help with contamination - (Jan/13/2008 )
hello
I have been seeing tiny black dots in flasks of PC12 cells. they do not move however. they seem to increase in numbers but very slowly. the cells look fine and they grow fine. no ph change or turbidity. do you know whether this is contamination? my colleagues have similar symptoms but they don't care and do not want to report it. I really don't know what to do it seems to be spreading to other cell lines as well. I am scared if I order new cells (which is v expensive) I just get the contamination again. 
is there a chance that they are cell debris? or dead cells floating ?
i thought so at the start. but now it just doesnt seem so. they seem to increase slowly and I cant get rid of them by centrifugation. cell debris should disappear after spinning down the cells and resuspending them in fresh media shouldn't it? these things dont they are just a nightmare and are keeping me from doing my experiments and am wondering whether all my previous work is all affected.
now i don't know whether i am being paranoid..but on my SHSY5Y cells I dont see things floating but the cells look like they have bits inside them! did anyone notice this before? what could it be?
please help!!!
This really seems like cell debris..
But, just to make sure, have you tested the possibility that your cells may be contaminated with mycoplasma?
But, just to make sure, have you tested the possibility that your cells may be contaminated with mycoplasma?
hello madirus
I have plated some cells on slides to check them with DAPI, is it 100% reliable? as in if i see no dots around cell nuclei it means there is no mycoplasma contamination?
also do you think the mycoplasma may be causing the cell debris?
Hmm
I do not know how our cell technician tests our cultures for mycoplasma. I will have to ask her.
As for the cell debris, the only fact that cells are growing and that some of them die can explain cell debris.
my understanding is that if is cell debris it will precipitate together with your cells when you spin them down. you could try dilute them out, and see if the black particles still expand.
if you can be bothered and have the time, you could try a ficoll gradient to separate your live cells from the other bits (this is assuming is debris and dead cells, not sure if it will separate contamination)
as for mycoplasma, i've never tested it myself but there are PCR kits that work really nice.
does this black bits appear in freshly thawed cells? if not, there's no need to buy new stocks, just revert to your old ones. Try aliquots frozen on different dates, with long times in between, they'll probably come from different cultures and if its a contamination hopefully you'll find one of them iss clean.
if you can be bothered and have the time, you could try a ficoll gradient to separate your live cells from the other bits (this is assuming is debris and dead cells, not sure if it will separate contamination)
as for mycoplasma, i've never tested it myself but there are PCR kits that work really nice.
does this black bits appear in freshly thawed cells? if not, there's no need to buy new stocks, just revert to your old ones. Try aliquots frozen on different dates, with long times in between, they'll probably come from different cultures and if its a contamination hopefully you'll find one of them iss clean.
yes they do appear in freshly thawed cells..lots of them! i tried all batches!! the thing is i have only obtained these cells few months ago and I think whatever this thing is it affected all of them since the very start.
Is it possible, that what you have been observing is normal for these cells?
if you can be bothered and have the time, you could try a ficoll gradient to separate your live cells from the other bits (this is assuming is debris and dead cells, not sure if it will separate contamination)
as for mycoplasma, i've never tested it myself but there are PCR kits that work really nice.
does this black bits appear in freshly thawed cells? if not, there's no need to buy new stocks, just revert to your old ones. Try aliquots frozen on different dates, with long times in between, they'll probably come from different cultures and if its a contamination hopefully you'll find one of them iss clean.
yes they do appear in freshly thawed cells..lots of them! i tried all batches!! the thing is i have only obtained these cells few months ago and I think whatever this thing is it affected all of them since the very start.
Oh dear not this again.
Mycoplasma contamination is not visible under the phase contrast microscope. Hoescht staining dfor mycoplasma detection is only visible when there is a massive mycoplasma contamination, and should be used in conjunction with Direct growth method for mycoplasma dectection. PCR IDENTIFICATION IS NOT RELIABLE...i.e the ATCC no longer offer PCR as a method for mycoplasma detection, as it is so unreliable.....it is also not an FDA approved method.
Some cells produce debris as normal i.e. DLD-1 and Caco-2.2 cells. The higher the cell density, the higher the number of black specs/dots.
My advice to you is take a sample of the growth media. Spin that down and resuspend the black specs in PBS and streak this onto an agar plate and leave in an incubator for 48-72 hours. If it is bacteria then you should be able to see colonies.
Kindest regards
Rhombus