While treating the adherent mammalian cells in a T-25 flask with an antibiotic (just after changing the media) can we use the same tip for adding the same antibiotic to another flask? Is it ok? Why am I assuming this is because:
Only one thing is possible that the antibiotic gets diluted and gets some media in it and that is also very less because the pipette is set to 2 ul.
Kindly suggest what you think. Thank you
it is better to add the antibiotic to the media before you change the media. Just calculate the volume of media you need for your cultures, aliquot it into a suitable sterile flask and add the antibiotic, then change the media of the cultures using the antibiotic-containing media. Adding components directly step by step to the cultures is not recommended because it increases the dangers of mistakes and contaminations.
Good luck!
Christoph Leder
If you are adding to another cell line, better to use another tip . It is the only way to be sure that nothing "bad" will happen
Bit if it is the same cell line in all falsks it would not matter. I wonder however whether I understood well your question.
I agree with Christoph. I always make the correct volume of media plus antibiotics first and then add to each flask. Using the same tip between different cultures is never advisable because you get used to doing things that way and then if you are passaging different cell lines (or the same cell line with different manipulations) it is all too easy to cross-contaminate your cultures.
Thank you all for your suggestions but my curiosity is won't the mammalian cells (be any) die when they get into the antibiotic vial?
It's not just contamination of the antibiotic with your mammalian cells I'd worry about. If your first culture is contaminated with mycoplasma or any other ampicillin resistant organism you will spread it across all your cultures. Turning the small problem of a single contaminated flask into a disaster by contaminating all your cultures.
It depends why you're using the antibiotic. If it's a selection agent e.g. for a stable cell line then, if the mammalian cells get into the vial, they may die due to being exposed to toxic levels of the drug. If it's bog standard pen/strep to kill off any bacteria that might be present in your culture, then the mammalian cells shouldn't die if they get into the vial (because the antibiotic targets the bacteria). Either way it's best practice to add antibiotic to the media first, and then add to the flask as others have suggested, so there's no risk of cross-contaminating cultures. If you do need to add it to each flask separately, use a new tip each time - it's not worth the risk! Bear in mind that routine cell culture shouldn't require the use of antibiotics/antimycotics long-term.
In that way, you may contaminate your cells with strangers. Imagine just one cell of something that is dividing faster. After a few passages, the contaminants will have outgrown your actual cells and you will work unknowingly with the wrong cells and get unreproducible and misleading results.
Besides that, by adding concentrated solutions directly, you will have high local concentrations and an inhomogenous distribution which both could cause unpredictable effects .
Simply prepare as much full medium as you need.
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