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Protocol for SYBR Green I Counts of Viruses and Bacteria

(including all the little tricks) updated July 2001

CITE: Noble, R.T., and J.A. Fuhrman. 1998. Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquat. Microb. Ecol. 14: 113-118

NOTE: Same protocol used for Viruses as well as Prokaryotes for Microscopy

• 0.02 um pore size, 25 mm diameter Anodisc filters (from Whatman; made of aluminum oxide).

• 0.8 um, 25 mm diam. AA Millipore mixed-ester membrane filters.

• glass 25 mm diameter filter holder with 15 ml funnel, Millipore-type.

• Sample (PRESERVED with 1% 0.02 um filtered formalin), plastic Petri dish, pipettes and tips.

• SYBR I solution from Molecular Probes*: make a stock solution by diluting 1:10 of the original concentration with 0.02 um filtered deionized water, and stored frozen for a week or so. From this stock solution, make a 2.5% working solution (final 2.5 x 10 -3 dilution of stock) just before use - preferably on the dish as described below.

• Antifade mounting solution: 50% glycerol, 50% PBS (120 mM NaCl, 10 mM NaH2PO4 pH 7.5), 0.1% p-phenylenediamine (made fresh DAILY from the glycerol/PBS + frozen 10% stock of the phenylenediamine).

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• Place the Anodisc filter over a pre-wetted 0.8 um Millipore filter (reusable many times, as long as it is completely intact and flat), in the glass filter unit.

• Apply the vacuum just after placing the Anodisc on the moist Millipore filter, and the Anodisc should moisten and stick in place completely flat and smooth with no air bubbles beneath.

• Prepare a 100 ul drop of SYBR (best made fresh e.g. : 2.5 ul of 1:10 diluted stock + 97.5 ul water) on the bottom of a plastic Petri dish (concn. is 2.5/1000 of manufacturer-provided stock).

• Try double the concentration of SYBR (i.e. 5 ul + 95 ul water) if there is a visible orange precipitate in the 1:10 diluted stock and/or the viruses do not look bright in the slides.  One standard Perti dish can hold up to 4 filters, each on separate drops of SYBR.

• Sample volumes to use: Oceanic seawater volumes typically 2 ml; deep seawater may need 5 ml or more.  Filter the preserved seawater sample through the Anodisc filter at approximately 20 kPa (or 7" Hg). The  filter funnel (or tower) should be removed shortly after the last liquid passes through, and the vacuum left  on while removing the filter.

• Any seawater on the back of the Anodisc filters or top plastic rim are blotted with a Kimwipe or paper  tissue (do not touch the top surface), so that the filter is uniformly dry and looks opaque when held up to the light for examination.

• IT IS IMPORTANT TO DRY IT COMPLETELY, best done by rubbing the bottom surface very gently on a dry Kimwipe for at least 2 minutes (in humid labs, a dessicator may be needed after rubbing the bottom dry on the Kimwipe).

• The Anodisc filters are laid sample side up on the drops of the staining solution for 15 minutes in a dark drawer or box (NOTE: 10 min. is OK, but fluorescence fades more). 

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• After the staining period**, pick up the filter with forceps so as to leave most of the stain on the dish, and any remaining moisture is then carefully wicked away from the back side of the membrane (or on the top plastic rim) with a Kimwipe, again until it looks uniformly opaque when held up to the light.

• Do not touch the Kimwipe to the top filtration surface (but the plastic rim is OK). AGAIN, DRY IT COMPLETELY FOR A FEW MINUTES (as above). Complete drying both before and after staining is important for bright fluorescence and to prevent fading.

• Place the completely dried filter on a glass slide, then put a 30 ul drop of antifade mounting solution on a 25 mm cover slip and invert it over the filter.

• Push the cover slip to be sure the mounting solution fills the square space under the cover slip.

• View with blue excitation. If viruses are in more than one focal plane, or appear to move, remake the slide.  If they fade rapidly, the filter probably was not dried well enough during preparation.

• Count at least 200 each viruses and bacteria in at least 10 fields. The field size for the viruses can be much smaller than for bacteria, perhaps 3 small squares (out of a typical 100 square grid).

• To calculate total abundance, consider that the viruses appear to be filtered through the entire 20 mm diameter area enclosed by the plastic rim (check this yourself, as it may change with different filter holders).

• Store slides FROZEN and dark.  Even 3-year-old slides stored this way retain bright fluorescence in our experience.

* SYBR formula and MW are proprietary. Our tested stock from Molecular Probes had an O.D. at 494 nm of 0.42 when diluted 1000-fold in water.

** If you see a film or spot of liquid on TOP of the filter after the staining step, it was not prepared properly (or the filter is cracked or defective) and it should not be used.

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