I'm doubtful that the source of contamination is from Liquid nitrogen. Because all the cells what i stored have bacterial trace in it and the LN2 sharing lab also have the same kind of trace. Cross checking is done in CO2 incubator, LAF also. Is there any possibility of LN2 contamination and how to get rid of, Kindly help regarding the same
Hi Priya, the contamination looks like yeast. So, in addition to P/S, use Fungizone in culture medium (final working concentrations between 0.25ug/ml and 2.5ug/ml).
As they are Primary cells, the contamination can occur during isolation.
Filter media, sterilize solution.
Shanmugapriya Shanmuga Sundaram
If these are primary cells of 2 to 5 days in culture, then it could be the cell debris. Looking at your image I doubt it could be bacterial contamination.
Probably if you are very sure about the contamination, I mean if you see the movement of bacteria, then the best option is to change the antibiotic from P/S to either gentamycin or Amphicilin, wash it with antibiotic PBS solution twice, and incubate it with media containing GENTA or AMPHI. This way you can slowly reduce the bacterial load and will be able to rescue your cells. Also include amphotericin B as antimycotic.
There is evidence of cross-contamination using LN2, this happens when the LN2 chamber is overfilled. This can be avoided by maintaining the appropriate LN2 level i.e., the cryo boxes inside the LN2 are in the vapor phase without getting in contact with the liquid LN2.
Cheers
Microbiological contaminations can be introduced into cell cultures by unsterile working techniques, contaminated water baths, media, plasticware, etc.
Please check this link for a trouble shooting guide with more detailed information:
https://www.promocell.com/app/uploads/2018/01/Trouble_Shooting_contamination.pdf
You can try to save the cells following the suggestions from Chandra Somasundaram and Ashis Kumar. But it's really tough and a success is not guaranteed; so, unless the cells are extremely valuable and/irreplaceable, it's not worth it. Most likely Hamadi Madhi will be right.
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