Enumeration of viruses

3 Enumeration of Viruses Rachel T N o b l e Southern California CoastaIWater Research Project (SCCWRP), 7171 Fenwick Lane,Westminster, CA, 92683 USA

CONTENTS Introduction Method Troubleshooting Applications Conclusions

~,~,~,~,~,~, I N T R O D U C T I O N Viruses are now known to be the most numerically abundant component of marine plankton (Bergh et al., 1989; Bratbak et al., 1990; Fuhrman and Suttle, 1993; Hennes and Suttle, 1995; Noble and Fuhrman, 1998; Fuhrman, 1999). In the late 1980s, some of the first reports were published documenting the high abundances of marine viruses at 10 '~' particles per liter of seawater, exceeding the typical abundance of bacteria (Proctor et al., 1988; Bergh et al., 1989). Since then, studies have demonstrated high numbers of viruses in all types of marine environments, from eutrophic coastal waters to deep blue open-ocean waters, from the sea surface to the depths of the sea, and from the polar to the tropical regions (Bratbak et aI., 1990; Cochlan et al., 1993; Guixa-Boixareu et al., 1996; Steward et al., 1996). Multiple groups of researchers have identified important roles of viruses in the mortality of heterotrophic bacterioplankton, cyanobacteria, and phytoplankton. They also play a role in biogeochemical cycling and control of species diversity (Fuhrman, 1999; Wilhelm and Suttle, 1999). Specifically, it has been shown by a number of researchers that viruses are capable of causing a significant portion of the heterotrophic bacterial mortality in certain marine environments (Fuhrman and Noble, 1995; Guixa-Boixareu et al., 1996; Steward et al., 1996; Weinbauer and H6fle, 1998). Current research in the fields of marine microbiology and marine microbial ecology requires the ability to rapidly enumerate viruses and bacteria. In the past, counting microbes in seawater samples by transmission electron microscopy (TEM) was the standard method (Bergh et al., 1989; Borsheim et al., 1990). This method is tedious, expensive, involves METt tODS IN MICROBIOLOGY, VOLUME 30 ISBN 0 12 521530 4

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time-consuming preparatory steps, lacks precision, and requires expensive ultracentrifugation and electron microscopy equipment not available to many researchers. In recent years, other stains such as DAPI (4'6diamidino-2-phenylindole) and Yo-Pro I (Molecular Probes, Inc.) have been used for enumeration of virus particles by epifluorescence microscopy (Suttle et al., 1990; Hara et al., 1991; Proctor and Fuhrman, 1992; Hennes and Suttle, 1995; Weinbauer and Suttle, 1997; Xenopolous and Bird, 1997). However, DAPI is not sufficiently bright to be used with direct visual observation on many microscopes. Therefore, it has been necessary for some researchers to use photomicrography or image intensification in order to count and size virus particles (Hara et al., 1991; Fuhrman et al., 1993). Newer microscopes may allow direct visual counts with this stain, but many labs do not possess high-powered microscopes and have turned to the use of brighter stains (Weinbauer and Suttle, 1997). Yo-Pro I has recently been used for seawater studies (Hermes and Suttle, 1995; Weinbauer and Suttle, 1997). The stain intensity is bright, but the stain is not compatible with aldehydes (such as formaldehyde and glutaraldehyde), it requires extra dilution and rinsing steps to remove salts, and the staining time is 2 days. Improvements have been made on the Yo-Pro I method originally reported by Weinbauer and Suttle (1997), which involved microwaving of the samples to permit penetration of the stain (Xenopolous and Bird, 1997). A newer stain, SYBR Green I, has been developed. The aim of this chapter is to provide the details necessary for enumeration of viruses and bacteria in seawater using the nucleic acid stain SYBR Green I (Molecular Probes, Inc.). This stain was originally used for research using flow cytometry by Marie et al. (1997). SYBR Green I is a viable tool which yields virus counts comparable to TEM in a broad variety of samples, and seems to be more easily applied to the analysis of seawater samples than some of the previously mentioned stains. SYBR Green I has the advantages of being usable in conjunction with seawater and commonly used fixatives and a short staining period. SYBR Green I stained viruses and bacteria are intensely stained and easy to distinguish from other particles with both older and newer generation epifluorescence microscopes. In addition to the methodological advantages that SYBR Green I offers, it is inexpensive and its manufacturer claims it to be less carcinogenic than other typical nucleic acid stains. It has recently been noted that another nucleic acid stain, SYBR Gold, also made by Molecular Probes, Inc., can be used interchangeably with SYBR Green I. This stain appears to require a slightly shorter staining time (12 min), and is less expensive. Although the author has not performed a quantitative comparison between SYBR Green I and SYBR Gold, it appears that this latter stain can be used interchangeably with the protocol listed here, with the minor change in the staining time (Chen et al., in press).

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ENUMERATION GREEN I

OFVIRUSES

USING SYBR

Principle The SYBR Green I m e t h o d is used for easy and rapid enumeration of both marine viruses and bacteria in seawater samples. Seawater samples are collected and fixed with formalin, which cross-links the proteins found in cell m e m b r a n e s and viral coat proteins. Marine bacteria and viruses are very a b u n d a n t in seawater, sufficient that w h e n seawater samples are filtered with an ultra-fine pore size filter (Anodisc, 0.02 ~m), they can be counted by epifluorescence microscopy. The material on the filter can be stained with a variety of stains, but for quick and inexpensive viral and bacterial counts, we have found the nucleic acid stain SYBR Green I to be the most advantageous. By diffusion, the stain permeates the filter and stains any particles containing DNA or RNA. Convenient quantification can be achieved by m o u n t i n g the filter on a glass slide, with the use of an anti-fade m o u n t i n g solution u n d e r the cover slip, and counting tile fluorescent particles by epifluorescence microscopy. The average n u m b e r of viruses and bacteria counted per r a n d o m l y selected field is multiplied by a conversion factor which represents the total n u m b e r of microscope fields that fit into the total available filter area. This conversion factor is then used to calculate a total n u m b e r of viruses or bacteria per filtered volume, often expressed as virus particles or cells ml ', respectively.

Equipment and reagents • • • • • • • • • • •

Formalin (37 % formaldehyde solution, Sigma Chemical, Inc.) 50 ml polypropylene conical tubes for sampling (Fisher Scientific, Inc.) Whatman Anodisc 0.02 ~m Membrane Filters (Fisher Scientific, Inc.) Millipore 0.8 ~m filters (Fisher Scientific, Inc. or Millipore, Inc.) Filtration manifolds and towers (Fisher Scientific, Inc.) SYBR Green I stain (Molecular Probes, Inc.) Sterile, 0.02 ~m filtered, deionized water Plastic Petri dishes (VWR Scientific Inc.) Eppendorf pipets and tips (VWR Scientific, Inc.) Slides and cover slips (VWR Scientific, Inc.) Mixture of 50% Phosphate Buffered Saline (0.05 M Na2HPO4, 0.85% NaCI, pH 7.5, Sigma Chemical Co.) and 50% glycerol (Sigma Chemical Co., should be made in advance and stored in the refrigerator) • p-Phenylenediamine (dihydrochloric acid, 10% w/v, Sigma Chemical Co., should be made in advance and stored frozen, in the dark)

Assay Sample collection 1. Collect seawater samples using Niskin bottles or triple acid-rinsed bottles (5 ~ hydrochloric acid), or by bucket and transferred into

45

acid-rinsed and sample-rinsed 50 ml polypropylene tubes (Fisher Scientific, Inc.) . A d d 0.02 ~m filtered formalin to the sample(s) to a final concentration of 1%. If the samples are not going to be filtered immediately, they should be stored in the dark at 4°C. Fixed samples can be stored chilled for up to a week, but optimally should be processed as soon as possible. Slide preparation 1. Whenever possible, preparation should be done under subdued light (dimmed room light is suitable). When ready to begin, perform a dilution of SYBR Green I stock from Molecular Probes, Inc. to 1:10 of the supplied concentration with 0.02 ~m filtered, sterile, deionized water. For example, dilute 5 ~1 of the SYBR Green I stock solution with 45 ~1 H20. Put unused stock immediately back at -20°C. Immediately prior to sample filtration prepare the anti-fade mounting solution. To do this, mix 990 ~tl of the 50% PBS/50% glycerol mixture with 10 ~tl of 10% p-phenylenediamine. Store this solution on ice, in the dark, while working. When removing the 10 ~tl of p-phenylenediamine, thaw, vortex, remove the 10 ~1 and then refreeze immediately. Also, for each filter to be stained, place a 97.5 ~1 drop of 0.02 ~m filtered, sterile, deionized water inside a clean plastic Petri dish. To each drop of water, add 2.5 ~I of the 10% SYBR Green I working solution (final dilution 2.5 x 10 ~). Keep the Petri dishes with the drops of stain in a cool, dark place during the course of the filtration. Filter a I to 10 ml formalin-fixed seawater sample through a 25 mm, 0.02 ~m pore-size, Anodisc membrane filter (Fisher Scientific, Inc.), backed by a 0.8 ~m cellulose mixed ester membrane (type AA, Millipore, Inc.) at 15-20 kPa vacuum. The volume of seawater to be filtered depends upon the type of seawater used, eutrophic estuarine samples will require filtration of only about 1 ml, whereas open-ocean seawater samples m a y require filtration of up to 10 ml in order to provide for statistically meaningful bacterial and viral counts. Filter the Anodisc to dryness and remove it with forceps with the vacuum still on. Lay the filter, sample side up, on a drop of the staining solution in the Petri dish for 15 rain in the dark. After the staining period, pick the filter up with forceps and carefully wick away any remaining moisture by touching the back side of the membrane to a Kimwipe (any droplets on the top plastic rim of the filter should also be blotted). 6. Place the filter on a glass slide. Onto a 25 mm square cover slip, place a 30 ~1 drop of the anti-fade mounting solution (50% PBS / 50% glycerol with 0.1% p-phenylenediamine). Invert the cover slip, drop-side down, onto the filter. Press d o w n on the cover slip with a 2.

.

.

.

46

Kimwipe to be sure that all bubbles are displaced. If the SYBR Green I stained filters are to be counted immediately, place a drop of immersion oil on top of the cover slip. 7. For reading the slides, r a n d o m l y select 10-20 fields to count a total of >200 viruses and >200 bacteria per filter u n d e r blue excitation on an epifluorescence microscope equipped with a 100 W Hg lamp. Virus particles will appear as distinctly shaped 'pinpricks' and fluoresce bright green, and bacterial cells will be much brighter and should easily be distinguished from viruses because of their relative size.

Troubleshooting SYBR Green I slides should be counted immediately, but can be stored frozen for 2-3 weeks. W h e n preparing the anti-fade m o u n t i n g solution, r e m e m b e r that p - p h e n y l e n e d i a m i n e is quickly oxidized at room temperature. Tubes of p - p h e n y l e n e d i a m i n e can be t h a w e d / f r o z e n only about three times before they need to be discarded. If a b r o w n color is noted in the p - p h e n y l e n e d i a m i n e solution, discard it, and immediately make a fresh solution. If, w h e n the slides are being counted, the viruses appear to be in more than one focal plane, or appear to be floating, remake the slide. Also, if the background fluorescence makes it difficult to count the slide, i.e. the slide appears washed out, remake the slide, staining the filter for the prescribed a m o u n t of time.

Applications Seawater samples stained with SYBR Green I and observed u n d e r an epifluorescence microscope demonstrate bacteria that are intensely stained, and virus particles that are brightly stained and countable (Plate 1). Detritus has not been significantly stained by SYBR Green I in past observations. Previous analysis of coastal samples in Noble and Fuhrman (1998), d e m o n s t r a t e d bacterial counts by SYBR Green I that were essentially identical to acridine orange counts, with an r -~of 0.99. Virus counts by both SYBR Green I and TEM are highly correlated (r: = 0.93, p < 0.001, Figure 3.1). There is a tendency for tile SYBR Green I counts to be higher, as indicated by the slope of the linear regression being 1.10 (Figure 3.1). In Noble and F u h r m a n (1998), virus counts by SYBR Green I and TEM s h o w e d very similar patterns in seawater samples from all depths, with SYBR Green I counts about 25% higher than TEM counts. Freshwater samples stained with SYBR Green I demonstrated viruses and bacteria that appear to be even more intensely stained than those from seawater (Fuhrman and Noble, 1998). In recent studies by Hermes and Suttle (1995) and Weinbauer and Suttle (1997), Yo-Pro 1 based virus counts were found to average about 2.3 and 47

3.0

C

E

2.0

"J

x

a3 1.0

•

(3 n>-

•

0.00.0

•

1.0

i

2.0

3.0

TEM (x 10 7 virus ml "1)

Figure 3.1. Comparison of virus counts using SYBR Green I and transmission electron microscopy (TEM) for a diverse set of marine samples. Error bars indicate the standard deviation of duplicate samples; where they are not seen, the standard deviation was less than the size of the symbol. Line indicates linear regression (r2 = 0.92, a subset of the data used for this graph appears as part of Figure 3 in Noble and Fuhrman, 1998).

1.5 times higher than counts by TEM, respectively, over a wide range. It has been demonstrated that at lower viral densities, TEM counts were generally similar to those by SYBR Green I, and at higher viral densities, TEM counts were clearly lower than those for SYBR Green I (Noble and Fuhrman, 1998). This trend is also consistent with work published by both H e n n e s and Suttle (1995), and Weinbauer and Suttle (1997) w h e n comparing Yo-Pro I and TEM. Both of these studies have also m a d e comparisons with an alternative stain for epifluorescence microscopy, DAPI. However, DAPI is relatively dim, and requires high-quality optics for quantitative visualization of viruses. SYBR Green I can be used to stain virus and bacterial particles in m a n y different types of samples, marine, freshwater, and sediment included. It is apparently not inhibited by the use of fixatives, the staining period is short, and SYBR Green ! is reported to stain both RNA and DNA viruses. In certain environments, it m a y be necessary to increase the recomm e n d e d concentration of SYBR Green I (2.5 x 10 ~ dilution) to yield brighter and more stably fluorescent viruses. Fading of the samples is best retarded with the use of the prescribed anti-fade m o u n t i n g solution. SYBR Gold (Molecular Probes, Inc.) is another sensitive fluorescent stain for detecting double- or single-stranded DNA or RNA. According to the product information from Molecular Probes, Inc., SYBR Gold has been s h o w n to be more sensitive than SYBR Green [ and II for staining nucleic acids in electrophoresis. A parallel comparison between SYBR

48

Gold and SYBR Green I was recently carried out by staining the same seawater sample (Chen et al., in press). It was found that the epifluorescence signal of SYBR Gold stained viruses lasted longer than that of SYBR Green I stained viruses w h e n the final concentration (2.5x) was used for both SYBR stains. Without using any anti-fade m o u n t i n g solution, the fluorescence of SYBR Gold stained viruses was stable for more than 2 min, while the SYBR Green I signal faded within 30 s. When a higher concentration (25x) of SYBR Gold or Green I was used, some bacterial cells in natural samples appeared to be overstained and their fluorescent halos could overcast the fluorescent signal of stained viruses. SYBR Gold is a less expensive nucleic acid stain than SYBR Green I, and can be a good alternative fluorochrome for fast staining and accurate counting viral particles in various types of aquatic samples. Based on these results, the author r e c o m m e n d s the use of the presented protocol with either SYBR Green I or SYBR Gold in conjunction with 0.02 ~m pore size Anodisc filters for determining the viral and bacterial abundance in seawater. Nucleic acid stains such as SYBR Green I and SYBR Gold m a y be used increasingly in the future for more efficient approaches, such as automated counting of viruses. Chen et al. (in press) have recently also used digital image analysis and flow cytometric analysis for rapid counting of SYBR Gold stained viral particles. Flow cytometric analysis s h o w e d that fluorescence per virus stained with SYBR Gold was about 2 times higher than that stained with SYBR Green I (Chen et al., in press). They also found that digital image analysis could detect some weakly stained viruses in natural samples that could not easily be detected by the h u m a n eye. A further advantage of the use of fluorescent stains is that digital images of both SYBR Gold and SYBR Green I stained samples can be quickly captured and saved in the c o m p u t e r for later processing. In the past, it has been quite difficult to incorporate viruses and virusmediated processes into research on aquatic food webs. This m e t h o d allows reasonably e q u i p p e d microbiology laboratories to perform rapid counts of virus particles in natural samples. Virus counts with SYBR Green I or SYBR Gold can be p e r f o r m e d easily in the lab or on board ship and m a y help elucidate the roles of viruses in aquatic systems.

References Bergh, O., Borsheim, K. ¥., Bratbak, G. and Heldal, M. (1989). High abundance of viruses found in aquatic environments. Nature 340, 467-468. Borsheim, K. ¥., Bratbak, G. and Heldal, M. (1990). Enumeration and biomass esfimarion of planktonic bacteria and viruses by transmission electron microscopy. Appl. Environ. Microbiol. 56, 352-356. Bratbak, G., Hetdal, M., Norland, S. and Things{ad, T. E (1990). Viruses as partners in spring bloom microbial trophodynamics. Appl. Environ. Microbiol. 56, 1400-1405. Chen, E, Lu, J. R., Binder, B. J., Liu, Y. C. and Hodson, R. E. (2000) Enumeration of marine viruses stained with SYBR Gold: Application of digital image analysis and flow cytometry. Appl. Environ. Microbiol. M press. 49

Cochlan, W. P., Wikner, J., Steward, G. E, Smith, D. C. and Azam, E (1993). Spatial distribution of viruses, bacteria and chlorophyll a in neritic, oceanic and estuarine environments. Mar. Ecol. Prog. Ser. 92, 77-87. Fuhrman, J. A. (1999). Marine viruses and their biogeochemical and ecological effects. Nature 399, 541-548. Fuhrman, J. A. and Noble, R. T. (1995). Viruses and protists cause similar bacterial mortality in coastal seawater. Limnol. Oceanog. 40, 1236-1242. Fuhrman, J. A. and Suttle, C. A. (1993). Viruses in marine planktonic systems. Oceanography 6, 51-63. Fuhrman, J. A., Wilcox, R. M., Noble, R. T. and Law, N. C. (1993). Viruses in marine food webs. ln: Trends in Microbial Ecology (C. Pedros-Alio and R. Guerrero, Eds) Spanish Society for Microbiology: Barcelona, Spain), pp. 295-298. Guixa-Boixareu, N., Calderon-Paz, J. I., Heldal, M., Bratbak, G. and Pedros-Alio, C. (1996). Viral lysis and bacterivory as prokaryotic loss factors along a salinity gradient. Aquatic Microbial Ecol. 11, 215 227. Hara, S., Terauchi, K. and Koike, I. (1991). Abundance of viruses in marine waters: Assessment by epifluorescence and transmission electron microscopy. Appl. Environ. MicrobioI. 57, 2731-2734. Hennes, K. P. and Suttle, C. A. (1995). Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy. Limnol. Oceanog. 40, 1050-1055. Hennes, K. P., Suttle, C. A. and Chan, A. M. (1995). Fluorescently labeled virus probes show that natural virus populations can control the structure of marine microbial communities. Appl. Environ. Microbiol. 61, 3623-3627. Marie, D., Partensky, E, Jacquet, S. and Vaulot, D. (1997). Enumeration and cellcycle analysis of natural-populations of marine picoplankton by flow-cytometry using the nucleic-acid stain SYBR green I. Appl. Environ. Microbiol. 63, 186-193. Noble, R. T. and Fuhrman, J. A. (1998). Use of SYBR Green i for rapid epifluorescence counts of marine viruses and bacteria. Aquatic Microbial Ecol. 14, 113-118. Proctor, L. M. and Fuhrman, J. A. (1992). Mortality of marine bacteria in response to enrichments of the virus size fraction from seawater. Mar. Ecol. Prog. Ser. 87, 283-293. Proctor, L. M., Fuhrman, J. A. and Ledbetter, M. C. (1988). Marine bacteriophages and bacterial mortality. EOS Trans. Am. Geophys. Union 69, 1111-1112. Steward, G. E, Smith, D. C. and Azam, E (1996). Abundance and production of bacteria and viruses in the Bering and Chukchi Sea. Mar. Ecol. Prog. Ser. 131, 287 300. Suttle, C. A., Chan, A. M. and Cottrell, M. T. (1990). Infection of phytoplankton by viruses and reduction of primary productivity. Nature 387, 467-469. Weinbauer, M. G. and H6fle, M. G. (1998). Size-specific mortality of lake bacterioplankton by natural virus communities. Aquatic Microbial Ecol. 15, 103-113. Weinbauer, M. G. and Suttle, C. A. (1997). Comparison of epifluorescence and transmission electron microscopy for counting viruses in natural marine waters. Aquatic Microbial Ecol. 13, 225-232. Wilhelm, S. W. and Suttle, C. A. (1999). Viruses and nutrient cycles in the sea. Bioscieilces 49, 781-788. Xenopolous, M. A. and Bird, D. E (1997). Virus a la Sance Yo-Pro: Microwave enhanced staining for counting viruses by epifluorescence microscopy. Limnol. Oceano% 42, 1648-1650.

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