When using a tissue culture treated plate, I started to run into these weird black stringy/shards. If it were contamination, my best guess would be that it is a fungal contamination.
However I do not think this is the case for a few reasons:
- It doesn't seem to resemble fungal contamination very well
- All of these shards/debris seems to be on the actual lid of the plate (seeing them requires lower focus), rather than being on the same plane as the cells
- Since their emergence, any factor or variable that may have caused contamination was eliminated and aseptic technique has always been on point
- Color of the media does not change drastically and appears to be relatively normal
I have not tested the pH of the media, however I suspect it is within the normal range given that the cells in culture seem to be happy. FYI the cells in culture are H9's on iMEF's.
I just want to get to the bottom of this, and figure out whether this could just be debris or if it actually is contamination.
Amer,
If these shards/debris you are seeing require lower focus to see and are not seen on the same plane as the cells, then in my experience they are most likely just scratches or something on the culture lid. If you think they are on the lid of the plate, you might try putting a new sterile lid on your culture and see if they are gone.
It's always good to be super vigilant when you see anything that might be contamination. Hopefully in this instance it is nothing you need to worry about.
Amer,
If these shards/debris you are seeing require lower focus to see and are not seen on the same plane as the cells, then in my experience they are most likely just scratches or something on the culture lid. If you think they are on the lid of the plate, you might try putting a new sterile lid on your culture and see if they are gone.
It's always good to be super vigilant when you see anything that might be contamination. Hopefully in this instance it is nothing you need to worry about.
Agreed with Mauring.
Don't hesitate and change your culture dishes and aseptic everything again and look this thing will be appeared or not but don't use this culture as a stock till you be sure they are clean. Something that suspicious me is you have it in different part of plate and it's really look like contamination but why it hasn't grown completely? So you can culture it on bacteria medium like BHI agar and fungal one.
Best wishes
Hi amer, as the plan of focus is different than the cells these might just be scratches on the plate. your cells look healthy and any contamination like that lets say if fungal would be visible to naked eyes overnight. So this must be artifacts only.
These are very interesting...I have never seen anything like this.
I recommend wiping down the bottom of the plate and very top with a KimWipe with a little bit of ethanol. Maybe wiping the inside of the lid with a clean KimWipe (no ethanol) and then looking at the plate again. If it still there, try marking with a permanent marker the top and the bottom of the plate to figure out where in the magnification are the "black strings". You can focus in on the black sharpie mark and work from there.
Are the "black strings" still there when you change the media? It seems like they are floating on top. Try changing the media and see if they are still there.
Unfortunately, they do not look like scratches to me. I think it is mold (95% sure), but it potentially could be threads from clothing. I have dealt with fungal contamination and then being paranoid that scratches are fungus enough to know this. If you have more cells, I recommend just throwing this plate out and starting fresh. Clean out your water bath and add fresh, clean water and water bath treatment. Mop the floor with diluted detergent, and clean your hood and counters well. You should be rid of it after this.
Please keep us updated on what you find.
Thank you for the input everyone. I am doing a little experiment with all of the different media I've used to investigate whether this emerges from contamination or if it really is just debris/scratches. If anyone else has any more to add, I would very much appreciate it.
I will keep you updated on what I find!
Amer, those are fungal hyphae. It is unfortunately contamination. Change the media and plate new cells with everything fresh, even the PBS. Don't waste any time brooding.
Hello all. Thank you very much for your input. So upon further inspection, I found that these ‘artifacts’ actually reside on the outside of the tissue culture plates (i.e. there is nothing actually in the media, which was confirmed with further testing). I discovered this by rubbing some ethanol on the bottom of the plate, after which they disappeared.
However, these artifacts do re-emerge in random spots. Although it is not inside of the plate and not affecting colony growth, this is frustrating me as their morphology is suspicious and their re-emergence in concerning.
For next steps, I intend on replacing and decontaminating absolutely everything (pipets, lab coat, sleeve etc.). I also intend on doing a steri-run on the incubator, which includes a 2 hour heating phase, followed by a 1.5 hour sterilization phase at 180 °C, and a final cool-down phase of approximately 8 hours.
Now I have done this before to try and get rid of these artifacts, but as you can see they still emerged. So does anyone have advice on any extra measures I could take when decontaminating the incubator?
@Amer Samir
Well done!!!
Regarding your stat about the sterilizing incubator, the machine do it automatically?
If yes, it's good but if it's no don't cool with me.
You sterilized your incubator before you found the reason? So your incubator is still contaminated. It's the point that I didn't take correctly from your reply.
Another question is important here that you are only person using the incubator?
Anyway it seems contamination agents including spores so choose the right protocol.
Finally reason of disagreement of mine by your sterilization program of incubator is such a high temp can damage your incubator filter especially but as I mentioned if it's manufacturing program, there's nothing to worry but if it is not use lower temp, 60 Celsius, at least twice or third times.
Best Regards
Alireza
Hi Amer! I talked with a lab technician in my building who also deals with cell culture (Cathy Parsons). She is thinking that this potentially could be dust, so to also wipe down your microscopes regularly with ethanol and a KimWipe. She told me that dust can often look very strange under the microscope and can come from pretty much anywhere. Just make sure you are mitigating dust anywhere in your cell culture room.
But, the fact that some of the structures look very concentrated in the center is a little concerning..so there is still a possibility that it is some type of fungus although neither of us have encountered something that quite looks like this (even though we've both seen many different types of fungi).
I suggest wearing a long palpation glove (one that goes up to your elbow at least) under a regular pair of nylon gloves every time you deal with cell culture so you can ensure that your entire arm is clean. These could be coming from your sleeves even if you are wearing a lab coat and even if you're not hovering over anything things can fly off your arm and contaminate something. I often do this if I encounter a period of time when dealing with contamination and it helps curb it a lot. Hope this helps.
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