It seems that you have a serious contamination. I suggest to discard your cultures, clean everything, get rid of cheap stuff and small aliquots ( trypsin ...). Then start again by incubating medium in your incubator and then with a new aliquot of one of your cell lines.

Although mycoplasm are a big problem in cell cultures, this is not the described acute problem. For mycoplasms see:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584481/

Microbial contamination may happen through multiple ways during cell culture procedures, as you can see here:

Stacey GN. Cell culture contamination. Methods Mol Biol. 2011;731:79-91. doi:

10.1007/978-1-61779-080-5_7. PubMed PMID: 21516399.

Even with the help of photos, it would be really hard to identificate with precision the type of contaminating microbes. Do you see any signs of molds and yeast contamination? There are no conventional signs of mycoplasma expansion in cell cultures, they may cause a panel of effects depending on the culture type/condition, as the authors of this paper stated:

Drexler HG, Uphoff CC. Mycoplasma contamination of cell cultures: Incidence,

sources, effects, detection, elimination, prevention. Cytotechnology. 2002

Jul;39(2):75-90. doi: 10.1023/A:1022913015916. PubMed PMID: 19003295; PubMedCentral PMCID: PMC3463982.

So, my suggestion is to test your culture medium for the presence of mycoplasma using one of the several available methods:

Uphoff CC, Drexler HG. Detection of Mycoplasma contamination in cell cultures.

Curr Protoc Mol Biol. 2014 Apr 14;106:28.4.1-28.4.14. doi: 10.1002/0471142727.mb2804s106. PubMed PMID: 24733240.

Hope this helps.

Looks like you have serious contamination issue! I am sorry about that. It can be really frustrating. If I was you, I would test for mycoplasma contamination first. Since all your cell cultures are behaving the same way, it could be that your FBS or DMEM/RPMI stock is contaminated. Also, decontaminate your incubator before you start another culture. 

Any photographs?

hi, 

It would be helpful with the snaps of your cells if possible..

Just discard everything and make fresh media then only revive the new vial of your cell line.

It would be nice to have some pictures to really comment on your problem. However, I do believe that you seems to have a contamination issue. It could be mycoplasma, although, you won't see exponential growth and cell death within 24 hours. It could also simply be the case of yeast infection (you can observe typical yeast infection under the microscope- small cluster of floating cells smaller then your cell line). I suggest you should throw away all the growing culture, opened media bottles, serum aliquots etc. I would really recommend you to decontaminate your incubator, safety cabinets, water bath (use sigma clean after decontaminating in your water bath) and of course thaw a fresh batch of cells. Try not to use any previously aliquoted serum and/or other culture medium. Hopefully your problem will be solves, fingers crossed! If you still experience the sane problem, test your cell stock for infections.

Hi, 

The fast growing cells must be a sign of immoralization . You have immortalize your cells by stressing them ,e.g low number of  seeded cells and high percentage of the FBS and of course mycoplasma help too by introducing mutation.

Hi,,

If you see many floating cells so it seems to be dead cells

also it could be a bacterial contamination, and you will see small rod like structure under the microscope and the media will be yellow and turbid

Thank you all for your suggestions,  Problem is solved, We prepared fresh media, Opened new FBS bottle, Washed the water bath, fumigated the entire room. Now there is no problem. Thank you very much.

Hi Raghuram Kandimalla! Thank you for the comments and suggestions above. I am experiencing a strong mycoplamsa contamination. In my case, the growing was the contrary, cells started to grow slowly and the morphology changed along time. However, the problem persists after throwing away all the FBS and trypsin aliquots, all the opened medium bottles, autoclaving the incubators, cleaning the air condicioning machine, water bath and flow cabins, etc. I am wondering if you could explain how did you fumigate the room? It would be really important for me to solve this undesired problem. Many thanks in advanced. 

Hi Laia Reverté, fumigation of room done by KMNO4 and formaldehyde 

(Around 5 gm of KMNO4 take in petri dish and pore 10 ml of formaldehyde in it), keep the petridish on any  tile or big glass plate before poring formaldehyde in it  (Other wise it might spoil your cell culture room floor). Do not enter into the room for 24 hours, after fumigation. Next day remove the total fumigation setup from room and follow normal UV sterilization, before you start working. Hope this will work.   

Thank you very much for your quick reply. I will do it for sure. I hope this  will work, too. Thanks again.

Hello everyone,

I was following this question. I would add one problem i had and try to have an answer from you.

Some weeks ago I had a strong contamination of two different cell lines. This first point was making me think that probably my medium was not good and I changed everything, Strangely all the solutions were incubated at 37*C to test if something was growing inside but I did not see anything.

Then I changed the medium anyway, I took two old stock of cells I prepared before the contamination (at least 4 passages before) and I put in culture. They grow at confluence the first day, I split them and they grow one day... Then they stop to grow and they become super slow. Usually I split confluent cells every 2 days. Here after 3 days cells are still at 40 50% of confluence... but they don` seem to grow more.But this time I dont see contamination. I would check for mycoplasma but as I told you I trashed the medium and all the other solutions and, again, I took a very old stock that I used in the past with no problem. So if there is a new contamination, I would say is coming from hood or incubator and not from media or cells. Then incubator or hood? In our culture room we are three / four workers... and just me with the contamination and grow problems.

Then I don`t really know what to think more.

Media are fresh

Stock cells is an old one used in the past without problems

incubator and hood is working for the other guys

me two different cell line contaminated (first time I have a contamination in 4 years of cell manipulation)

I work as properly I can: I clean everything every time with EtOH70%.

If someone of you could give me an idea, it would be great!