I am doing plaque assay on 6 wells plate, I usually allowed the virus to adhere on the cell surface for two hours, then addition of agarose overlay medium. I am using 200ul of inoculum. Where I am making mistakes? Whether in calculation or in protocol.
What is the volume of cell medium that you add to the wells? We usually use 1ml, as this is necessary for cells not to dry out.
For poliovirus, we first wash the wells with three washes of complete PBS (i.e. PBS containing 100 mM calcium and magnesium) then we apply 100 ul of diluted viral stock in complete PBS. We rock the plates by hand every ten minutes during a 30-min adsorption period to ensure distribution of the virus across the plated cells (and to keep them from drying out). The adsorption is carried out at 37C. We then overlay with 4 ml of complete DMEM containing 0.72% agarose, incubate an additional 48 hrs to develop plaques, and then stain with Crystal Violet. Hope this is of some help.
I usually add 1.5-2ml medium. I allow the virus to adhere for two hours, I am using 12ml RPMI medium with 1% of agarose and 2.5% of FBS (agarose overlay medium). I use to shake the plate every 45 minutes during adherence of virus. But still I am not getting good plaques. Where I am stuck then?
What is the purpose of your plaque assay? Quantification of infectious particles only?. If so, you may want to try TCID50. This technique is easier, you do not have to worry about secondary plaques, overlay agar or disrupting the cell monolayer, and gives you the chance to know your system better. Once you master it, you may find easier to set up your plaque assay.
you can use 0.5 ml of the virus for adsorption for 45 min in incubator with timely shaking the plates and use CMC instead of agarose. prepare 0.9ml of CMC and mix equally with 2X MEM along with 0.2% FCS and after proper mixing, overlay it onto each wells. 4-5 ml is sufficient for 6 well plate. keep it for incubation depending upon the virus infectivity and CPE. DO NOT REMOVE THE PLATES TIMELY FOR OBSERVING THE PLAQUES. because it may disturb the plaque formation and you will not get the required contable plaques... best of luck for the assay
Priyanka;
What cell line are you using?
What virus are you trying to titer?
For what purpose are you trying to titer the virus?
How are you shaking your plates?
How many cells do you add to each well and how confluent does that end up being before you add virus?
How long do you wait after infection before counting plaques?
Actually, I have to standardized Plaque assay for JEV. I am working on BHK-21 cell line, In the first dilution, i am getting few no of plaques, which is not correct. In the first dilution like 10 power -1, No of plaques should be around 30-40, or more than that, but I am getting only 11-17 plaques. That is my problem. In other protocols, they are showed some 50-70 plaques in first dilution and I am getting only 11-17 plaques. Why I am getting less plaques? What can i do to improve that.?
For getting more number of plaques use fresh vials of viral inoculum, not vials already thawed, as the virus titer falls. you can also add more quantity of viral inoculum for adsorption purpose or shake the 6 well plate a little in every fifteen minutes, which increases virus adsorption. Again try to add virus inoculum at 70-80% confluency as high cell density interferes with the development of visible plaques.
1. 200 ul of inoculum is not precis you must know the MOI of your virus
2. Two hours of adsorption is perhaps long for your cells and the cells risq damage
I think you must define the ideal time of adsorption, and the diluant of your virus ( Medium can be prefer to buffer if your cells are very sensitive during the time of adsorption ( beging to retract, cell became spheric)
Its better if we know the kind of virus and the cells us for your work, answers perhaps better
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