It is hard to determine from the image as I don't see plaques from dilution -1 but see some plaques in dilution -6. I would recommend infecting each well with 200 ul of each dilution instead of 100 ul and infect each dilution in two wells. You may also need to compare infectivity of your p1 virus in parallel with other positive control!

I do not understand your protocol "After this I did the standard agar (1% low melt agarose with 50/50 media or purified water) overlay and then added 1mL of media to feed the cells and incubated them for 4 days."

we overlay the cells with .8% low melting agarose in 1x (final concentration) media let solidify and then after few days colors the plaques with neutral red. If you put liquid media on the agarose layer it will diffuse under the agarose layer the virus will diffuse and infect all the cells...

Hello Paula Nazarena Arrías , Didier Poncet and Abdou Nagy ,

Thank you all for your insightful comments. I will give your suggestions a try and see what happens. Unfortunately, this is a new project so I don't have a positive control yet (a known baculovirus) but hopefully I will in the future.

All the best,

Sabrina

You can try to mix 10 ml melted agarose (low melting one) and 30 ml SFM900 (1.3X) (or regular media that you use for culturing cells) together which is plaquing medium and overlay cells with 2ml/well (of 6-well plate) plaquing medium. And wait more than 4 days to see the dead cells clearly by eye under a microscope then add neutral red maybe day 7 or day 9. I would not recommend to mix the neutral red and plaquing medium together and add it on cells on day 4.

I agree with Dr. Didier Poncet response. It's very suitable. So, you should apply it to obtain good results.

With best regards.

Hi Sabrina,

I suggest the following improvements to apply:

1. The virus inoculum is only in one part, while the other part of the monolayer is dry. I recommend shaking the plates every 10-15 minutes during your 1-hour incubation.

2. As mentioned above, 6-well plate could be better since it is more flat than a 12-well plate.

3. The neutral red may have contained crystals; it is important to dissolve it for a long time (preferably overnight) and then to filter it.

All the best

Thank you all for your help! I have redone the titration several times now. The monolayer seems intact now, after using 200uL of virus instead. I have also stopped adding media after the plaque overlay, as suggested. I was wondering if anyone would be able to provide feedback on these? I have circled some of the areas I hope is a plaque.

Thank you in advance for your time,

Sabrina

Hi

it looks that you got one ! you can pick it with a pasteur pipette (or a 1ml tip) and amplify it ... then you will have a cloned baculo...

the only pb I see is if you did serial 10x dilutions of your inoculum you should have 10 plaques (or at least several) in the preceeding dilution...