I have revived an adherent cell line from second day I noticed a whitish cloudy layer in media. When i disturb the media the cloudy layer get easily dispersed into the media (more no. of dead cells floating from day3) unlike bacterial contamination which usually won't get dispersed as i noticed in my previous batches of culturing same cell line. my question is whether it is dead cell floating like cloudy layer or is it contamination.
Hi, you can try this to see whether it's contamination or not:
1) Take media supernatant and leave some to grow with FBS at 37°C in your incubator. If you have some bacteria, this should make them grow and you should be able to see it under the microscope/media color change/turbidity...and time to decontaminate your cell culture equipment etc.
Alternatively, you can also take some supernatant to spread on an agar plate and leave at 37°C in your incubator dedicated to microbiology if your lab has one.
2) Wash cells 1-2x with PBS (shake the plate well) before changing medium. Perhaps you have a lot of cell debris/death after thawing your vial which may stress the remaining living cells etc... (dead cells generally accumulate in the centre of the wells). If you have contamination, the turbidity will come back anyways, but if this gets rid of your problem, it may be just due to the thawing (or prior freezing) process.
Do you have any images of what you see under the microscope?
Good luck!
If the dispersed cloudy layer doesn't show any bacterial cells, it could potentially be cryopreserved cells that have surpassed their storage lifespan. Typically, if these are dead cells, they might follow the movement of the shaken plate. However, if they are bacteria, they usually exhibit flexible movement even when the plate is moved in a specific direction. Dead cells would often appear as shattered cell debris.
I've had it too. I did as Olga Roman said in the second version. I washed the DPBS well and added the medium again. Unfortunately, the turbidity returned and all the cells died, from which I concluded that it was contamination (although the medium contained streptomycin and penicillin). I conducted a number of experiments - I added a separate serum in which I perform cryopreservation, DMSO, to an empty medium, and held the tips from the dispenser for a while. It turned out three vials of which the one with the tips was subsequently contaminated (they were not sterile due to the fact that the laboratory assistant forgot to do this).
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