I am trying to do a plaque assay for the virus we use in our lab. We purchased through ATCC and diluted it with PBS per a protocol in the lab. Now I am trying to run a plaque assay to get the titer of the virus so we can give the proper amount of virus for infection. I have done the assay off of a collaborator's (from another institution) protocol, and have done it numerous times with no success. Does anyone have any tips/tricks/hints of any sort? I am doing 1:5 serial dilution of the virus in PBS/0.5mg/mL BSA. The virus is incubated for 2 hours at 37C before being removed then washed 2x with PBS. Lastly 3 mL of agarose overlay is added, incubated at room temp for 5-10 minutes then placed in a 37C 5% CO2 incubator for 3-5 days. The overlay for a 6 well plates consists of 10 mL 1.5% agarose in MQ water, 10 mL 2x MEM, 10 mL regular MEM, Trypsin (2ug/mL final concentration) and 5uL Gentamicin. Any tips would be appreciated.