protocol_72.txt

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.