Constant Infections(?) - I am in need of some serious help (Jan/20/2011 )

I may be wrong, but I've been told you can't filter media once serum is added, as many of the vital components of the sera won't be able to pass through the filter.


UncleRudder, I understand that you think your aseptic technique is good enough but I doubt it is, or you wouldn't be getting contamination. You're inexperienced with cell culture and may not have been taught properly to begin with. As someone else said, you can read all the protocols and instructions you like, but sometimes there are little things that need to be learnt from an experienced person.

It may be some small thing you've overlooked or bad habit you've unknowingly picked up- I can't stress enough how important it is to have somebody experienced watch you and see if they can see any problems.

It has to come from somewhere and you've said yourself that everything you used (for your last attempt) was new and sterile- pipette tips, flasks, media, even cells.

I'm not meaning to sound harsh, but if you aren't willing to consider the possibility that it is something you are doing wrong, you may never resolve your problem.

Of course I'm not saying the contamination can't be from somewhere else, that is a possibility too. Suspect everything!!

Good luck!!

Thank you for all the responses. I can't stress enough how grateful I am that people are willing to help!

So here is what I've managed to accomplish in the last 2 weeks that I've been culturing RG2 cells (a cell line that we have a massive stock of):

I had a 2nd co-worker observe my technique for errors. He told me that he didn't see any errors but he suggested that when I alcohol up my gloves, I roll my sleeves up and apply alcohol to my forearms as well. Following his advice I went 11 days without any problems! After 7 days I felt like I had possibly solved my problem, so I again began culturing the fibroblasts (since my supervisors have been getting understandably a bit impatient with my lack of data). After completing 1 successful MTT the cells developed a weird "infection" a few days later. It wasn't the same thing that I've seen before, it looked like floating "chunks." Not a very good description at all, but I can't think of any other way to describe it. Regardless, the transparent "chunks" didn't kill the cells, so after several washes with PBS, I transferred them into 2 new culture flask. In 1 flask I added antibiotics and antimycotics, and the other flask did not receive anything except the regular culture media.

Thoughts on the rolled up sleeves and alcohol'd arms as well as the mystery floating bits?

Great news that you've managed to sort out your original problem!

Hmmm do you keep your sleeves rolled up whilst working? Perhaps the source was your lab coat. Do you change it regularly or use it for lab work other than cell culture?

Not sure about the "chunks". Did they disappear after your washing? Could you post up a picture?

I keep my sleeves rolled up while working in the hood. I do wear my lab coat for work other than cell culture.

I'll post a picture if the "chunks" return.

The chunks may well be FCS/FBS precipitate. You shouldn't need to ethanol your arms if you are wearing a lab coat and have the sleeves tucked into the gloves. You should be changing your lab coat on a regular basis, and if you are working with bacteria or yeasts, keep a separate lab coat just for cell culture.

My arms are disturbing long so my sleeves are not long enough to tuck into my gloves, even when I use those super long gloves.

I'm the same Uncle Rudder, I can never find a lab coat with long enough sleeves. I wouldn't worry too much, I quite often culture with no lab coat at all and its never been a problem for me.