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
