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.
