Cell culture contamination - (Jun/05/2008 )
Hi all,
im having quite a major problem in my culture..my monocyte cell is contaminate with sumthing that i dont familiar..it have been a while i cant do my culture work..the media is not viscous but there are debris at the botton of the well...i can see the tiny cells moving and sumtime they are like streptococcus with 400X magnification...ive started with new cultures,media and serum but same thing happen again
by the way, do you think newborn calf serum(NBCS) is the source of contamination?ive incubate the serum and i can see the thing, but the media is clean
i use NBCS from gibco and it is stated that the serum is virus and mycoplasma tested but didnt stated bacteria tested... could it be the source of my problem?
Does the debris get more with time? Bacteria should be growing quite rapidly. If it does not grow, it is more likely to be some kind of precipitation from the components. Sometimes cells can also make some debris/blebs/something, although I know nothing about monocytes. The movement can also be brown movement of particles, they are not necesserily alive.
Yes it does..but it takes longer time than normal bacterial contamination..the media is clear..and yes at first i thought it's only some kind of debris but i can see only the thing that have the brownian movement, and cells just stood still (the size of the unknown :1/10 from the size of my cell)...if my culture is more than 5days, the cells will look very sick (sumtime will burst out) and the replication is very slow if not didnt replicate at all...
By the way, do you did cell culture too? is it primary cell or cell lines?my labmate use cell line but the problem didnt strike her...
Yes it does..but it takes longer time than normal bacterial contamination..the media is clear..and yes at first i thought it's only some kind of debris but i can see only the thing that have the brownian movement, and cells just stood still (the size of the unknown :1/10 from the size of my cell)...if my culture is more than 5days, the cells will look very sick (sumtime will burst out) and the replication is very slow if not didnt replicate at all...
By the way, do you did cell culture too? is it primary cell or cell lines?my labmate use cell line but the problem didnt strike her...
1.Start culturing some other cells line in your media, even if cells don't grow, if there is no contamination there, it is in your frozen vials or there is really no contamination issue.
2. Autoclave and disinfect incubator/shelves.
3. Thaw a new vial of your cells.
4. Filter every single reagent that you add to the cells.
5. Serum is sold sterile, heating it is not to make it sterile. So, if you suspect it to be the cause, filter it.
6. If you want furhter reading: http://search.vadlo.com/b/q?sn=158621799&a...lture&rel=0
..
1/10 of cell size and slower growth suggest some kind of yeast. Although yeast is not supposed to move actively. I think cellcounter had some good ideas, it is probably wise to follow them.
I do some cell culture on both primary cells and cell lines. I've also had occasional contamination, but never a long-lasting one.
1/10 of cell size and slower growth suggest some kind of yeast. Although yeast is not supposed to move actively. I think cellcounter had some good ideas, it is probably wise to follow them.
I do some cell culture on both primary cells and cell lines. I've also had occasional contamination, but never a long-lasting one.
thanks for the suggestion...but from my reading throughout the forum i can see that im facing a common problem in tissue culture..tiny black dots that move like rushing ants...some do suggest maybe that's only a brownian movement, some says bacteria from Mycobacteria genus or Acinetobacter..rare type of bacteria but whatever it is there are still no confirmation what exactly the thing is which i cant focus on how to get rid of the contaminant..really frustrating...
but the good news is my faculty will upgrade a room to become a clean room..an A grade tissue culture lab...expected to use in October this year..so i hope this can solve the problem as i also suspected that the environment is also one of the culprit....
in the mean time i just focus on reading and maybe i can come out with a journal about contamination in tissue culture..hehe 
If you have a microscope, you could try to take a picture of it, and post it in the forum.
Hi kupac..i use different id now..ok i'll try to take pix of monocyte culture....hope that'll help
agree.
my friend use to grow some human macrophage.
she also see some dots that vibrate.
She was panic when the first time she saw it.
But later found that the dots do not multiply or make the media cloudy. the cells turns out to be fine.
we were too paranoid then, i guess.
Cell culture is the process by which prokaryotic, or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.
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