Why can I not see any plaques when performing a viral plaque assay with MHV?

29 December 2020 2 6K Report

Hi, everyone!

This is my first question posted to Research Gate, so I hope I’m doing this right. Please correct me if I need to change anything in future posts.

I’ve attempted performing a viral plaque assay three times now without any plaques forming in any of the trials. I have little to no background in microbiology, so it’s possible that I’m making a simple and obvious mistake, but I’m open to any and all feedback. I am using the mammalian cell NCTC clone 1469 in a 6-well plate and infecting it with MHV. I’ve outlined the general procedure used below.

Begin growing NCTC clone 1469 cells in 6-well plates. DMEM with 10% horse serum is used as growing media, and cells are left to grow for ~24 hours.

Prepare virus dilutions by placing ~1 mL of undiluted virus solution (of unknown concentration) in 1 vial. Take 500 μL from the first vial and place in a second vial, adding 500 μL of NCTC 135. Repeat the same dilution process four more times to obtain dilutions of 10-1 to 10-5.

Remove growing media from the prepared 6-well plate and rinse with PBS.

Add 500 μL of each viral dilution to a separate well.

Incubate the infected monolayers at 37oC for 1 hour, shaking plates occasionally to distribute viral dilutions in wells.

Overlay inoculated cells with 2 mL of NCTC 135 containing 1% bacto agar, 5% horse serum, and 10% tryptose phosphate broth.

Incubate well plate at 37oC for ~24 hours, or until plaques become visible.

Discard the agarose layer and stain the culture with a solution of 0.1% crystal violet and 20% ethanol for 10 minutes to 1 hour.

Count plaques in each well and calculate the concentration of initial suspension in PFU/mL.

Is it possible that an Endpoint Dilution Assay would be more effective in determining viral concentration for this specific cell and virus combination?

Thank you!

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