Hi
I have AGS cell lines in culture for our experiments. Recently, I started facing fungal/ yeast contamination of the AGS cell line in culture continuously, once they are 90% confluent. The contaminations kept appearing even while using new vials of frozen cells. I tried to find out the source of contamination by incubating the RPMI 1640 media, FBS, PBS, trypsin and antibiotics in the incubator we keep the cell lines under the same conditions used for the culture of cell lines (37C, 5% CO2) but couldn't find any trace of contamination. I sterilized the safety cabinet thoroughly, and used the UV sterilization before starting the work and after finishing. I used amphotericin B in the culture but the contamination reappeared after few days. Have anybody faced similar incidents with AGS cells? Please advise on what to do about the contamination.
Did your contamination happened after storage?
With considering your explanation it seems your stocks are contaminated.
Hi Alireza Mordadi
I suspected the same and so we purchased fresh stocks from ATCC. But the contamination repeated with the new stocks also after few weeks in culture.
We have other cell lines in culture that are not affected by contamination and we share the incubator with other people who do cell culture. They had no contamination issues even while we were getting contamination in AGS cell line. So we are not sure of the contamination source.
Do you use filter flask, right?
In such cases we allow another person from out of lab observe the processes and do it and we observe.
It is very helpful to finding the source of contamination.
Alireza Mordadi Yes. We use filtered flasks.
Our other lab members tried to culture the cells, but the contamination returned after few days.
Put a flask with just media in your incubator without cells.
And check for contamination.
Have you tried making a fresh media bottle with fresh FBS and PenStrep? I agree Alireza, given the conditions you provided, one of the only possibilities left is the media. Also, you may also want to change the water bath eith new water and Aquaguard. Do you coat your flasks with any attachment factors? Also, when you describe contamination, if you zoom in at 40X on a light microscope, do you notice rod shaped structures (bacterial) and/or branched structures (fungal).
Hi Alireza and Jack
I have kept indicator flasks containing media and all the other reagents in the incubator. But theres no trace of contamination. Each time we get the contamination we discarded the used bottle of the media and made everything fresh.
I see spores and branched structures when observing under the microscope.
@Jack
We change the water in the incubator every week. I do not use any attachment factors for the cell.
So your source of cell is totally contaminated. Try provide your cell from another source.
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