After we thawed the cell line from liq N2 storage, we saw some floating things in the media after 24hrs, but after we change the media on that day we didn't see so much on the next day and didn't cause any problems for the cell growth also. My question is when we see such floating things how could we distinguish whether its a contamination or just cell debris (there is no change in color of media)?
If its getting less then its not contamination....the serum in freezing mixtures often produces a slight precipitate plus there may be debris from dead cells if your freezing/thawing technique isn't so good. After about 25 years of tissue culture you will be able to distinguish bacteria from debris just by looking down the microscope! I work with several connective tissue derived cell lines and primary cells that produce lots of rubbish in the media of a similar size to bacterial cells
In general its not a contaminant if:
1. There is no odd media colour change and the particles don't increase in number
2. The particles are uneven in shape and size
3. There is no directional movement at high magnification....... all particles jiggle at high magnification due to Brownian motion but a lot of bacterial contaminants do actually swim! If you see a particle moving any distance at all in a straight line or circle, its probably a contaminant
From your queries it seems like debris from dead cells. When you freeze down the cells some percentage of cells die. So when you thawed them debris remains in the media. So, after the cells attach, remove the old media with fresh one. Recheck for fungus and mycoplasma contamination. The possibilities are
1. Precipitates from media
2. Dead cells
3. Very early fungal infection
Sometimes, in some cells lines, black dot like bodies can be seen. Though the cells behave normally and morphology also looks normal. Lymphoprep can be used in that case.
Let me know.
If the "particles" don`t come up again after medium change then you should be on safe side for contamination.Next time you could take of the culture medium after thawing up cells and plate it on another dish, cultivate it at 37°C and check in the microscope. If there is no increase in number of particles there is no contamination.
Just for checking if your N2-stock is ok I would thaw up a second vial of cells and culture it without medium change. The cells would look not very nice with dead cells around them but the number of floating things should not increase.
I think size shape and movement can dtermine whether its infection or not although some fungus might not move but, definitely it will be growing rather than lessening as is ur case
Hi Rubina,
Was your vial frozen with DMSO (freezing solution), and did you centrifugate your cells after thawing them (DMSO is toxic for cells at room temperature) ? A light centrifugation is recommended for the thawing procedure, before plating.
Bacterial or yeast contamination can be checked directly on microscope, but for mycoplasm contamination, you need to use a detection kit on a medium sample when your cells are nearly confluent, such as this one :
http://www.rndsystems.com/Products/cul001b/AssayProcedure
Like Leanne and Ranjan, I believe that it's only some cell debris !
Hope it helps :)
If its getting less then its not contamination....the serum in freezing mixtures often produces a slight precipitate plus there may be debris from dead cells if your freezing/thawing technique isn't so good. After about 25 years of tissue culture you will be able to distinguish bacteria from debris just by looking down the microscope! I work with several connective tissue derived cell lines and primary cells that produce lots of rubbish in the media of a similar size to bacterial cells
In general its not a contaminant if:
1. There is no odd media colour change and the particles don't increase in number
2. The particles are uneven in shape and size
3. There is no directional movement at high magnification....... all particles jiggle at high magnification due to Brownian motion but a lot of bacterial contaminants do actually swim! If you see a particle moving any distance at all in a straight line or circle, its probably a contaminant
An obvious, inexpensive and fast method to visualize debries and dead cells which might be floating in culture media is the trypan blue count.
Basically you pick a small amount of culture media (as low as 10 microliters) and add 1:1 (volume : volume) of trypan blue staining solution (it costs 10 euro from SIGMA). Then load 10 microliters of your sample in a haemocytometer and check if your "unknow bodies" turn blue. If the answer is yes it's all about dead cells and debries.
If they do not turn blue it might be some other kind of contaminant, like protein clots from FBS (if old but still in use).
I'd exclude bacteria or fungi because otherwise your medium would have turned yellowish.
Enjoy, and good luck!!
It is probably dead cell debris since it is decreasing, but you should centrifugate the cells in order to remove the deade cells before continuing cell culture.
In case of contamination you would see a change in culture medium colour which would probably also become opaque (not a clear liquid) and you would observe a slower than usual cell growth. Also, if you performed a cell count you would see a bigger percentage of dead cells than usual.
Yes that is probably dell debris as it is not increasing after the media change. During cryopreservation with DMSO, it is natural that some of the preserved cells get stressed and ultimately dead and on thawing these dead cells comes in form of cell debris.
However if you are preserving the contaminated cells then you get contamination along with the thawed cells and that contamination does not decreases by mere media change.
Since your media colour does not changes, it means that there is debris along with the live cells.
Rubina, the answer is very simple. Many get confused by the so called "moving" particles in the culture medium. It is important to distinguish non-living particulates and contaminating bacteria. There are two kinds of movements that you should learn to distinguish - the "Brownian" movement and "Active" movement. cell debris and any non-living particulates exhibit Brownian movement where they exhibit some movement, but pretty much stay in the same place. In the cases of bacterial contamination, you will see active movement where these particles move actively and move away from the current position. Next time, you see particulates, look at the movement patterns. As others mentioned above, contamination will explode in a day or two and of course you can, very often, smell it when you open the incubator. Hope this helps.
I agree with John. Just check if the junk you see in the media gets diluted over time. If the amount keeps decreasing every time you pass your cells, it's very likely to be cell debris.
Like Sebastiano I recommend trying trypan blue staining-- it is a diazo, vital dye that will be excluded by living cells and stain dead cells and all sorts of debris. Even better it is inexpensive and easy. It is also routinely available in labs that do cell counts via haemocytometer so if you do not have some a lab down the hall might be able to lend you a few microliters.
Take a small sample of your culture--- 10-50ul, add 10ul trypan blue, make up a wetmount slide and view. If it is debris and you are working with an attached cell line the clean up is easy. Let the cells attach, remove the growth media, rinse with PBS or HBSS to dilute and wash away clinging dibris, then add fresh growth media.
Hi!
You can cultivate their cell line without antibiotics. This is indicated for amplifying the cells, as a quarantine. If it is contaminated, you will have no doubt in a few days. Antibiotics do not prevent contamination only control the growth of microorganisms. You may be working with infected cells but not the middle muddy due to the antibiotic, although this does not seem to be your case.
Have a good luck!
I agree with the answer of John Wardale and Sakthikumar Ambady. This would be better understood while good practice and experience.
cultivate your cell line without antibiotics. If it is contaminated, you will have no doubt in a few days. Antibiotics do not prevent contamination only control the growth of microorganisms is also prohibited.
Thawing from frozen vial to re-suspension should be done very quickly (one will learn through experience!), as DMSO is toxic to cells if not processed quickly! It's a nice practice to re-suspend the thawed cells into 10-15 times the volume, and do a slow centrifuge to remove the floating debris then re-suspend the pellets and then plate the cells, if you are not comfortable doing that, you can always directly plate the cells. If they are adherent, make it a point to change the medium after 24hrs of plating.
If it is a primary cell, you can plate the cells, remove about an ml of the re-suspended cell and do dye exclusion test to estimate dead/live or contaminations, if any. G'Luck!
Use 100X optics and watch for the floaters to moved throughout focus. If they do you have a bacterial contamination. If there are persistent little black floaters you probably have mycoplasma contamination. Bacteria will usually start to change media color and get turbid but mycoplasma may not.
Barb Potts
Potts and Nelson Consulting, LLC
i have same problem but after wash the cells and after one day the cell debris come back again and if leave the cell for one day with out change the medium the cell debris become more and i have doubt in fbs but i don't know what i should do
i guess simple way is to check for directional movement and waiting for 24 hrs
Dear Rubina,
I have the same problem for my HepG2 cell line. After thawing or passaging cell using Trypsin-EDTA I ussualy see black dots after 24 hours. But after washing well by PBS and adding new medium, black dots are fully removed indicating cell debris. you can check the following comments for your bacterial contamination:
1. At first check your flask medium appearance. If it is clear, ussually there is no contaminations.
2. Check your black dots movement. If there is no movement so no contamination is happened.
3. Wash your cells using PBS and check for black dots after 24 hours.
4. And finally for more troubleshooting, you can perform a bacterial culture on blood agar and or EMB/Mckankey agar medium.
If there is turbidity in the media, then there is definitely bacterial contamination right?
First in bacterial contamination the media colour is different. Then view under microscope. The bacterial movement clearly seen in definite direction. If you have any confusion about how it moves refer videos about contamination and observe the pattern of bacterial movement
What about this one?
Recently passaged cells John Wardale & Barbara Potts
I have the same question-what it is in the picture from Anjali. I have the same problem in some of very old cultures
Hi Anjali
Did you get rid of this problem ad what was the solution for it do have the same isue now.
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