh solution by heating to 50 - 55°C in a water-bath. Filter-sterilize solution separately.
2. Please note: Soluble ferric pyrophosphate must be kept dry and in the dark. Do not use if chemical looses its green colour and becomes brown or yellow.