# 780: MIDDLEBROOK MEDIUM WITH MYCOBACTIN


## Main sol. 780

2	mg	Mycobactine J	
4	ml	Absolute ethanol	
1000	ml	**Main sol. 645**	

1. Reconstitute the contents of one vial of 2 mg Mycobactine J with 2 ml absolute ethanol (analytical grade). Shake carefully so that the product is completely dissolved. Pour the contents of the bottle into 1 litre of medium 645 kept melted in a water bath at +56°C, taking care to pour out all of the product. Rinse out the vial that contained the Mycobactine J with an additional 2 ml of absolute ethanol and pour into the culture medium. Mix the Mycobactine J alcohol solution and the culture medium very thoroughly.


## Main sol. 645

19	g	Bacto Middlebrook 7H10 agar (Difco 262710)	
5	ml	Glycerol	
900	ml	Distilled water	

1. Sterilize for 15 min by 121°C cool to 50 - 55°C. Add 100 ml Middlebrook OADC enrichment (Becton Dickinson 212240) under aseptic conditions and mix thoroughly.
2. If you can´t order the commercial media then prepare the media in this way: