Hello everyone and thank you in advanced for any help given. I have some cell culture contamination problem (stem cells under normal culture medium): Macroscopically, they look like small white patches attached to the culture dish that do not move; Microscopically, as the attached pictures. I have discarded all the materials used while performing this culture and performed exhaustive cleaning.
I believe it is a fungal contamination. However, I would sincerely appreciate any second thoughts or any other recommendation to avoid having this problem again. Thank you so much!
Hello Maria
Fungus and mold spores are ubiquitous in the environment and generally infect cultures via an airborne route. Heating and air-conditioning systems are notorious for having high concentrations of spores. To prevent fungal infection you should adopt standard practice in cell culture laboratory namely, routine environmental monitoring. For this purpose, equipment like biosafety cabinets and HEPA filters should be checked regularly every six months to ensure proper functioning. Maintaining proper sterility in the laboratory becomes essential. To prevent the development of severe infection it is therefore critical that the cells be scanned regularly, macroscopically and microscopically, during routine media changes or passaging, for the potential presence of low-level infection which can be controlled.
The two most common antimycotic agents used in cell culture that are effective against fungi or molds are Amphotericin B (Fungizone) and Mycostatin (Nystatin). Fungizone may be used in cell culture media at final working concentrations between 0.25ug/ml and 2.5ug/ml. Please note that Fungizone is typically very toxic in cell culture and therefore should be used conservatively. Nystatin may be used at final working concentrations between 100U/ml and 250U/ml. However, if the fungal infection is severe, the best way out would be to discard the culture and start again with fresh frozen stock culture.,
Good aseptic technique and careful handling of cells and reagents will help you work better without facing any problem of contamination. Once or twice a month you should fumigate the tissue culture laboratory taking necessary precautions.
Best.
I see large masses of cells plus cell outgrowth on the dish, but no contamination. Could you place an arrow next to, or circle, the contamination?
I agree with Ellen A G Chernoff this looks like overgrown cells. fungi should be very large compared to cells and look more stringy. next time you can place some of each reagent only in a dish and see if things grow to check for contamination
I am sincerely grateful for all your answers and guidance. To be honest, my experience on cell culture is not vast. I have uploaded the pictures showing what I thought was a fungal contamination.
Ellen A G Chernoff Julie Ann Dougherty could I please ask you, If these are cell outgrowths, why are the cells next to them detaching? Again, extremely grateful for your help, it is relieving to know it might not be contamination. Blessings!
The cell aggregates are detaching (or moving) because the cells are more strongly adherent to each other than to the culture substrate.
Hello Maria,
Those are not contaminations.
Those are micromasses that consist of cells.
I usualy get the same pictures as yours when I culture mesenchymal stem cells, and I keep culturing after they are confluent. The cells spontaneously differentiated into chondroblasts that form micromasses that will stain positive with alcian blue. The smooth brown color is the chondrocyte matrix. In my experience, this fact happens to umbilical cord, adipose tissue and bone marrow derived MSCs, and may be also other tissue derived MSCs.
Thank you again, for your explanations, answers and guidance! Sincerely appreciated.
Hello Maria,
I agree with Dr. Ellen A G Chernoff , Dr. Julie Ann Dougherty and Dr. Jeanne Pawitan . It is something like a mass of rapidly dividing cells. So please ignore my previous comment which is meant for cell cultures with fungal contamination.
Fungal contaminations will usually cause change in the media colour and cause the media to be turbid. You can also observe fungal hyphae under light microscope.
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