We are working with mammalian cells and we observe the contamination above all in haematological cell lines (K-562, TF-1 and also in CD34+ cells isolated from cord blood).
-- Normally, these cells are round but, after contamination, we observe really big cells, cells that look like fibroblasts, cells with protrusions (pictures attached).
Particles do not seem to move a lot but it is difficult to determine whether this movement is "autonomous" or not. They also seem to adhere to the support.
We try to observe if something grow in fresh media but we did not observe anything. It is a good idea to remove the cell line but we do not know how to distinguish these particles from the debris.
We do not have the material to study bacteria but we sent some samples to several labs:
- Nothing grew on their media
- 2 labs dit not observe anything with gram staining but one found gram + bacteria (but the staining was not good). These Gram + bacteria are found inside and outside the cells (pictures attached)
- We hypothesised that coud be mycobacteria. Then, we sent samples to a another lab to perform Ziehl staining. However, this test appeared negative.
- We do not know exactly whether microbiological tests were done on our media our on cells.
- We did mycoplasma test in our lab and the results were negative.
- We do not have the material to perform the universal PCR.
- Concerning cross-contamination, we don't think so because we are also working on primary cells, which are also contaminated.
Le jeudi 30 juillet 2015 17:32:58 UTC+2, Mike Horwath a écrit :
A few questions to help with diagnosis:
What cell lines are they? (mammalian, insect, etc)
Are these particles actually motile/swimming, or just drifting? It is hard to tell from the video.
If you remove the cell line (straining, hypososmotic lysis, etc) is the contamination able to replicate by itself in fresh media?
If you streak the culture on agar, do you get colonies?
What did you mean by this: "At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative." Mycoplasma will NOT show up on a gram stain. Was there another test the lab did for mycoplasma?
Dakota's suggestions to check for contamination in your media and check for 16S RNA are good ones.
One of the hardest kinds of contamination to detect and get rid of is contamination from another cell line. It's possible you could have a 2nd cell line mixed in, that has a different morphology and might be producing more apoptotic blebs...
Mike
On Thursday, July 30, 2015 at 10:54:26 AM UTC-4, Dakota wrote:Check some controls. Look at just the growth media fresh, then after a few days. Aerosols from pipettes could be building up but I imagine you use filter tips.
Also, just spin everything down, extract DNA, and run 16s or ITS bacteria/fungal barcoding to look for a band. Sequence it if you have one and you could get the exact species
On Jul 30, 2015 1:35 PM, "Cindy GRANDJENETTE" <tupa...@gmail.com> wrote:Thanks for your answer.--We have this problem for 4 months. At the beginning we thought that the particles we observed were mycobacteria. But the gram tests from the microbiological labs appeared negative. Of note, our cells look grainy.
We are not able to determine whether these particles are the source of contamination or just a consequence.
We use penicillin-streptomycin and fungizone (Amphotericin B) in our culture media.
We already cleaned the lab (laminar flows, incubators, etc), autoclaved everything, and threw our cells away. But the problem is still here. And we do not know how to solve it.Cindy
Le mercredi 29 juillet 2015 23:06:07 UTC+2, Ravasz a écrit :Hi,
No idea what this is, but I can tell you what its not, maybe that helps:
- It's definitely not yeast, that looks very different. Yeast would consist of small groups (4-10) of oval particles, smaller than a mammalian cell, with a well defined, smooth outline. They would also outgrow your cells and kill the culture in a matter of days. This is not like that.
- It's not mycoplasma or a virus as both are too small to resolve in a light microscope. They would simply not be visible even in a rampant infection.
- It is not usual cell debris as it is moving.
My best bet is that it is some form of bacteria. Were you using penicillin-streptomycin in your medium? If not, try adding that. You can also add some Fungizone too if you are desperate, as it might be some fungus.
However, such "resurrected" cultures are never really perfect as the cells were put under stress and they might be permanently altered, even if appearing completely cleaned at some point. I would just discard the whole culture along with any media and buffers that are in use, autoclave any equipment that came into contact with it and thoroughly wash incubators and culture hoods with Virkon, or your disinfectant of choice.
If this is showing up in multiple cultures, then the source of contamination is not within the culture itself.
Maybe this helps.
Mate
On Wednesday, 29 July 2015 14:21:08 UTC+1, Cindy GRANDJENETTE wrote:Dear all,We have problems of contamination in several of our cell lines. Cell viability is pretty good, however, cell morphology is affected whatever the cell line. In addition and importantly, we observe a lot of small particles in the medium. Their number increase over time.Mycoplasma tests were negative. We also sent our samples to 2 microbiological labs. Nevertheless, they were not able to identify the source of our contamination. We are wondering whether these contaminations are due to (myco)bacteria, viruses, or even yeast.You may find attached movies showing how these particles "appear". The morphology of this cell line is normally homogeneous with round cells.Does somebody have an idea concerning this contamination ?Thanks for your help,Cindy
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