Blood sample was used to isolate cancer cells by AC electric field. Isolated cell suspension was then cultured in RPMI 1640. After few days moving round objects were found and keep increasing in their numbers. It is possible that during isolation process there might be contamination from surrounding workplace. I had sterilised the equipment with 0.5% Virkon for 5 min before isolation. Is it possible that there will be contamination during blood collection or sample transfer? Just want to have an idea which moving objects they could be?
I have also seen small round objects in primary cell cultures. They look like swimming and vibrating around cells. They may be microparticles from dying cells doing Brownian motion. If the pH remains fine and the medium does not turn turbid then it is less likely to be contamination.
I have also seen small round objects in primary cell cultures. They look like swimming and vibrating around cells. They may be microparticles from dying cells doing Brownian motion. If the pH remains fine and the medium does not turn turbid then it is less likely to be contamination.
I would also bet on debris. Contaminations are easy to recognize by ph-drop (as Ru-Jeng told) and more importantly: bacteria are "swimming" directed and very fast. In addition their size is rather small and the numbers will increase rapidly.
The increasing numbers is worrying, but I can't tell for sure from the video if they're bacteria (but thanks for including the video, it's usually very helpful for people to actually *see* the problem).
Can you transfer a small drop into some fresh medium and another into some fixative, and see if they increase in numbers in the medium?
Also, can you see if they stain for DNA?
Yes, it should be the dead cell debris, floating around the cells. If it is bacterial contamination, I think it can be easily recognized under the micropscope. I agree with Ru-Jeng and Bjorn, pH is also an good indicator, if it is unchanged there should be no need to worry about.
I see the same even when using three different antibiotics. I haven’t detected any bacterial nor yeast DNA by means of DAPI stain or PCR. In my case I think it's cell debris because when I increase light intensity under microscope the particles increase their movement with random brownian-like movements (please excuse my English).
i agree that it is debris from dead cells; organelles are released and persist after a cell has died and ruptured. I've had the same worry with primary cultures from time to time. However, if those particles were bacteria, the culture would be completely filled, obviously turbid without the need for microscopic examination, and with a very low pH, by the next day.
Possible sources of contamination could be from blood collection, outer surfaces, techniques but if it was sterilized properly than it shouldn't happen. If it’s contamination, you should be able to see it under the microscope, debris can occur if the material is not digested properly. To eliminate mycoplasma growth you can carry out Hoescht assay, If it is fungus, (branch like), clean with ethanol, the lids of the material, and thing’s then use an anti-fungal agent such as Nyastatin.
Biologicals. 2010 Mar;38(2):273-7. doi: 10.1016/j.biologicals.2009.09.006. Epub 2009 Nov 18.
Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant.
Gray JS, Birmingham JM, Fenton JI.
Hi have you checked your culture for mycoplasma. Would suggest doing that
I used to see the same situation in cultured neuroblastoma cells. When I tested the cells and media I found no sign of mycoplasma, which was a relief. I too go along with the concept of cell debris, because there was no change in pH and no turbidity in the media.
Me too sow these vibrating particles in my cultured cells, also I am with the debris description
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