Question

can u please help me with question 1 and 3

Describe in general each step for cell culture "splitting cells" and then ve use each reagent in each step, i.e Trypsin, Growth Media ... tting cells" and the reason When working in the biological safety cabinet (hood) is important to: Please explain your answer a. Make sure the hood is on and working b. Wipe the hood down with 70% alcohol c. Minimize quick movement d Not introduce contamination (by not wiping everything that goes inside, not wiping your hands, not using gloves, and sneezing) e. All the above 2 In the event of a bacterial contamination in cell culture the easiest thing to do is and why? Discard the cells and have a new thaw frozen vial of fresh cells b. Simply add antibiotics to kill the contaminant c. Keep the culturing like nothing happened d. Wash the cells several times with PBS

Answer 1

ANSWER 1:

1. Take DMEM media, PBS and Trypsin.
2. Pre-warm all the reagents in 37^o  C water bath.
3. turn on the laminar air flow and wipe it with 70% ethanol for sterilisation. Run the UV light and air flow for 30 min.
4. Look at the cells under microscope to ensure they are in healthy condition.
5. Aspirate off the old media. Rinse gently with 10 ml PBS.
6. Aspirate off the PBS. Add 2ml Trypsin.
7. Put it in the incubator for 4 min. Prepare centrifuge tube.
8. Tap the dish to help release cells.
9. Examine under microscope (fully trysinized cells should appear which are not attached to the surface of the dish).
10. Add 10 ml of fresh media. Resuspend cells in the media.
11. Pipet up all the cells into the centrifuge tube. Centrifuge cells for 3 min at 300*gm.
12. Prepare and label a new culture flask. Put 10-15 ml of fresh media into the new flask.
13. Aspirate the media above the cell pellet. Resuspend cells in 10 ml fresh media.
14. Dispense the proper split volume of the cells to the new flask.
15. Incubate the flask at 37^o C in CO2 incubator.

DMEM- It is a basal medium used for supporting the growth of different cells.

PBS- It is a balanced salt solution used in cell culture for the washing of cells before dissociation and also in transporting of the cells.

Trypsin- It is a proteolytic enzyme which break the protein to dissociate the cell.

ANSWER 3:

b) Simple add antibiotics to kill the contaminant.