Viral and bacterial isolates , propagation and preparation of stocks
Pipette 1.7 mL amplified 0.22 - µm filtered virus stock into 1.7 - mL screw cap tubes .
Float the microcentrifuge tubes in long ultracentrifuge tubes ( 14 × 95 mm ) using water until they are just flush with the top of the ultracentrifuge tubes ; balance them to within 1 g .
Load the ultracentrifuge tubes into the SW40 rotor and spin them at 133,000g for 1 h .
Recover the microcentrifuge tubes .
Remove the supernatant and resuspend the pellet .
Perform all subsequent steps under subdued light since the stain will fade if exposed .
Thaw the SYBR Green I , then add 1 µL stain to each concentrated virus tube and incubate for 15 min in the dark .
Verify the staining ( and monodispersal ) of the viruses by pipetting 1 µL of the suspension onto a microscope slide .
Add an 18 × 18 mm coverslip and observe the slide under the epifluorescence microscope .
Add 1.65 mL water or seawater medium to each tube and respin as above .
Remove the supernatant and resuspend as above ( completes first wash out of stain ) .
Repeat steps 10 and 11 .
Repeat steps 10 and 11 again .
