Hi everyone,
I've been recently culturing 4T1 cells. The cells looked happy and healthy after thawing for a few days (i changed the medium every other day); however, after trypsinization, they had abnormal morphology and all died after a day. I've attached two images of the culture just before throwing them away. The culture was also a bit bad-smelling and somehow turbid.
I had the same problem with my other cell line NIH3T3. The cells are in good health initially but develop small particles and brownish clusters after trypsinization, and die after a day or two.
I assume this might be a contamination problem, probably with yeast.
Can anybody tell me what type of contamination they think this is, and how we can get rid of it for our future cultures?
Dear Mina,
It seems that the problem relies on your tripsinization step.
Could you please provide more details on your tripsinization protocol? Is the trypsin new or from an old stock? How long do you incubate your cells with trypsin? Do you add medium after your incubation time?
All these steps can impact your outcome.
Best,
Hi Wolfgang,
Thank you very much for your reply.
I do the trypsinization with 0.25% trypsin-EDTA solution, adding about 1.5 cc of trypsin and incubating at 37°C for about 2-3 minutes. Then I add twice the volume complete RPMI (3 cc), and centrifuge at 200g for 5 minutes to settle the cells and throw out the trypsin.
I am really confused because I have checked these steps a thousand times. I am getting suspicious to the quality of the trypsin.
Hello Mina Hosseini
Yes, it is yeast contamination. Yeast is a much rarer type of contamination found in cell culture. The incidence of yeast usually increases in spring and summer in labs that have an inferior hygiene concept. They are also introduced by errors in aseptic techniques. Yeast contamination in cell culture is generally via an airborne route but yeasts can readily colonize an incubator and can then spread to other cultures by contact of contaminated flask or dish surfaces during cell culture manipulation. Yeast cells multiply faster than mammalian cells and slower than bacteria but usually a contamination becomes clearly obvious within 2-3 days under the microscope, or it is indicated by the color change of the medium.
You need to re-check your hygiene plan as yeast contamination is usually brought to the lab from outside.
For more information on yeast contamination and the required treatment is provided in the link below.
https://unclineberger.org/tissueculture/contaminant/yeastcontam/
Best.
In my opinion the photographs give an impression of yeast. In order to confirm whether this is yeast, you may please inoculate the suspected culture on Sabouraud dextrose agar or Pal sunflower seed medium ,and study the macroscopic as well as microscopic morphology of the suspected yeast colony. Pal sunflower seed medium was developed by us in 1975 as a selective medium for the rapid isolation and presumptive identification of Cryptococcus neoformans from clinical and environmental materials.Now this medium is widely used for the isolation of yeasts and also non-dermatophytic filamentous fungi, such as Aspergillus, Fusarium, Alternaria, Curvularia, Mucor, Rhizopus etc.
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