I am referring to current protocols in Microbiology 15D.3.1-15D.3.4 for plaque assay of West Nile Virus. As per the protocol the agarose should be diluted at 1:1 ratio with 2X MEM+8%FBS. However I do only have DMEM and not MEM. I would like to know whether DMEM can be used instead of 2X MEM for this step.
Hi there, the use of growing medium depends on how the cells grow. In my experience some cell lines tolerate more than one type of growing medium, while others do not. You should try your cell line with the two types of medium and change it only if they have the same growth rate and the same morphology in both. If you do not have this possibility, you can only try with your control virus and hope that works.
In my experience, most epithelial cell lines (epi lines are typically what are used for plaque assays) are very robust, and will grow in just about any medium.
DMEM is more nutritious than MEM, so if your plaque assay lasts longer than a few days, you might have some concerns with cells overgrowing during the assay, making it hard to detect cytopathic effect separate from overcrowding of cells.
Be sure to use the DMEM at 2X concentration, however, just as you would with MEM; otherwise when diluted with the agar the cells won't have access to enough nutrients, and will starve (at least somewhat, slowing progression of virus plaque formation, as well as rounding up cells and making detection of CPE difficult). Hope this helps!
Hi Soham
I am continuously using 1:1 ratio with DMEM+5%FBS and 4% Methyl cellulose in my experiment to know the titer of Chikungunya virus. As you are using West nile virus and both Chikungunya virus as well as West Nile Virus belongs to Alphavirus. Therefore i think you should use the same which iama using.
I use regular DMEM + 4% FBS for plaque assay on HEK 293 to titer Adenoviruses. Other thing that you should consider is the concentration of your agar/agarose. In my case, I mix 3 mL of 5% agar with 12 mL of DMEM 4% FBS to get a mix with a final concentration of 1% agar. Regards the equivalence of MEM and DMEM like Anna says, it depends of your cell line.
Good luck.
Is there any advice on removing the agar without disturbing the cells underneath? I have used 1% agarose in DMEM and I am unable to remove the agar overlay without scraping cells underneath. I have tried 10% formalin, "carefully" removing with a spatula, turning it upside down and banging on it...I have yet to successfully be able to count plaques.
Hi Gregory..I would advice you to use carboxy methyl cellulose instead of agarose, which forms a viscous medium mixed together with 2X MEM..the plates should then be left undisturbed until you want to count the plaques. This medium can then be pipetted or sucked out (using a suction pump), followed by a PBS wash and then stained with crystal violet or amido black.
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