Hi, all,
I cultured PC3 cells with a Quasi-vivo plate. The plate was rinsed 15min with ethanol. After one night culturing, the bottom of the wells were covered with one white layer visible by naked eyes, and when observed under phase contrast microscope, the image gets very dark and increased exposure time doesn't help at all.
It seems that something smaller than cells were growing, if I make a scratch on the bottom of well, and shake it a little bit, the white thing can be detached and pipette out of the culture medium.
So, what is it?
Thank you all,
Kind regards,
Rose
usually, contamination will be floating. you can stain the white with DAPI, if can be stained.then it's some contamination.if not, it's some element from medim or the plate.
It is contamination probably. Start again with fresh cells and fresh medium, clean incubator, decontaminate,
Check if you added antibiotic/antibiotic mixture in medium,
I'm not sure exactly what your protocol is as I've never washed cells with ethanols but I believe ethanol can precipitate proteins in the media - are you washing the ethanol off with PBS or something before replacing the media?
I use ethanol as fixative, when I want to stain the cells, but before I fixed the cells, I washed the cells with PBS, otherwise all proteins in the culture medium will be coagulated, forming a very thin layer floating in the medium, which appear brownish under the microscope, and white with the naked eye. the thin layer can be attached to the cells when you aspirate the liquid, and then it will be difficult to be removed.
I used to stain cells directly on wells, and take photograph of the cells.
I never wash the cells with ethanol, if I am still culturing the cells, as ethanol will kill the cells.
If you are using polypropylene plates the acetone and ethanol causes a reaction making it black and rought
Hi, all,
Thank you for the replies. I am sorry for a late response.
I didn't add ethanol to the cells. I meant that I sterilized the plate by 70% ethanol, and let it dry. Then I seeded cells into the plate and let it incubate overnight. The second day, I observed the white layer on the bottom of the wells.
I more believe the white layer is because of contamination, but I don't know what kind of contamination it is. Kind regards, Jing-Huan
Hey! I concur with most comments above that suggest that the ethanol may have caused proteins to precipitate from the cells. Tissue culture dishes normally come sterile and do not have to further sterilised. We sterilise glass cover slips with 70% etoh, but make sure to run them over a flame briefly before using them for culture purposes. You could maybe try to obtain some pre-sterilised dishes and culture your cells in them without using etoh at any step.
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