Hello everyone, I recently found small white dots around the cells as shown in Fig01 and Fig02 while culturing HeLa cells. The 60X magnification is shown in Fig03 and Fig04. It seems that most white dots are cell debris.
However, I observed that there are small particles floating around the cells, about 1 to 2 um in diameter. It can be checked in Video01. Is this contaminated? Can anyone identify what these small particles are?
The culture medium I used was DMEM + 10% FBS + 1% P/S. The Hela cells can't grow normally these days, hence I'm trying to troubleshoot.
Hi Di Yin
Seems some bacterial infection, might be micrococcus. You can check further by Gram's staining.
Hi, it looks like bacterial contaminations from your low power pictures. however, from your 60x zoomed in pictures, it doesnt seem to have bacteria growing. Since you have antibiotics in your medium, it would be less likely if you are having bacteria in your Hela cells.
I would suggest to take out a new vial of cells from your stock, use all freshly prepared media to grow the cells again to see if the cells behave better.
It takes several days to learn what and the effect of floating particles. If it is contamination, it will increase and result in damage and death of cells .
The possibility of it being from cell residues and the remnants of their debris. Also, it may be the remnants and sediments of the materials used in the culture medium, especially FBS.. Therefore, it is preferable to use 0.22 micron filters when preparing the culture medium to purify the used materials, especially FBS.
Hi Salequl Islam , Thank you for your reply. I would like to know whether Galanthus will proliferate in large numbers if the culture lasts for 5 days.
Another factor I wasn't sure about was the fact that, despite the poor growth of the cells, I didn't find a large population of bacteria after a long period of culture.
Hi Di Yin
I do not have experience of checking Galanthus effects there. To avoid the situation, you can take a fresh lot of cells with freshly prepared media. Sometimes cellular senescence (when cell passage number is very high) can make this type of granular debris. To avoid it, you can go for single-cell cloning (dilute you cells so that 100 microliter contains 1 cell, then allow each cell in a 96-well plate to grow further. Then upscale cell growth from 96-well to flask gradually). This newly formed clones will remain healthy for several subsequent passages. I experienced it.
It seems like contamination with either bacteria or yeasts. You can check whether it is true with fluorescent microscope and staining of cell culture with DNA-binding agents (like Hoechst or DAPI). If you will see that these small particles are glowing in blue, be sure that this things are alive.
Hi, it seems a contamination by bacteria, Sow the medium of your HeLa culture on blood agar, if there was bacterial growth proceed with the tests for identification. Sow the medium also on sabouraud for any growth of fungi.
No question, your cells are not happy.
The cell morphology and debris suggests cell death by necrosis.
I see no evidence for yeast (large refractive bodies), fungi (filamentous hyphi) and little evidence for the usual bacterial suspects (Staph, Bacillus, Coccus)
Based upon my experience, I suggest that you probably have a slow growing bacteria that secretes exotoxin (probably a soil pathogen, possibly Pseudomonas). It is the exotoxin that is killing your cells, not the pathogen!
The small black dots jiggling around your cells are proteinaceous aggregates, likely debris from dead/dying cells, exhibiting Brownian motion.
You should check the colour of your media. If it is changes rapidly to yellow, then definitely bacterial contamination. If the colour does not change yet cells continue to die, it is most likely exotoxin (this can included residual detergent from stock bottles!).
No solution, other than discard and start with fresh culture from LN2 master bank.
And make sure that you wash your hands!!
Hi David Ian Leavesley, Sergey A. Tsymbal, Elena Fujiwara
,Thanks for your kind reply. To further identify the source of contamination, I stained the cell with DAPI and took bright-field images and fluorescence images at 10X/40X/60X respectively.
The specific pictures are shown in the attached file. Can you identify whether it is the bacteria (mycoplasma) contamination?
It looks like bacterial contamination, But if you can send video clip it will give more clarity.
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