Hello,
Recently I performed lentiviral transduction on several cell lines. On day one with the lentivirus, I observed black aggregates moving (dancing, vibrating) around my cells. These are only in cells to which I added virus.
When I harvested my lentivirus in the supernatant of my packaging cell line, I spun the supernatant (3000RPM for 15 minutes) to remove cellular debris, filtered it with a 450nm sterile filter, and then froze it at -80C. These steps should have removed bacteria, correct? Moreover, the aggregates are resistant to the 1% Pen/Strep in my culture media. They do not turn the media turbid, and they never seem to reach log phase.
Could I be seeing the virus itself?
Can lentiviruses form visible aggregates?
Could these be abiotic aggregates exerting Brownian motion?
I have seen this phenomenon before and as of now, no one has given me a satisfactory answer regarding the identity of the dots.
Thank you.
I just have a suggestion for you.
It seems your culture contaminated with fungi.
Culture your media on sabouraud dextrose agar.
Regards.
Very interesting! I hope we will not return to the days of small beginnings of Microbiology; the days of animacules. Please try the filtration step again, this time with .22 micron. If you check my publications, you'll discover I don't take chances. I centrifuge, filter with .45 micon and follow up with .22 micon. Plating on different culture media as suggested by Alireza Mordadi is recommended for quality checks. What kind of microscopy did you adopt, please?
It's not advisable to filter lentivirus with a 0.22um filter - you'll lose some. That's why a 0.45um filter is generally recommended.
There are companies that will analyse cells and culture media for you to look for Mycoplasma and many other contaminants - that's probably the best way to know for sure.
Viruses are too small to be visible under the microscope, so the dancing dots are probably bacteria or unicellular fungi. They could be just bits of inanimate debris but if they were, you would expect to see other signs of cell damage that could give rise to the debris.
I'd streak out onto a nutrient agar plate (or any other general microbial growth medium) and see what grows. If it's bacteria, you may be able to prevent the growth with antibiotics. If it's fungi, your options are limited. However, fungi seem less likely as they will not pass through a 450 nm filter.
I experienced very similar situation as well. Those little ones won't grow to make the medium yellow or turbid even I incubate the culture to 14days. I inoculated my culture into those mediums for sterility test. And I got negative result, nothing grew up in the sterility test medium 30days after inoculation.
I am curious what are them as well. Anybody can cue me what I can do now?
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