The movie is real-time video (not timelapse). Phase contrast, 40X objective, 0.6 NA. Field of view is ~200 microns in width. Organisms were found in isolated regions of a primary neuronal culture and seem to oscillate mainly in place without any net linear displacement. It is unclear whether they are proliferating if at all. They can be found day after day in roughly the same location and with the same fast movement. Solid lines in background are neuronal processes.
Brain tissue obtained from a third party was dissociated. The neurons were obtained by letting the cells attach to a PDL-coated flask. At first the cells were allowed to attach for 5 min after which the medium (along with any cells that were still unattached) was replaced with neurobasal medium. The noise in the background is a noisy refrigerator closeby in the room.
the reason for my query about the background noise is the possibility of vibration from another source, or of electromagnetic field from an electric appliance
There are no antibiotics in this culture. The vibration issue of course came up. However, the speed of the movement observed is much faster than the vibrations caused by the equipment. Moreover, the same moving particles can be found repeatedly in the same location day after day and whether the fridge is on its On cycle or not. The compressor turns on and then off after a while. This does not rule out the effect of electromagnetic fields though.
Do you have a slow-motion video? It's difficult to see what's happening clearly when they're moving so fast.
Also, have you been able to get any images under 100x?
If you let the cells grow in liquid media (or just suspend them in liquid media) it's possible you could separate the unknown cells with centrifugation and further analysis.
Unfortunately, I do not have a slow motion video. I agree that some of the particles are moving really fast. Some seem more stuck and are moving slower. The slow ones give you an idea of what's happening. Thanks everyone for trying to figure this out.
I also need to add that what everthey are, they do not seem to be proliferating. This is leading us to think that they might be some aggregated nanoparticles and not any biological organisms at all! At some point during the dissociation, we used medium from another lab for a 5 minute incubation, and that lab uses magnetic particles coated with oligonucleotides for some other downstream procedure in their lab. We think that perhaps the magnetic nanoparticles got into the medium that we used and got stuck to the bottom of the flask and remained subsequently stuck (For over 4 weeks and after several media changes in all new medium!). The magnetic particles would need an electromagnetic field to move/rotate, but perhaps if one undergoes Brownian motion then it can cause adjacent ones to start rotating thus setting up the motions that I am observing. It is far-fetched but I can't think of anything else at this point.
If they are nanoparticles they might fluoresce based on what they're made out of; so maybe try looking at them with a fluorescent microscope?
Nanoparticles is a farfetched thought, because the size seems larger - probably micro-particles. Scale is not provided, but with a 40X objective, the sies look as if these are around 10-20 microns. I am thinking this might be some sort of parasites. I see some sort of appendages around the body of moving object. Looks liek cilia. Bacterial flagella are difficult to observe without staining but cilia can be observed easily.
I would predict it to be parasites! keep us posted what you find out. If these are magnetic particles, use a simple magnet to see if you can drag them. When you place a strong magnet close to your samples, you will observe a different movement/oscillation! Interesting!
A few updates on the nanoparticles hypothesis:
1- We did try to bring a magnet in proximity of the flask and nothing happened. This was one of the strong magnets used to position mounts on an optical bench. I also tried to keep the flask on top of a magnet for a few days in culture, but the location of the particles did not change.
2- The full FOV in the video is about 200um in width and I agree that nanoparticles would have had to form aggregates to look like this. The moving particles are on the order of a few microns (including the "tentacles"-like structures attached to them.
3- Following with the magnetic particles hypothesis, I obtained an aliquot of the magnetic particles the other lab uses, Dynabeads 11533D (CELLection kit from Invitrogen), and added them to another culture dish to see what they look like. Dynabeads seem to come in either 2.8 um or 1um in diameter but the spec sheet for this particular part number did not specify the diameter (will call invitrogen next). In any case, immediatelly after adding to the culture the beads look bigger, are much more circular and smooth, and do not move after settling to the bottom.
4- So as of today I do NOT think the moving "organisms" are beads from the other lab. i.e. I still do not have a good hypothesis as to what they are. As of today, they are still there, in the same location and moving the same way at the same "frequencies". The neurons in the culture (now at DIV 59!) look mostly dead at this point, but this does not seem to affect the movement or location of the "organisms".
5- Thanks for suggesting looking at the sample under fluorescence. Perhaps other synthetic particles may be present that we are unaware of at this point. Will try this soon.
If possible micro-organisms, a traditional gram stain might be revealing. If they stain by gram, you will be able to see their morphology (and gram reaction). If they do not stain by gram, it is very likely that they are not micro-organisms. I realise that this is a simple approach, but it is quick and might be useful.
I am following these question because I believe in cell cultures are not sterile as the same way microbiota studies reveal that many tissues of our body are not sterile and if you( Nada Boustany ) can prove it, it maybe change the way of studies on cell cultures.
Here are couple more movies.
1- The magnetic Dynabeads are 4.5 microns in diameter. Here is a movie of them (MVI_8132). As far as I can tell, they are completely different from the moving structures I am observing. I am attaching a movie of the Dynabeads dropped on a old culture of Bovine Aortic Endothelial Cells (BAEC). Same optical conditions as in the first movie.
2- I am also attaching another movie (MVI_8134) of fast moving particles in a different part of the flask that I found later at the left edge of the flask. I did not not say this before, but this is a large T175 flask, and for now, I seem to only find these things in a few locations around the flask. Due to the size of the flask, I cannot really tell how many such locations I could also be missing. In this movie, I am noticing a slowly moving "organism" and a couple of non-moving structures that look like still versions of the moving ones. I recorded this movie on Dec. 18 2018. The first movie was recorded on Nov. 13 2018.
3- I could not detect any particle (or moving) fluorescence under 514nm excitation or 546nm excitation, although the surrounding material (cell debris/aggregates of old/dead cells) autofluoresces a bit. Will need to swap filters to look under blue or UV light. I like the idea of running a gram stain. However, I am reluctant to run any stains yet as I may have to fix everything in the flask and I am not sure how to isolate the structures from the middle of the flask without destroying the sample.
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