my cells doubling time around one day and i cultured them four days ago and they did not double or increase in number. they did not die neither. may be the medium is not enough? .
As Rose said, it could be a mycoplasma infection, or cells have reached replicative senescence and won't proliferate further. If these are primary cells then I suggest not using them at higher passage (depending on cell type, primary cells shouldn't be used beyond P5-9).
Dear Heba Huessin
Pls. find the attached files, may help to answer your question, good luck.
http://www.pjps.pk/wp-content/uploads/pdfs/27/3/SUP-10.pdf
http://vlab.amrita.edu/?sub=3&brch=73&sim=1105&cnt=1
http://aem.asm.org/content/75/9/2712.full
http://www.math.unl.edu/~jump/Center1/Labs/What%20Affects%20Yeast%20Growth.pdf
http://www.jove.com/video/3064/aseptic-laboratory-techniques-plating-methods
http://www.biotechniques.com/BiotechniquesJournal/2005/July/Factors-affecting-double-strand-break-induced-homologous-recombination-in-mammalian-cells/biotechniques-45499.html?pageNum=2
Kenneth D. McClatchey . 2002.Clinical Laboratory Medicine.LWW Doody's all reviewed collectionMcClatchey: Clinical Laboratory Medicine Lippincott Williams & Wilkins,Pp. 1693. ISBN 0683307517, 9780683307511
http://www.vetmed.ucdavis.edu/whatsnew/pdfs/chicken_march_2015.pdf
https://www.dairyscience.info/index.php/enumeration-of-lactococcal-bacteriophages/factors-affecting-plaque-formation.html
Regards
Prof. Houda Kawas
Did you check for mycoplasma contamination? I have found that Mycoplasma contaminated cells grow very very slowly
As Rose said, it could be a mycoplasma infection, or cells have reached replicative senescence and won't proliferate further. If these are primary cells then I suggest not using them at higher passage (depending on cell type, primary cells shouldn't be used beyond P5-9).
To add another option to those mentioned above: there could be something wrong with the serum - too old, low concentration, zero concentration etc. Serum contains growth factors and other nutrients that allow cells proliferation. Without serum, cells will not die immediately but won't proliferate either.
In addition to mycoplasma and/or serum starvation, if your cells are primary cells they might have hit a senescence crisis
both cell lines died. each one had two flasks so total four flasks died so i dont think it is senescence. i think medium was fine as they were grown for one week well. so, may be mycoplasma but dont know how to detect mycoplasma infection.
Unfortunately mycoplasma is very common, with I think about 80% of the population carrying it. It is the nightmare of any cell culture lab; it is a wall-less bacteria tthat does not cause cloudiness in the media and is so small that it cannot be eliminated by filtering through 0.2um filters. Once in a flask it can easily spread to all other culture in the same facility. If aseptic techniques are not tip top, it can be introduced in the culture by the operator. In addition, if culture have been derived from primary tissues, it could have been already present within the tissue. The most common way to check for contamination is to perform a PCR on the media in which cells have been grown for at least 48 hour. There are several kits commercially available and easy to use which are fairly exhaustive in terms of the different strain of mycoplasma. Alternatively, there are several fluorescence based assay that can detect the infection in culture - really depends on your lab set up what is best to use for you.
As for the decontamination, similarly to detection, there are several commercially available antimycotic that can be used simply by adding to the culture (like you do for pen/strep). Amongst many company that provide kits there are Thermo Fisher and Lonza
Just google mycoplasma detection or mycoplasma decontamination and several option will come up.
As a rule of thumb, it is best practice to check all culture in the lab for mycoplasma every six months minimum
Good luck
The simplest method to detect mycoplasma infection is DAPI staining exactly like for staining of nuclei in your cell culture. Just if there is mycoplasma infection you will see not only nuclei of your cell but also additional dots, usually close to the cell membrane of your cells. If infection is great the genetic material of mycoplasma will be visible even as some filaments between your cells.
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