Frank Crittenden

If adding antibiotic to prevent contamination, you want to add it when you are making your medium so you can filter it through a 0.2 um filter. I have ran cell culture experiments with new students in the past and there are two sides to the coin on this.

1) You do not use antibiotics, so if a contamination starts you can address it right away but you teach proper aseptic techniques

2) Use antibiotics which will help minor chance of contamination but doesnt account for great aseptic technique

3) A third option is have anyone new with cell culture work in there own incubator (Might not be possible) until they can maintain cells without contamination

I have been in labs that did not use antibiotics in cell culture because you can see an issue right away and I have been in labs were we did use antibiotics because it was a common use area. The choice is yours, as long as you stick with one or the other for experiments. Another suggestion is if you have a variety of skill levels in cell culture, I would give every individual their own Media, Trypsin, and PBS (Or Hanks Buffered Solution or w/e you use for washes). That way if something happens to one, there is less of a chance that everyone's cells are effected. Hope this helps a little bit.

If adding antibiotic to prevent contamination, you can add it in your medium after sterilization, if the melting point is more than sterilization temperature then you can add it before sterilization. If you want to add after sterilization then you have to filter it through a 0.2 um filter.

Add your antibiotics alongside other reagents such as FBS when you are making the aliquotes.