Protocol for Middlebrook 7H11 Agar (Difco) with Glycerol

Protocol for Middlebrook 7H11 Agar (Difco) with Glycerol

Material: Middlebrook 7H11 Agar 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 21 g of the powder in water and stir it
4.     Add 5 ml of glycerol (Pipette slowly) and stir it
5.     Autoclave the medium at 121 ⁰C for 15 minutes
6.     After Autoclaving cool the medium up to 50 ⁰C (The temperature of container which can be tolerated by skin).
7.     Then, aseptically add 100 ml of OADC to the medium*
8.     Add 2.5 ml of 20% Tween 80 to the medium *

* Steps 7 and 8 must be done in a safety hood
* Temperature higher than 50⁰C can denature OADC and Tween 80, on the other hand low temperature will cause solidification of agar. Therefore, add OADC and Tween 80 at 50⁰C  and pour the medium on plates as soon as possible before solidification.

Protocol for Complete DMEM (1000 ml) preparation from 5X basal DMEM

Protocol for Complete DMEM (1000 ml) preparation from 5X basal DMEM

1.     Autoclave the Filtration apparatus before using in filtration

2.     Then, put 690 ml 3A water in Filtration apparatus

3.     Add 200 ml 5x DMEM basal media in Filtration apparatus

4.     Add 100 ml FBS in Filtration apparatus

5.     Add 10 ml clear L-Glutamine in Filtration apparatus (Before adding keep it in water bath at 37⁰C for 5 to 10 minutes to be clear)

6.     Filter the mixture

7.     Pour filtrated media in a Autoclaved Glass Bottle (Complete DMEM is prepared)

8.     Preserve Complete DMEM in Fridge at 4 ⁰C

Protocol for 5X DMEM

5X DMEM

1. Take 900 mL of 3A water in a Beaker 
2. Put it on a magnetic stirrer and turn on the machine
3. Add whole bottle of DMEM Glucose (67 Gram) slowly in the Beaker
4. Stir it up to soluble completely into 3A water  
5. Add 10 gram of Sodium-bicarbonate (NaHCO3)
6. Then, add 17.9 gram of HEPES
7. Adjust P^H 7.4  by adding 10N NaOH
8. Then transfer the medium in measuring flask and make the volume 1000mL by adding 3A water
9. Filter the medium in Biosafety cabinet
10. Preserve medium  in 4⁰ C