Tissue Culture Laboratory (BIOE342) - Protocol

Feeding Cells


Mammalian cells need to be fed every 2-3 days. Without changing/replenishing the media, the cells will die. Since cells require supplements every 2-3 days, scientists working with cells have to be conscientious and deliberate in remembering to replenish the media of all their in vitro cell cultures.


Because of the routine feeding requirements, each person will need to come in Friday (M/W group) or Saturday (T/Th group) to feed his/her cells. While this is an acknowledged inconvenience (for the students, TAs and faculty), it is necessary for the future success of the experiments and well as learning to appreciate the complexity of tissue culture. Feeding cells is a rapid process and should take no more than 15-20 min.



Ethanol squirt bottle and kimwipes

Aspirating pipets

10 mL sterile pipets

Cells in T-75 flask

Media with 10% serum and antibiotics

Automatic pipetters



1.      Wear disposable latex gloves at all times.

2.      Wear safety glasses at all times.



1.      Warm media in 37C waterbath.

2.      Check cells in T flask under microscope to confirm that the cells are healthy and not fully confluent. (If cells are 90%-100% confluent, they need to be passaged, not fed.)

3.      Clean hood with ethanol. Spray ethanol liberally over surfaces and wipe clean with kimwipe.

4.      Sterilize all materials, bottles, etc. which are loaded into the hood. Spray hands with ethanol. Jars of liquid need to be sprayed with ethanol . Sterile pipets may be placed in the hood directly. Automatic pipetters should enter the hood WITHOUT sterilization.

5.      Spray hands with ethanol. Remove T flasks from the incubator and quickly place in hood. (As general guidance, do not spray flasks with ethanol.)

6.      Attach a aspirating pipet or sterile glass Pasteur pipet to the tube attached to vacuum. Turn on vacuum system by opening vacuum valve in hood.

7.      Using the aspirator, empty liquid media covering cells. Be careful to not touch the pipet to anything outside of the T flask. Turn off aspiration.

8.      Add 10 mL of media to cells in T flask. Lightly swish media on base of T flask.

9.      Replace flask in incubator.

10.  Dispose of liquid and solid biohazards wastes properly.

11.  Clean hood with ethanol. Spray ethanol liberally over surfaces and wipe clean with kimwipe.