Assay to Determine the Virus Infectious Dose of Drosophila C Virus in Cultured Insect Cells

To determine the average virus tissue culture infective dose, first, seed 1 x 10⁵ S2 star-cells in 100 microliters of complete medium into eight wells per column in 12 rows of a 96-well plate. Then, return the plate to the 25-degree Celsius incubator.

Randomly select five tubes of virus from minus 80 degrees Celsius storage. While the cells are settling, fill 10 sterile 1.5-milliliter microcentrifuge tubes with 450 microliters of sterile complete medium, and add 50 microliters of virus stock to the first 1.5-milliliter tube containing 450 microliters of sterile complete medium, diluting the suspensions tenfold per step to the 12th well.

At the end of the incubation add 50 microliters of each dilution to the appropriate well in each column for that tube of virus, and add 50 microliters of culture medium without virus to one well per column, as the negative control well. Then, place the plate in the 25-degree Celsius incubator for three days, and assess the cytopathic effect of each virus stock under a bright field microscope at a 20 times magnification daily.

Classify a well in which the cells look blurry and the medium is full of fragments as a positive well, and a well in which the cell morphology is normal as a negative well.