Somehow my Plaque Assay for LCMV does not work in a reproducible manner.

I have followed a protocol established in a collaborative lab but sometimes it works and sometimes it doesnt.

The cell layer is always intact but either the well appears completely blank or there are "stains" rather than plaques showing.

Could anyone help me out? Or maybe somebody knows what might/could be the issue?

The protocol is as followed.

I use it mostly for spleen & Small intestine which I homogenize thoroughly .

I put 200µl of the tissue suspension on 400µl of MEM(5%FCS) in a 24well plate and make 6 dilutions/sample.

I then transfer 200µl of that virus dilution into a new 24well plate & add 2.5x10^5 Verocells and let it incubate 5hrs at 37°C with 5%CO2.

Then I overlay it with 300µl of a 1:1 micture 2xDMEM:2%Methylcellulose

THen incubation 60-72hrs in 37°C 5%CO2 incubator.

after incubation fix with 250µl PFA 20-30mins-wash

add 250µl 0.5%Triton X100 20mins incubation-wash

add 200µl 10%FCS PBS 60mins wash

add 200µl VLA-4 ab (1:100) 60mins incubation-wash

add 200µl peroxidase labeled goat anti-rat ab (1:100) 60mins incubation-wash

add 300µ of developing substrate (12.9ml 0.2M Na2HPO4 +12.1ml 0.1M citric acid + 25ml ddH2O+20mg OPDtablet+20µlH2O2)