Dear all,

Most PFU assay protocols instruct to embed the cells in a feeding medium enriched agar. This provides the cells with nutrients and prevents further growth and virus dispersal.

I was wondering whether it is possible to provide the cells with new nutrients/chemicals while they are embedded in the agar (without affecting the assay).

I thought to pipet new medium onto the agar and rely on diffusion to bring the nutrients/chemicals to the cells. Is this feasible/sensible with say a 5mm layer of 1% agar? Or could it lead to the viruses spreading through the added medium and ruin the assay?

I realise this question might depend on the size of the viral particles, so let's say a 30nm diameter spherical particle.

Janaki Narayanan et al. 2006 state that the pore size of 1% LM agarose is 600nm, plenty for the viral particles to pass through. So the plaques are not necessarily dependent on cell-to-cell contact and viral particle migration within the agar might contribute.

Thus, to summarise, would adding medium onto the agar allow the viral particles to spread beyond the distance usual for plaque formation and thus ruin the assay.

Many thanks in advance for your insight.