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 step 8 and 10 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 .
