Extinction dilution cloning for isolation of viruses infecting protists
Prepare 9 tubes (numbered #1–#9) with 4.5 mL medium.
Add 500 μL of the virus filtrate (virus size fraction) to tube #1 and vortex.
Change the pipette tip and transfer 500 μL of suspension #1 to tube #2 and vortex.
Repeat the procedure to tube #8.
Pour vigorously growing algal host culture into the reservoir tray, fill the pipetter and add 150 μL culture to each well in lines 1–9 of a 96-well cell culture plate.
Fill the pipetter and add 150 μL culture to each well in lines 1–9 of a 96-well cell culture plate.
Empty the tray.
Pour dilution tube #9 (control medium, no virus) into the reservoir tray.
Fill the pipetter and add 100 μL of to each well in line 9.
Empty the tray.
Repeat steps 7 and 8 for dilution tube #8–#1 and fill the respective well lines in the culture plate.
Put on the lid and seal tightly with plastic tape to avoid drying.
Incubate under appropriate conditions.
Use an inverted microscope and inspect the culture plates for signs of cell lysis at regular intervals.
Mark wells where lysis is observed and continue the incubation with daily inspections until no more lysis occurs.
Prepare a second extinction dilution with virus from the most-diluted well.
Propagate virus clone from the most diluted well in a larger volume and store appropriately.
