Hi all,
I have been struggling with a contamination that looks like black dots in my CHO cell cultures (See photos).
It does not change the colour of my media or make it go cloudy. The cells in my T75 flask seem to grow (but sometimes slowly) but once I seed some cells into a 6 well plate (so having a decreased number of cells and smaller volumes) the black dots appear rapidly and the cells start to not look good and do not transfect well. These black dots particularly appear in the DMEM/F-12 nutrient mixture than standard DMEM
Does anyone have an idea of what this could be and any possible solutions?
I’ve done all the obvious things, new cells from frozen, new TC and transfection reagents, deep clean of the equipment etc but they still keep appearing
Hello Joanne Clark
In the following paper, the investigators report the identification of a bacterial cell culture contaminant, a member of the Achromobacter genus. Cell cultures with this type of contamination had qualitative growth differences, such as slow growth and reduced cell viability.
The bacteria in this genus can survive in a wide range of environments including tap water, waterbath, FBS, new born calf serum, and many common laboratory solutions. In addition, commercial cells are found to contain this contaminant. It is important to be aware of this multi-drug resistant bacterial contaminant. Selective killing of these bacteria in eukaryotic cell culture is successful only with a combination of two antibiotics, piperacillin and ciprofloxacin, both at a concentration of 10 µg/ml each.
Please refer to the article attached below for more information.
Article Got black swimming dots in your cell culture? Identification...
Best.
If you see these dots moving, it is probably bacteria,
This can be verified by DAPI coloration under microscope (but this will destroy the sample).
Have you added antibiotics & anti-fungal in your culture medium?
Best
You can filter sterilize everything you use on these cells; complete media, PBS, trypsin, and anything else filterable. If you still have contamination, it is probably the cells themselves. If so, you need to thaw a different passage or find a new source of cells.
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