Method 6

Background

SYBR Gold has been demonstrated to be a bright and stable stain for counting viruses and bacteria in aquatic environments (Chen et al. 2001). SYBR Gold stains both DNA and RNA. Viruses or bacteria stained with SYBR Gold emit yellow-green fluorescence (with blue-green light excitation) that can be enumerated under epifluorescence microscopy. Typically, viral and bacterial densities in aquatic samples are approximately 107 VLPs/ml and 106 bacteria/ml, respectively. The protocol described here is mainly for aquatic samples, can also be used for counting viruses and bacteria in culturesNote 1. In some cases, if the viruses-to-bacteria ratio is stable, both can be counted from the same filter (0.02 µm pore-size Anodisc filter), we recommend counting viruses and bacteria from separate filters because the viruses-to-bacteria ratio may vary dramatically.

(Right: a typical view of marine microbial community stained with SYBR Gold 2X)

Fixation and storage of samples for viral counting

For natural water samples, fix 1-2 ml of water sample with paraformaldehyde (0.02 µm filtered) at the final conc. of 1%, for 10 min at room temperature.

Place the fixed sample into liquid nitrogen for at least 30 min. Once frozen, samples can be stored at -80oC or -20oC for long termNote 2.

For viral counting, thaw the sample on ice and move to step 5 as follows.

Procedures for counting viruses

Be sure to wear your lab gloves since you don’t want to stain your own DNA. Prepare the SYBR Gold working solution (2X) right before you start filtering samples (see Step 2 below). One should also estimate how many samples need to be stained and prepare an appropriate amount of working solution.

Preparation of SYBR Gold working solution: Original SYBR Gold stock (Invitrogen) comes in concentrated form (10,000X). A working solution with 2X SYBR Gold is ideal for staining viruses and bacteria. We recommend using freshly prepared 2X working solution because SYBR Gold at low concentration is not stable. Typically, a secondary stock solution (100 X) is prepared by diluting 1:100 of the original SYBR Gold stock with 0.02 µm filtered TE buffer (10mM Tris-Cl; 1 mM EDTA, pH7.4-7.6). The secondary SYBR Gold stock can be stored at -20 C for 1-2 weeks. The 2X working solution is prepared from the 100X solution by further dilution with TE buffer. A 100 µl drop of 2X SYBR Gold solution is pipetted onto a Petri dish (for each filter). Up to 4 filters can be stained in one Petri dish (diameter: 10cm).

Place a 0.02 µm pore-size Anodisc filter over a pre-wetted 0.8-2.0µm Millipore filter. The Millipore filter serves as a support and can be reused as long as it remains intact and flat.

Apply a vacuum so that the Anodisc filter will stick to the backing filter. Try to avoid air bubbles.

Filter 200-500 µl of water sample through the Anodisc filter with vacuum pressure no greater than 20 kPa (or 150 mm Hg).

Remove the filter tower when the sample has passed through the filter, and keep the vacuum on while removing the filter.

Any remaining solution on the back of the Anodisc filter or top plastic rim should be removed by wicking the filter with a Kimwipe or paper tissue (do not touch the sample area). The drying step here is critical in order to obtain a good staining result.

The Anodisc filter is laid with sample side up on the drop of staining solution (2X SYBR Gold) for 15 minutes in the dark.

After staining, pick up the filter with forceps and wick away remaining solution on the filter with a Kimwipe tissue. Again, the drying step is important here.

Place a drop (10 µl) of mounting solution onto a clean slide, and mount the filter on top of the drop. Spot 5-10 µl mounting solution onto a clean cover slip, flip it over to cover the sample filterNote 3.

Add one drop of immersion oil to the top of the cover slip, and examine the slide under blue-green light excitation

(Fitch filter, excitation at 480-495nm) using an epifluorecence microscopeNote 4.

At least a total of 300 VLPs or bacterial cells should be counted from 10-20 random view fields.

Procedures for counting bacteria

Follow steps 1 to 4 as described above.

1-2 ml of aquatic samples (~106 bacterial cells per ml) is filtered through a 0.2µm pore-size polycarbonate membrane filter (Millipore)Note 5 with vacuum pressure ca. 150 mm Hg.

Release the vacuum pressure to zero after all the sample filters through. It is important to double check that vacuum is completely released before SYBR Gold stain is added.

Add 300 µl of 2X SYBR Gold solution into the filter towel, and cover the towel with aluminum foil to avoid light. Stain bacterial cells for 15 min in the dark.

After staining, remove the stain by applying the vacuum.

Follow the steps 9 to11 as described above.

Materials

Anodisc filters, 0.02 µm pore size, 25 mm in diameter (Whatman)

Polycarbonate membrane filters, 0.2 µm and 0.8-2.0 µm pore size, 25 mm in diameter (Millipore)

Filter holder for 25 mm diameter filters, with 15 ml funnel (Millipore)

A vacuum flask (see the image)

Petri dishes, pipettors and pipette tips

Forceps

Glass slides and cover slips

Kimwipes or paper towels

SYBR Gold stain, 10,000X (Invitrogen)

Mounting solution: A mixture of 50% glycerol and 50% TE buffer (TrisCl: 10mM, EDTA 1mM, pH 7.4-7.6)Note 4.

Pumps (a hand pump from Nalgene is handy and quite)

Notes

1) It is noteworthy that filtration volume needs to be adjusted accordingly if viral or bacterial density is very different from those in the natural environment.

2) The liquid nitrogen quick-freeze treatment here is modified for viral counting. With this treatment, viral particles stained by SYBR Gold or other dyes are brighter and easier to count compared to those without. If no liquid nitrogen available, fixed samples in 1% paraformaldehyde can be stored at 4oC in the dark for a few months.

3) The stained samples can be stored at 4oC in the dark for up to 3 months without significant loss of fluorescence signal. SYBR Gold is a stable stain and can be used without antifade treatment. Antifade agent, 0.1% phenylenediamine in glycerol:PBS (1:1), is recommended for SYBR Green I stain (Noble and Fuhrman 1998).

4) For some samples, a high background signal may interfere with counting. In this case, two remedies can be tried to reduce the background fluorescence. One is to reduce the concentration of working solution to 1X or 0.5X. The other way is to rinse off the extra stain from the filter by laying the filter onto a drop of TE buffer for 1 min.

5) Alternatively, bacterial cells can be retained on a 0.2µm pore-size Anodisc filter (Whatman). Anodisc filters are nearly three times more expensive than membrane filters. However, Anodisc filter is easy to manipulate and provides a flat and smooth surface for counting.

References

Brussaard, C. P. D. 2004. Optimization of procedures for counting viruses by flow cytometry. Appl. Environ. Microbiol. 70:1506-1513.

Chen, F., J. R. Lu, B. Binder and R. E. Hodson. 2001. Enumeration of viruses in aquatic environments using SYBR Glod stain: application of digital image analysis and flow cytometer. Appl. Environ. Microbiol. 67:539-545.

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.

Acknowledgement

We thank Kate O’Mara for her photographic arts and editorial changes.

Selected photos showing the key steps for preparing SYBR Gold staining

1. Basic setup for SYBR Gold counting 2. Placing a filter on top of filter tower

3. Adding a drop SYBR Gold solution (2x) 4. Placing the filter on top of SYBR Gold

5. Stain the filer in dark for 15 min. 6. Wick off the excess stain from the filter