Dear all,
What could these things be in a clean mammalian cell media? Yeast? Mycoplasma colonies? Media components sedimenting?
They are as big or a little bigger than lymphocyte cells. No organelles are visible, these oval things are very smooth and of different size. I am hesitant to use this media because it much more orange than the other vial of same media I made on the same day. It has been filtered through double 0.2 um filter.
I made a cell media and set up a mock plate with all medias I made and cells to monitor whether there is any contamination.
After 5 days, this media is not turbid, cells are growing well in it.
Not sure if these things are dividing.
I will submit it for mycoplasma testing.
What could it be though? Any ideas?
Thanks,
Maria
I've grown cells that were contaminated with mycoplasma, you won't be able to see it with light microscopy/phase contrast unless you do staining (DAPI and others). However, mycoplasma contamination is actually pretty widespread especially depending on the region of the world. It is a subtle contamination. If the pH changes quickly and turbidity increases rapidly, then I would try to see if it matches anything relating to bacteria as they divide rather fast.
Have a look at this source and see if it matches what you are seeing:
https://www.thermofisher.com/tr/en/home/references/gibco-cell-culture-basics/biological-contamination.html
I'm no expert, but they seem somewhat oddly shaped - they are not round, they are somewhat pointy. Maybe something from your media or some excretion?
Maybe you could try some staining. Maybe with DAPI to see if it contains DNA?
If it was (living) yeast, they'd definetly be dividing in a normal media and you would notice turbidity in the medium rather sooner than later. If you suspect any sort of contamination you should not use the culture media in an incubator with other experiments or use as a component in eg freezing media, that might be handed around in the lab. Also clean the incubator thoroughly after testing the media. The staining suggestions from Can and Tomás above seem indicated, a trypan blue staining will give you some additional insights and an occasional test for mycoplasma is not wrong when working in labs that are at risk. I would expect an occasional dead cell in a medium if somebody pipetted carelessly, but if they are occuring often, somebody would have needed to empty a lot of old supernatant medium into the bottle, or the cells would have died there after growing there for long enough to induce the colour change. However, most contaminants are sadly not that picky on where they grow and I expect you'd add Pen/Strep or similar right after opening a bottle and not after things already started growing. In general, if the medium has changed color, I would not recommend using it any further (even if cells would still grow in it), because the change in pH might introduce unnessecary variation into your experiments. Some other considerations: are you sure these come from the bottle with medium and are not present eg in you culture plates, pipettes or any other equipment? especially if you have filtered the medium, I would not expect a lot of debris. Have you opened the bottle freshly and has it been stored correctly? Did you have added any supplements to the medium (like FBS) that could have introduced these? If you are in a lab where people collect supernatant for various purposes, check with your colleagues whether this is an unlabeled case of collected supernatant. If the bottle has been opened freshly by you, I would check with the manufacturer for additional insights, as they know how the components behave under different storage conditions and usually reply rather fast.
Hi all,
Thanks for your recommendations, I will no use this media in cell prep and will follow your recommendations
It is not possible to recognise the creature without proper structure in your slide. Click a picture of any of the specific part of the individual colony in your culture.
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