Cell Feeding – General Notes
1.
Take the appropriate media out of the fridge and put it in the water bath at
37’C for about 20 minutes before you start. You want to maintain warm conditions for the cells as much as possible……they don’t like cold media!!
2.
Turn the light on the laminar flow hood to FLOURESCENT , and put the blower to the ON position. You must also raise the window for the blower to work.
3.
Spray down the surfaces of the laminar flow hood with 70% ethanol before you place anything in the hood. Make sure you get the back and side walls. If the spray bottle runs out of ethanol, there is more ethanol on the bottom right hand shelf of the fume hood in the main lab.
4.
Put on clean gloves and a lab coat, and spray your gloves with 70% ethanol.
5.
Open the incubator and carefully remove the cell culture flasks from the incubator. Be gentle with them, try not to tip them up too much.
6.
Place the culture flasks in the laminar flow hood.
7.
Take an autoclaved piece of glassware (beaker) to use for collection of the old media. Autoclaved glassware will have aluminum foil covering the top. The outside of the glass will not be clean though, so this needs to be sprayed with ethanol before being placed in the hood.
8.
Remove the now warmed media from the waterbath. Use a piece of blue roll or kimwipe to dry off the outside of the container. Again, the inside of the container is sterile, but the outside is not, so you must spray the outside with ethanol before putting it in the hood.
9.
Before you start the feeding, think about how you have things set up in the hood. If you are right handed, you want to have the media container and waste beaker on your right hand side.
10.
Feeding is done on a 50% basis: you should remove 50% of the media from the culture flask, and replace it with the same volume of fresh media.
11.
We use a few different size culture flasks in the lab, which hold different volumes of media. On the ledge of the laminar flow hood, you will see the different sizes, with their feeding volumes written on the sides.
12.
Keep the tip of the pipette away from everything. If you have a concern about contamination, use a new pipette.
Keep culture flask and media bottle lids turned upright. Don’t touch the outside of flasks or beakers with the pipette tips. Keep your hands in the hood at all times; if you have to take them out for more than a few seconds, re-spray them with ethanol before putting them back into the hood.
13.
When you have finished feeding, write “50% feed, today’s date” on the outside of the flask.
14.
Put the flasks back in the incubator.
15.
Replace the cap of the media bottle, and wrap it with a piece of Para film.
This is important, because it’s an indicator to everyone else that the contents of the bottle are sterile, and should be used only in the laminar flow hood.
Once the cap is wrapped, place the bottle back in the fridge.

16.
Dispose of all used pipettes in the red hazardous waste container. Take your beaker of waste media and place it in the sink. Add some bleach and let it sit for half an hour, before pouring it down the drain.
17.
Remove any other waste from the laminar flow hood, and re-spray the surfaces with ethanol.
18.
Close the window of the hood, switch the blower to the OFF position, and turn the UV light on. This is how you want to leave the hood. The UV light will kill almost all contaminants that could have entered the hood. Make sure you pull the sash all the way down.