Protocol for Middlebrook 7H9 Broth (Difco) with Glycerol

Protocol for Middlebrook 7H9 Broth (Difco) with Glycerol

Material: Middlebrook 7H9 Broth Powder, Glycerol, 20% Tween 80, OADC

1.     Take 900 ml distilled water in a glass flask

2.     Place the flask on a magnetic stirrer

3.     Add 4.7 g of the powder in water and stir it

4.     Add 2 ml of glycerol (Pipette slowly) and stir it

5.     Autoclave the medium at 121⁰C for 15 minutes

6.     After Autoclaving cool the medium up to 45⁰C

7.     Then, aseptically add 100 ml of OADC to the medium*

8.     Add 2.5 ml of 20% Tween 80 to the medium *

9.     7H9 broth is ready, store it at 4⁰C

* Steps 7 and 8 must be done in a safety hood

* Temperature higher than 50⁰C can denature OADC and Tween 80.