Good day to everyone,
I am quite new to cell culture and I hope someone could help with the problem I am facing. I recently started new cultures of K562, Daudi, and NALM-16 cell lines in RPMI1640 with 10% FBS. After 3 days, the cells grew well (density ~ 10^6/mL) with high viability, and the media had a "salmon" color. However, I observed that the Daudi flask had a certain level of cloudiness to it. When I moved the flasks around, I could see grainy particles moving. Under the microscope, I did not see any yeast or small moving organisms.
Could someone please tell if I am having a contamination?
Best regards.
Hi there,
Welcome to cell culture. Unfortunately, these are are hallmarks of contamination. Often contaminated cultures will form white-ish cloudy bands of dead cells. Under the microscope you will be able to see wispy projects of fungi, or small moving bacteria. However, when the cultures are cloudy this indicates severe contamination and actually makes it more difficult to identify under a microscope. If your media contains a pH indicator, a sudden shift in the media colour will make this obvious.
First steps are to discard any contaminated cultures. There is no point attempting to save these as they will have undergone any number of stressors. Next time you may look to practice with PenStrep antibiotics.
Just to rule out the media as the source of the contamination: I guess you are using the same batch of RPMI1640 and FBS for the other cell lines, where there are no signs of contamination?
Otherwise, as Lewis Williams already said, it looks like your Daudi cells have a bacterial contamination - that's what the "grainy particles" are, I suspect.
If your cells grow as suspension culture, cloudiness is not necessarily bad, just a sign of more cells growing in culture. Cloudiness alone is not a sing of contamination in a suspension culture as it probably is when cells grow attached to the plate. You should watch for other factors such as changes in the color of the medium and observe cells to see if any strange structure can be observed.
Thank you all for your inputs.
I transferred 1 mL of culture supernatants to LB broths and also did a PCR with 16S primers. I see no growth and the PCR came up negative. Maybe it is as Diego Raffo suggested that it was my cells being overgrown?
I will also do a mycoplasma test to be sure.
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