SYBR Gold Staining for viral enumeration using 13 mm Anodisc 0.02 μm filters
Cut a 3 ml syringe as a filter funnel : ( Fig 1 ) Take a gasket from 13 mm MILLIPORE filter holder ( FISHER , SX0001300 ) .
Fig 2 Obtain a clamp for 25 filter funnel .
Make dilution of virus prep in 0.02um filtered seawater to a concentration of ~ E + 07 particles ml - 1 .
Thaw the commercial stock of SYBR - Gold in the dark at RT and centrifuge at ~ 3000 rpm for 5 minutes because SYBR - Gold is in DMSO .
Centrifuge at ~ 3000 rpm for 5 minutes .
Dilute SYBR - Gold in 0.02 µm filtered TE buffer to 100x ( 10 µl in 990 µl TE buffer ) .
Add 1 µl of SYBR working stock in 49 µl 0.02 µm filtered TE buffer in a plastic Petri 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 and place a 25 mm 0.2 nitrocellulose filter ( the support filter ) on top of the water .
Fig 3 Switch on the vacuum , the support filter should be flat on the filter base .
Place a 13 mm Anodisc filter on the wet nitrocellulose filter .
Fig 4 Place a gasket on the 13 mm Anodisc filter .
Fig 5 Place the syringe filter funnel carefully on the gasket and apply the clamp .
Fig 6 Switch on the vacuum and add samples for filtration , leave the vacuum on for 1 more minutes after sample drained completely .
Leave the vacuum on for 1 more minutes after sample drained completely .
Take away the clamp and syringe filter funnel while vacuum is on .
Carefully push the filer to the edge of the filter base by tweezers while vacuum is on to remove the filter .
Fig 7 Dry filter membrane on Kimwipes in the dark at RT completely .
Remove membrane and place viruses - side up on staining solution in the Petri dish for 15 min , cover the Petri dish by aluminum foil .
Cover the Petri dish by aluminum foil .
Dry filter membrane again on Kimwipes in the dark at RT completely .
Pipet 10 µl antifade solution on a microscope slide and place the stained filter membrane on top of it .
Pipet 10 µl antifade solution on a coverslid and carefully place it on the filter to avoid bubbles .
Pipet 10 µl antifade solution on a coverslid and 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 .
