|Plaque assay for Synechococcus
Edited: 6 November 2009, Megan L. Larsen
18 x 150 mm glass tubes with metal caps, sterile
AN plates (1.5%)
AN soft agar (0.5%)
50 mM Tris-HCl, pH 7.8, sterile, for dilution blanks
13 x 100 mm tubes, capped and sterile
37°C water bath
WH7803, concentrated to 4.0 X 108 cells/ml (or Pbi or SAG-241-80)
1. Label and Pour AN plates1. Pour the plates either the day of titering or the day before. Fresh plates give more uniform plaque size; old plates (3 days or more) give more erratic results.
2. Melt the AN soft agar and aliquot 3.0 mL to each tube. Hold in the 37°C water bath. The soft agar can be made up in quantity in advance and sterilized in 200-250 ml aliquots and stored until ready to use. Melt enough for use. The soft agar can be remelted and used several times.
3. Quantify and concentrate cells. Use a hemocytometer to determine the concentration of cells per mL. This number will be used in determining how much AN should be used to resuspend cells after centrifugation. Concentrate the WH7803 Synechococcus to 4.0 X 108 cells/ml2 in the centrifuge at 5,000 rpm, 5 min, 4°C and resuspend the pellets with fresh AN.
4. Serially dilute the viral sample in 1/10 dilutions using 100 μL of virus and 900μL sterile 50 mM Tris-HCl adjusted to pH 7.8.
5. Add 0.1 ml of the diluted virus and 0.3 ml of the concentrated Synechococcus to each tube of molten agar. Remove 13 x 100 mm tubes of soft agar from the water bath to a test tube rack as needed. 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 (this needs to be done quickly, as the soft agar will solidify quickly once it has been poured onto the plate). Use a figure 8 motion to spread soft agar. Allow the agar to solidify (a few minutes), invert the plates with the lid down (so moisture condensation stays in the lid and off the agar surface), and incubate the plates at RT (25°C) in continuous light. Stack the plates only two deep, as the plaque size will increase if the plates are stacked deeper.