I am a first year Ph.D. student in Cell and Molecular Biology department, UT. I am going to start a project on Breast Cancer and for this I need to be skilled on cell culture. I never did cell culture in my undergrad and now after shadowing my PI and seniors in the lab, I started my own session. But unfortunately my cells are getting contaminated. I wash my hands, rub my hand with alcohol, sterilize my hood with alcohol, spray alcohol in every bottle of reagents I use, Using auto pipet ( Big pipet). What else I am missing? I am feeling so pissed off because my seniors are doing the same but they are not facing the contamination. Please help me to overcome this situation.
If you and your seniors are utilizing separate batches of the same reagents (i.e., each individual has their own set), it could indicate that your specific set is contaminated. However, if both of you are sourcing from the same batch and you’re still encountering issues, it’s possible that a step in your sterilization process was overlooked. This could be something as simple as your lab coat requiring sterilization, or perhaps the antibiotics were inadvertently omitted from your medium.
The first thing i am missing on your "getting readdy list" is the absence of : using gloves.
Moreover, i dont know if you used tips with filter on your pippet, and if those tips are sterilized (autoclave or buyed sterile).
It is also possbile that if u have some rings, bracalets, watch or anything like that, it could be the contamination focus if you dont take it out from your hands before starting working.
Hello Sadia Haque Khan,
Even if you had the opportunity to work along with members experienced in cell culture, since you are new to cell culture, it will take a while till you develop the skills of handling cultures.
Keep following the sterile practices learnt from your seniors. Observe your seniors when they handle cultures. You should record even the minute details such as the way they open the knob of the culture flask during feeding the cultures or subculturing. For instance, the knob of the culture flask should be kept in such a way that the inner side of the knob faces upwards and does not touch the surface of the laminar hood. The pipet tip should not touch any non-sterile surface, and so on and so forth. You will be able to master these skills with practice.
Till you master these skills, I suggest you add antibiotics to the growth media which will help prevent contamination. Generally, antibiotics are not supposed to be added to cultures. Once you get the confidence of handling cultures without any contamination, you may discontinue the use of antibiotics.
Good Luck!
Thanks All. I use gloves, no jewellery and we use the same batch of reagents. But today I found that my senior also get contaminated. :(
If your senior also get contaminated, i will discard all the media that are using and i will start with a new one, also of PBS and trypsine (if you use it for passing cells)
Hi Ruben, thanks for the suggestion. I do insect cell cultures too and there we do not need to use PBS and Trypsine. Still I found issues. Today did cell culture again from fresh. Lets see what is waiting for me.
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