I am facing a problem with cell culture for weeks now. The media of the cell culture turnes turbid (white little spots, see picture) but stays reddish/pink. First this happened after 2 weeks of culturing the cells, than after 1 week and then after a couple of days. Under the microscope cells looked normal, but there were tiny little black dots everywere (see picture). We think it is bacterial. To exclude a error in my technique, my collegue started a new culture with new media, but again we observed a contamination.
We then cleened the entire cell culture room, the wather bath, the incubator, the hood,... But the problem persisted: we started 2 cultures and experienced a very low number of muscle cells in one plate and in an other almost all cells died (even with DMSO in the media). I added a picture of that to.
We use pencicillin/streptomycin.
Does anyone have an idea about what this is? Any suggestions of what we can do about this?
May be use an earlier passage number cells to eliminate the chance of contamination in the cells themselves. Were the cells initially tested for mycoplasma?
Thanks for your answer! It is true that we used cells of passage 5-10 (préliminary tests). We now started a new culture with cells of an other subject to see if our problem is due to a contamination in the cells themselves.
We never tested the cells for mycoplasma. But I thought that mycoplasma was invisible under the microscope and here I observe clearly anormalities.
Hope to hear from you!
Hi, maybe the source of your cells is contaminated. have you ever tried new source of muscle cells?
It is possible that your cells are stress due to lack of some nutrients in the media. Stressed cells often release vacuoles as indicated.
But I think it is probably due to mycoplasma contamination. You can test it by ELISA or PCR commercially available kits. You cannot see mycoplasma under standard microscope, only indication is some of the changes you mentioned. If you can afford, it is then best to discard and start over. If not the are treatments available to get rid of mycoplasma which could take weeks and several passages. We sell this product - http://antibodyresearch.com/mycolytix-mycopalsma-inhibitor-solution-1000x/. You can also include this as a precaution when starting new culture. Thanks
Sanaz Hassani Thanks for your answer. This afternoon I'll look how the cells from another subject are doing. I hope they'll look healthy!
I looked at a contaminated dish this morning and I really think I am dealing with a bacteria instead of mycoplasma (I keep it on my bench because I'm curious to see how it develops). I added a picture and a video and hope that you will help me to identify this little b*st*rd :)
I also don't think that it's mycoplasma because you can not see them under regular microscope. I hope the new source of cells is good.
The cells from the new subject are looking fine! I'm so glad :) I hope they stay like that when I come back on monday. *fingers crossed*
If you use DMSO for freezin your cells make sure that it is not contaminated .
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