Hello! In recent days, I'm having problems with yeast contamination in my fibroblast culture. Then, I decided to use Amphotericin B (fungizone) at a concentration of 2.5 ug/mL.
This antifungal is kept frozen at -20 ° C and an aliquot is withdrawn from the stock to prepare a complete medium (DMEM with 10% FBS, 1% P / S and amphotericin at the stipulated concentration). When prepared, this complete medium is kept refrigerated at 4 °C.
When I`m going to change cells media (every 2 days), I need to warm the medium to 37 ºC, ideal temperature of the culture. After use, return the medium to the refrigerator.
Some manufacturer's manuals say that amphotericin B is only stable for up to 3 days at 37 °C. Thus, I fear that with this process of heating and cooling the medium, amphotericin B loses its stability and antifungal activity, although the stock of complete medium is maintained a few minutes at 37 °C until the medium is exchanged.
Thus, I would like everyone's opinion whether I should keep amphotericin B in the stock of complete medium because it don`t lose activity in this process, or should I withdraw an aliquot from the frozen stock of amphotericin and add directly into the culture bottle in the ideal proportion of use, avoiding to maintain at a higher temperature and takin risk to losing activity.
Hi, Caio.
First I recommend making concentrated stock solution of fungizone (~2000X stock solution of 5~10ul aliquots at -70C).
It would be nice not to use fungizone at all. We often have the same problems. We regularly do fume sterilization of the incubator, followed by 70% EtOH cleaning after shutting-down without any culture for a day. All trays and water bath, etc. If yeasts persist even after the sterilization, cleaning the lamina flow and precautions of sterile media preparation, EtOH spray onto the bottles of media, PBS, TE bottle and gloves etc, clean pipette dispensers by soaking in 70% EtOH and air dying completely. Despite all these efforts they persist, then check lamina flow filter to be replaced.
Finally and most likely, the cell stocks may already be contaminated with yeasts from the frozen stocks. Get new cell stock from company. They would not disappear with fungizone, they are so happy in someone else's medium.
Hello Caio!
I'm a beginner in cell culture and I just saw this question and I'm having the same concern If I should add AmpB directly to the media I want to store at 4'C for further use or use an aliquot for every single time while preparing the media? Wondering if you have resolved this and share your experience. Thank you.
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