protocol_70.txt

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.