Media Recipes for Cell Culture

The following procedures will be performed utilizing aseptic technique, as

described in Aseptic Techniques Used for Mammalian Cell Culture
General Media Rules
Do not use media older than two months. Glutamine breaks down when
exposed to temperatures higher than 10C causing an accumulation of
ammonia and a more basic pH.

To dispose of contaminated media add full strength bleach to bottle

equaling 2% of the total volume. Cap media and invert several times. Let
media set for 10 minutes then pour liquid down sink drain. Place media
bottle in a biohazard disposal container.

Unused and contamination free media will be poured down the sink drain
if it is no longer needed or older than two months.

Media without proper labeling will be disposed of by the lab manager.

o Proper labeling includes media name, initials, and date made.

Media Creation Procedure

1. Thaw frozen components in the water bath.
2. Prepare biosafety cabinet with a sterile receiving bottle and sterile 0.22
micron filtering unit fit to the opening of the bottle.
3. Pipet media components into the stock media bottle.
4. Cap and invert until ingredients are thoroughly mixed.
a. Try to prevent the formation of bubbles
5. Attach the vacuum line to the filter unit and slowly pour mixture into the top
of the filtering unit.
6. Once all media has been filtered or the receiving bottle is full, remove the
vacuum line, filter unit, and cap the bottle tightly.
7. Label with filtered bottle with:
a. Media name
b. Initials
c. Date made
8. Store complete media in a refrigerator.
9. Cap and return remaining Penicillin/Streptomycin and L-glutamine to -20C
storage.

10% DMEM Complete with antibiotic

Supplies

500mL bottle of DMEM Medium

50mL of Fetal Bovine Serum
5mL of L-Glutamine, 200mM
5mL of Penicillin/Streptomycin

10% RPMI-1640 Complete with antibiotic

Supplies

500mL bottle of RPMI-1640Medium

50mL of Fetal Bovine Serum
5mL of L-Glutamine, 200mM
5mL of Penicillin/Streptomycin