SYBR Gold Staining for Viral Enumeration (Case 2)
Prepare buffer glutaraldehyde: Make dilution of virus prep in 0.02 µm filtered seawater to a concentration of ~E+07 particles ml-1 Fix viral sample with 20% buffered glut to a final concentration of 0.5%. 
Incubate at 4°C for 30 min.
Prepare working solutions of SYBR Gold. 
Add 2 µl of SYBR working stock in 98 µl 0.02 µm filtered mQ in a plastic Petri dish, 4 drops in one dish. 
Cover the dish by aluminum foil.  
Set up the filtration unit, connecting it to a vacuum.  
Add a few drops of 0.02 µm filtered mQ on the filter base. 
Place a 0.2 nitrocellulose filter (the support filter) on top of the water.  
Switch on the vacuum, the support filter should be flat on the filter base. 
Add a few drops of 0.02 µm filtered mQ on the support filter. 
Switch on the vacuum to pull the water through.  
Apply a 0.02 µm Anodisc filter over the support filter.  
Apply the filter tower and clamp while vacuum is on. 
Switch off the vacuum and add viral samples. 
Switch on the vacuum and wash filter set with ~1 ml of 0.02 µm filtered mQ. 
Remove the filter while the vacuum is still on. 
Rinse tower in 1L Q-water in between samples. 
Blot onto paper towel to dry.  
Dry filter membrane on Kimwipes at RT completely. 
Remove membrane and place viruses side up on staining solution in the Petri dish for 15 minutes (cover with aluminum foil). 
Dry filter membrane again on Kimwipes in the dark at RT completely. 
Pipet 20 µl antifade solution on a microscope slide.  
Place the stained filter membrane on top of it.  
Pipet 30 µl antifade solution on a cover slide.  
Carefully place it on the filter to avoid bubbles.  
Place slide at -20°C to enhance fluorescence.  
Read slides using 100x oil immersion objective and inverted fluorescent microscope
