PBCV - 1 Virus Plaque Assay
Pour the plates either the day of titering or the day before .
Melt the MBBM soft agar and hold in the 50°C water bath .
Dispense 2.5 ml of soft agar to sterile 13 x 100 mm tubes and hold at 50°C .
Concentrate the NC64A chlorella to 4.0 X 108 cells / ml in the Sorvall centrifuge at 5,000 rpm , 5 min , 4°C and resuspend the pellets with fresh medium .
Concentrate enough so that 0.3 ml can be used for each plate .
Make up dilution blanks for the virus in the sterile 13 x 100 mm tubes with 50 mM Tris - HCl , pH 7.8 .
Dilute the virus in 1 / 10 serial dilutions .
Label the plates .
Remove 13 x 100 mm tubes of soft agar from the water bath to a test tube rack as needed .
To each tube , add 0.3 ml of the concentrated chlorella and 0.1 ml of the diluted virus ( when titering the NY - 2A virus , use 0.05 to 0.1 ml of the concentrated chlorella instead of 0.3 ml ) .
Mix briefly ( by rolling the tubes between the palms of the hands ) and pour the contents of the tube onto the plate .
Tilt the plate gently until the entire surface of the plate is covered with soft agar .
Allow the top agar to solidify and stick to the bottom agar ( maybe 20 minutes ) .
Invert the plates with the lid down .
Incubate the plates at 25°C in continuous light .
Stack the plates only two deep , as the plaque size will increase if the plates are stacked deeper .
Plaques will be ready to count after 3 - 4 days incubation .
