The following image uploaded is from our Sub-ventricular Zone (SVZ) derived neurosphere culture (mice), Primary culture - Passage 2 - Day 2. Thread like structures were observed at 20X and 40X. Cells generally looked fine. There was no turbidity observed in the media. These filamentous structures mostly were non-motile, but some small ones were swimming around and some were just moving at a single point. Kindly share your comments on what this could be.
It appears that your culture is contaminated with bacteria. It could be non motile rods (Bacillus species). You can confirm by doing Gram staining of culture supernatant.
If there's any sort of movement in the media then it is most likely bacteria. In addition to what Mrinalini said, make sure you clean the incubators out thoroughly. These infections are often very hard to get rid of once established. Watch out for other vials of cells coming out of liquid nitrogen storage too and check they aren't infected.
We did gram staining but it didn't worked out. What may be the other reasons ?
Hi,
I agree with the previous comments, most likely a bacterial contamination. Do you use antibiotics for your primary culture? If not, consider adding standard antibiotics (penicillin/streptomycin) at least for the first days after initiation of the culture, it is very hard to generate contamination-free primary cultures without antibiotic supplementation.
If you want to be sure, take a small aliquot of your culture and add it to fresh medium (without antibiotics). After a few days of incubation, it should look like in your picture (just without the cells) if you actually had bacteria in your culture.
We did gram staining but no such organism was observed. And we also tried inoculating the organism into bacterial culture medium and it also didn't worked out.
Hi,
don't use special bacteria medium (LB?) for the sterility test, but the same complete cell culture medium as in your culture, as you know that your potential contaminants will grow in this particular medium. They may require specific factors introduced e.g. by FBS. For example, certain bacteria (e.g. mycoplasma) need a lipid source (either from synthesizing cells or special blood agar) to proliferate, so also consider adding the aliquot to an indicator cell line of choice.
A further quick test is adding a DNA-specific, permeant dye (e.g. Hoechst 33342) either directly to the culture or the collected supernatant. If the particles become fluorescent, this is also a good indicator for bacterial contamination.
Hi,
If this is bacterial contamination you should see medium color change after several days of incubation without antibiotics or by testing in LB, also the amount of dots increase during the time tremendously. The movement can be due to vibration of microscope and it does not necessarily mean that you have a moving object, unless the active movement is obvious.
In general such a cloudy dots can be normal for primary culture especially from brain, and for cell population with high death rate. For primary culture it should be clear after several passaging or several medium change through adaptation of cells with new condition. Changing half of medium each time can be helpful for faster adaptation.
You can also check below links for more information regarding cell culture contamination:
https://www.researchgate.net/post/How_to_Identify_Contamination
https://www.researchgate.net/post/How_can_I_identify_and_distinguish_between_cell_culture_contamination_and_cell_debris
https://www.researchgate.net/post/Do_we_have_cell_culture_contamination_or_not
https://www.researchgate.net/post/How_to_identify_the_cell_debris_with_contamination_in_the_cell_culture2
Hi,
just a suggestion which popped up. If it's not a bacterial infection, it might be denaturated proteins. Did you use heat-inactivated serum? If yes, it might have been inactivated to long or too hot (standard is 30min 56°C). Then filter your serum over 0.2µm and try again. The movements of particles in your medium could be due to Brownian motion (https://en.wikipedia.org/wiki/Brownian_motion).
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