Hello, I’m currently suffering a problem with my cells. The cells in question were recently transduced with lentivirus to obtain cells stably expressing a construct. After several weeks of selection with antibiotics, I was ready to start with experiments but from one day to the next the media became cloudy and clusters of cells were dying. The cells that still were adherent looked healthy and were able to proliferate. We initially suspected a bacterial contamination, but the cultivating medium was unaffected. There we’re also no signs of yeast or fungal infection. Next, I’ve performed a test for mycoplasma which was positive, but normally mycoplasmas don’t cause turbid media so I’m doubting whether this is the only explanation for my problems. Is there anyone else that come up with an explanation? Many thanks in advance!
Dear Anneleen,
Regardless of the identity of the contaminant, my advise is to throw away EVERYTHING, clean up and decontaminate twice (and this goes also for your laboratory coat and the incubators, microscopes and all surfaces in the lab) and start all over again this time controlling every step. I attach a very useful booklet pulished by Conrning.
Feeling very sorry for you,
Agnieszka
If you use an electronic pipettor you should also check the filter. This item is missed many times in the hunt for possible cell culture contaminants. Have discovered that even with the use of serological (filtered) pipettes the contaminant can sometimes get into cultures.
Hi Anneleen.
In our laboratory we have got a problem with bacterial contamination, which presumedly was caused minimal contamination of laboratory plastic.
We always used medium without antibiotics, therefore contamination was found almost immediately after beginning using. But for a long time we found contaminated reagents, freesed cells etc.
For identification nidus we did some actions, which are the imitation of the usual operation with plastic, and included contact with media, and after that media was incubated for 2-5 days. Thus was identifed the most probable nidus. We tested 3-4 samples of each plastic units in each lot.
Only orderly execution of this tests may help you to find the nidus. If you have contamination in 2 and all the more in 3 lots, you will come to a conclusion, that the nidus is in the instruments, air, furniture etc.
Hello Anneleen,
I agree with Mr. Agnieszka. According to my experience working with human umbilical vein endothelial cells (HUVECs) and endothelial progenitor cells (EPCs), I would like to tell you that the most scientifically based option is to start the culture from the beginning! You must clean and sterilize very well your culture room and the incubator. You must also prepare new clean buffers and solutions and check media are free of bacteria. All the workers in the lab should use right cell culture techniques and keep culture lab clean. When everything is ok, take a vial of cells from the nitrogen and put them in a plate to grow. Finally, if you want to check whether your medium is free of bacteria or not, you put only growth medium in a sterilized plate without adding cells. Then, you put it in the incubator at 37o C and examine after 1 day the colour of your medium. If it will change and it is different, it means that it is contaminated.
I hope that I helped you!! Good luck with your experiments!
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