Hi all,
I am dealing with a very stubborn fungal contamination issue in my mammalian cell cultures. I could really use some pointers for how to get rid of the contamination at present and how to prevent it in the future. I have attached photos.
Measures I have already taken include:
- Deep cleaned the inside of the incubator and culture hood with a strong, broad spectrum, quaternary ammonium disinfectant.
- Clean the water reservoir, autoclave the water before refilling, and adding water conditioner.
- Threw away all disposable materials such as pipette tips, falcon tubes, culture flasks, well plates, contaminated reagents, etc., and replaced them with new, sterile materials.
- Autoclaved all non-disposable materials such as aspirator tips, micropipettes, beakers, shelving units in my incubator, aspiration flask and tubing, etc. and replaced them with new sterile materials.
- Washed all lab coats.
- Purchased new reagents and filtered all remaining reagents and media.
- Threw away my cell cultures and restarted with plating brand new frozen cells (passage 0, primary MSC line).
- I already use pen/strep in my medium.
- I plated 'contamination test' well plates with fresh medium, containing no cells, to see if I observed any signs of contamination growth for 1 week in the incubator prior to re-starting the cell cultures. I observed nothing at all. No media changes, no turbidity, no observations under the microscope or visible to the naked eye so I thought I was in the clear.
- All newly passaged cells were seeded on -sterilized sample discs, as can be seen in the photos, which were sterilized in samples containers, in the autoclave at 121C for 45 minutes.
- I have ordered (1) Normocin Antimicrobial Reagent and, (2) Amphotericin B Solution to add to the culture medium. I have yet to add these new antimicrobial bioreagents to the medium but will do so as soon as they are delivered.
With all the measures I have taken already-- I still have no clue where this contamination is coming from nor how to get rid of it other than completely starting over again as I had already done. This is the first time I have ever seen colony formation and hyphae in my cell cultures. Can anyone please give me some advise?
Hello Genesis Marrero
For the protocol on fumigation you may follow the link below.
https://www.researchgate.net/post/Fumigation_of_Tissue_Culture_laboratory_to_eliminate_air_born_contamination
Best.
I had a similar contamination issue. When I cultured the contam on an agar plate it turned out to be yeast. Nystatin is best for yeast contam.
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