A protocol for enumeration of aquatic viruses by epifluorescence microscopy using Anodisc™ 13 membranes
© Budinoff et al; licensee BioMed Central Ltd. 2011
Received: 5 May 2011
Accepted: 25 July 2011
Published: 25 July 2011
Epifluorescence microscopy is a common method used to enumerate virus-like particles (VLP) from environmental samples and relies on the use of filter membranes with pore sizes < 0.02 μm; the most commonly used protocols employ 25 mm Anodisc™ membranes with a built-in support ring. Other filters with small pore sizes exist, including the 13 mm Anodisc™ membranes without a support ring. However, the use of these membranes for viral enumeration has not been previously reported.
Here we describe a modified protocol for 13 mm Anodisc membranes that uses a custom filter holder that can be readily constructed in individual investigators' laboratories from commercially available Swinnex® filter holders. We compared VLP concentrations obtained from phage lysates and seawater samples using both Anodisc membranes, as well as Nuclepore™ small pore-size membranes (0.015 or 0.030 μm). The 13 mm Anodisc membranes gave comparable estimates of VLP abundance to those obtained with the 25 mm Anodisc membranes when similar staining methods were employed. Both Nuclepore membranes typically gave an order of magnitude lower VLP abundance values for environmental samples.
The 13 mm Anodisc membranes are less costly and require smaller sample volumes than their 25 mm counterpart making them ideal for large-scale studies and sample replication. This method increases the options of reliable approaches available for quantifying VLP from environmental samples.
Specifications of Whatman membranes used in this study
Filterable Diameter (mm)
Cost per filter (USD)
Results and Discussion
A practical limitation of the 13 mm Anodisc membranes is the lack of a peripheral support ring to facilitate handling of the membranes. To alleviate this limitation, we constructed custom filter holders and used modifications of traditional protocols for enumeration of VLP. The feasibility of using Nuclepore filters for viral enumerations was also revisited using modified protocols to reduce filtration times. In part, our motivation to reevaluate the feasibility of Nuclepore membranes for VLP enumeration was prompted by production problems of Anodisc membranes , which have been subsequently resolved but serve as a reminder that the availability of alternate protocols would be useful.
Construction of custom filter holders for 13 mm Anodisc membranes
Enumeration of VLP using 13 mm Anodisc membranes
Our protocol for preparing virus slides using 13 mm Anodisc membranes is based on that of Ortmann and Suttle (2009), with modifications of the staining procedure. Back-staining is the standard protocol for Anodisc 25 membranes and involves placing the membrane sample side up onto a drop of stain, incubating, then removing excess stain by either wicking  or applying vacuum . However, back-staining is technically challenging due to the small size and absence of a support ring on the 13 mm membranes. Thus, samples were pre-stained prior to filtration. The detailed protocol is as follows: i) A virus sample was brought up to a final volume of 900 μL using 0.02-μm filtered diluent (AN media or seawater). ii) 100 μL of SYBR Gold (25 ×, 0.02 μm filtered) was added to the sample and then incubated for 15 min in the dark. iii) A backing filter (0.2 μm, polyethersulfone, Pall Corporation, Port Washington, NY) was placed onto the screen of the Swinnex outlet and overlaid with sterile MilliQ water (~2 mL). Vacuum pressure (5 in Hg) was applied to pull the water through and stopped immediately so not to dry out the filter. iv) The backing filter was overlaid with MilliQ water (~2 mL) again and a 13 mm Anodisc placed on top of the water. v) The vacuum was then applied to pull the water through and sandwich the filters together. vi) With the vacuum still on, the modified Swinnex inlet (containing a gasket) was carefully screwed on and tightened with sufficient torque; excessive torque would crack the membrane and insufficient torque caused particles to be preferentially filtered towards the periphery of the membrane. vii) The sample was added to the center of the funnel. After all the liquid had visually disappeared, the vacuum was continued for an additional 30 seconds. viii) With the vacuum still on, the Swinnex inlet was carefully unscrewed, leaving the gasket and the two filters on the outlet. ix) The vacuum was cut and the three pieces (sandwiched filters and gasket) were removed as one and placed on Whatman (grade 4, qualitative) paper to dry for one min. x). Using forceps and a needle, the gasket was removed and the filters separated. xi) The Anodisc was mounted on a glass slide with anti-fade solution (50% glycerol, 50% PBS, 0.1% p-phenylenediamine). Filtration time was < 5 min per mL. Parallel samples were also prepared with a post-stain rinse, where 500 μL of 0.02-μm filtered media or seawater was added to the funnel and pulled through with the vacuum.
Enumeration was performed on a Leica DMRXA using filter cube L5 (excitation filter BP 480/40, suppression filter BP 527/30). For each slide, 20 fields and at least 200 particles were counted. To calculate the concentration of virus particles ml-1, the average number of particles per field was multiplied by the dilution factor and microscope conversion factor and then divided by the volume of sample filtered (in ml). The microscope conversion factor was calculated as the filterable area of the membrane divided by the area of each individual field. Variance in the filterable area using the meniscus loading method for the 25 mm Anodisc filters and the Swinnex filter holders for the 13 mm filters was 18.38 (± 0.115) and 9.61 (± 0.131), respectively.
Comparison of VLP counts using Anodisc membranes and evaluation of staining methods
Comparison of back-staining and pre-staining of Anodisc membranes in VLP enumeration of three sample types
1.32 × 106 (0.08)
1.32 × 106 (0.10)
1.63 × 106 (0.07)
1.54 × 106 (0.15)
1.29 × 106 (0.13)
1.26 × 106 (0.07)
9.59 × 105 (1.86)
1.66 × 105 (0.37)
Sargasso Sea water
7.50 × 105 (1.30)
1.75 × 105 (0.17)
5.93 × 105 (1.15)
2.28 × 105 (0.54)
14.99 × 105 (0.45)
3.22 × 105 (1.06)
Southeastern US coastal waters
4.41 × 105 (0.62)
3.28 × 105 (0.35)
2.58 × 105 (0.35)
2.75 × 105 (0.41)
Discrepancies in VLP counts due to staining method and post-rinsing are most likely a reflection of differences in concentration and composition of viral communities (in terms of size and fluorescence) as well as organic material in the natural samples. For example, coastal environments and other highly productive systems typically contain a higher proportion of eukaryotic algae in the plankton then do oligotrophic systems, such as the open ocean . Viruses that infect algae are routinely isolated and have been shown to be quite large in size (capsid, 100-220 nm) and contain large genomes [19, 20]. A higher proportion of smaller, less fluorescent viruses in the open ocean could contribute to lower VLP counts after post-rinsing. The issue of including a post-rinse in the processing of natural samples for VLP enumeration is environment dependent and beyond the scope of this report, which is designed to illustrate the comparability of sample processing with the 13 mm and 25 mm Anodisc membranes.
Analysis of Nuclepore membranes
Modifications of existing protocols allow the reliable use of Anodisc 13 membranes for enumeration of VLP using epifluorescence microscopy. In parallel studies, we found that Nuclepore filters (polycarbonate, 0.03 & 0.015 μm pore sizes) consistently yielded lower observable VLP. These low counts may be attributed to non-uniform pore sizes that were evident by scanning electron microscopy of these filters (Figure 2). However, more rigorous parallel comparisons of the Nuclepore and Anodisc membranes are necessary to determine this conclusively. Differences in VLP abundance estimates between Anodisc 13 and 25 membranes were evident with environmental samples if a post-rinse step was not included in sample processing. While rinsing of membranes gave the most consistent results across the two Anodisc membranes, it may result in loss of enumeration of VLP depending upon the environment from which the sample was derived. Given the heterogeneity of natural virus populations, individual investigators will need to consider the issue of applying a post-rinse on a case-by-case basis.
Sample collection and preparation
Viral lysate was made using cyanophage S-PWM1, which infects Synechococcus sp. WH7803 (aka DC2) . The lysate was filtered through a 0.2-μm Durapore™ filter and stored at 4°C - this filtered material served as the lysate standard. Open ocean water samples were collected from the Sargasso Sea (May 28, 2005; 36.343° N, 51.315° W) and coastal water samples were collected off the coast of Georgia, USA (Nov 18, 2007; 31.372° N, 80.561° W). Multiple seawater aliquots (2 mL) were uniformly distributed, fixed in 0.5% glutaraldehyde and frozen at -80°C at the start of this study to ensure reproducibility.
Enumeration of viruses using 25 mm Anodisc membranes
The protocol using 25 mm Anodisc membranes follows that published by Ortmann and Suttle (2009), with minor modifications. Briefly, filtration was performed on a Hoefer® filtration manifold (Hoefer, Holliston, MA) without chimney weights. After the backing (0.45-μm pore-size cellulosics; MicroSep™, GE Water & Process Technologies, Trevose, PA) and the Anodisc filter were mounted on the filter stage with the vacuum on, the sample (final volume 1 mL) was applied to the top, forming a meniscus. The filter was back-stained by placement sample side up onto 100 μL of SYBR Gold stain (25 × concentration, Invitrogen, Carlsbad, CA) and incubated for 15 min followed by application of a vacuum to remove the stain. Samples were also prepared with a post-stain rinse of 850 μL of 0.02 μm filtered media or seawater. For direct comparison to the Anodisc 13 membranes, parallel samples were also pre-stained in a microcentrifuge tube prior to filtration. Filtration time using the above protocol was < 5 min per mL of sample.
Determination of filterable area for Anodisc membranes
The filterable area of the Anodisc membranes was determined by passage of a cell culture of the naturally pigmented bacterium Synechococcus sp. WH7803 through them. Digital images were analyzed with Adobe® Photoshop® CS4 (Adobe Systems Incorporated, San Jose, CA) to calculate the area containing pigmented cells. The data reported is a range of the averages obtained from triplicate filters.
Enumeration of viruses using Nuclepore membranes
As pre-stained black Nuclepore membranes with pore sizes of 15 and 30 nm are not commercially available, membranes were stained using 0.2% Irgalan Black (Acid black 107, Organic Dyestuffs Corporation, East Providence, RI) dissolved in 2% acetic acid as previously described , with the exceptions that staining time was reduced from 3 hours to 15 minutes and filters were used immediately. Polyester drain discs (Whatman), which are designed to improve flow rate and provide a flat surface to eliminate rupturing were used as backing filters. Filters were placed in 25 mm Swinnex filter holders for filtration and processed using the same reagents and solutions described for the Anodisc membranes. The filtration time required for the Nuclepore 15 and 30 membranes using the above protocol was < 60 min and < 10 min per mL, respectively.
SEM imaging of Nuclepore membranes
To assess whether the filtration protocol could be damaging or altering membrane pore size, scanning electron micrographs of the Nuclepore membranes were taken before and after filtrating media (0.02 μM filtered AN) or seawater (0.02 μM filtered Sargasso Sea water) using a LEO 1525 field emission scanning electron microscope (Carl Zeiss Inc., Thornwood, NY, USA). Avoiding lateral stress, the membranes were cut, mounted on a stub and viewed. No coating was applied so as to not obscure the pores. At least 3 regions of each filter were viewed and at least 50 pores measured from each filter. Filtration did not appear to damage the filters or change pore size. Initial attempts at preparing the filters for SEM did suggest that lateral stress (excessive stretching or twisting) of the membranes could drastically increase pore size (data not shown).
Statistical comparison of virus counts from the Anodisc membranes
The statistical software package SPSS was used to compare the VLP counts between the technical replicates (repeated-measures ANOVA, C.I. of 5%) and between the membrane types (2-tailed paired t test, C.I. of 5% or repeated-measures ANOVA, C.I. of 5%). Counts obtained from the individual fields of each slide were first evaluated using the Shapiro-Wilks test. Data sets that failed the Shapiro-Wilks test (having p-values < 0.05) were transformed using the Box-Cox transformation. The resulting transformed variables were consistent with a normal distribution. Mauchly's test of sphericity was performed and if the test was found to be significant (having p-values < 0.05) either the Huynh-Feldt (for epsilon values > 0.75) or the Greenhouse-Geisser (for epsilon values < 0.75) correction was applied.
This work was funded by the National Science Foundation (OCE-0550485 to AB and OCE-0825405 and OCE-0851113 to SWW). The authors would like to thank J. Dunlap for assistance with SEM.
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