An Inexpensive, Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Abstract

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely-used method to accomplish this in aquatic samples is the 'filter-mount' method in which samples are filtered onto costly 0.02 μm-pore-size ceramic filters for enumeration of viruses with epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold lower materials cost) alternative virus enumeration method in which fluorescently-stained samples are wet-mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations <5×10(7) mL(-1). The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had equivalent precision to, those from the filter-mount method across concentrations ranging from 2.17×10(6) to 1.37×10(8) viruses mL(-1) when tested using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter-mount method, and is appropriate for rapid, precise and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1×10(6) viruses mL(-1)) encountered in field and laboratory samples.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Full text

An Inexpensive, Accurate, and Precise Wet-Mount Method for
Enumerating Aquatic Viruses
Brady R. Cunningham,
a
Jennifer R. Brum,
b
Sarah M. Schwenck,
b
Matthew B. Sullivan,
b
Seth G. John
a
Department of Earth and Ocean Sciences, University of South Carolina, Columbia, South Carolina, USA
a
; Department of Ecology and Evolutionary Biology, University of
Arizona, Tucson, Arizona, USA
b
Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through
infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral
abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the
“filter mount” method, in which samples are filtered onto costly 0.02-␮m-pore-size ceramic filters for enumeration of viruses by
epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration
method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of vi-
ruses in samples with viral concentrations of <5ⴛ10
7
viruses ml
ⴚ1
. The concentration of viruses in the sample is then deter-
mined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus
concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated
with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 ⴛ10
6
to 1.37 ⴛ10
8
viruses ml
ⴚ1
when tested by using cultivated viral isolates and natural samples from marine and freshwater envi-
ronments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate
for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (>1ⴛ10
6
viruses ml
ⴚ1
)
encountered in field and laboratory samples.
Viruses are the most abundant biological entities in aquatic sys-
tems, and their infection of microorganisms has substantial in-
fluences on microbial ecology, biogeochemical cycling, and gene
transfer in aquatic environments (reviewed in references 1and 2). An
accurate method to quantify aquatic viruses is thus essential for use in
field and laboratory studies to investigate the roles of viruses in
aquatic environments. Enumeration of viruses in aquatic samples has
previously been accomplished by using transmission electron mi-
croscopy (TEM) (3), epifluorescence microscopy (reviewed in refer-
ence 4), and flow cytometry (reviewed in reference 5).
While each of the above-mentioned methods requires the use of
relatively expensive laboratory equipment, the per-sample cost of the
widely used epifluorescence microscopy method has recently in-
creased dramatically. This method involves filtering the sample onto
0.02-␮m-pore-size ceramic filters, staining viruses on the filters by
using one of several available nucleic acid dyes, mounting the filter
onto a slide, and visually enumerating the deposited viruses by epi-
fluorescence microscopy (reviewed in reference 4). However, the fil-
ters used for this “filter mount” method have risen in cost to ca. $10
each in the United States (with increased costs in some other coun-
tries), creating a significant financial burden for researchers pursuing
studies of environmental viruses. To address this, we have developed
a new, less costly “wet-mount” epifluorescence microscopy method
to enumerate aquatic viruses, in which fluorescently stained samples
are wet mounted directly onto a slide, with quantification of viral
concentrations based on the relative abundance of viruses and silica
beads in the sample.
MATERIALS AND METHODS
Comparison of the wet-mount and filter mount methods for virus enu-
meration. The wet-mount method was tested by comparing viral concen-
trations obtained with the wet-mount and filter mount methods in trip-
licate samples collected from a variety of marine and freshwater
environments as well as in cultivated viral lysates (described in Table S1 in
the supplemental material). Briefly, field samples included those from a
6-depth profile (5 to 300 m) from the Eastern Tropical North Pacific
Ocean (using whole seawater samples); 8 surface ocean locations through-
out the Pacific, Atlantic, and Southern Oceans chosen for their range of
chlorophyll concentrations (collected on the Tara Oceans Expedition [6],
using 0.2-␮m-filtered samples); and a freshwater location in South Caro-
lina. All field samples were preserved with glutaraldehyde (0.5% final
concentration), flash frozen in liquid nitrogen, and stored at ⫺80°C until
analysis. Lysate samples included the Synechococcus virus S-WHM1 (7),
two dilutions of the Synechococcus virus S-SM1 (8), and the Prochlorococ-
cus virus P-HM2 (9). Triplicate independent 1-ml samples were processed
by using each of the filter mount and wet-mount methods, as described
below. Statistical comparison of viral concentrations obtained by using
each method was then performed by using two-tailed ttests and Pearson
correlation (SigmaPlot v12.5; Systat Software Inc.).
Filter mount sample preparation and analysis. The filter mount
method was performed according to methods described previously by
Received 4 November 2014 Accepted 12 February 2015
Accepted manuscript posted online 20 February 2015
Citation Cunningham BR, Brum JR, Schwenck SM, Sullivan MB, John SG. 2015. An
inexpensive, accurate, and precise wet-mount method for enumerating aquatic
viruses. Appl Environ Microbiol 81:2995–3000. doi:10.1128/AEM.03642-14.
Editor: K. E. Wommack
Address correspondence to Seth G. John, sjohn@geol.sc.edu.
B.R.C. and J.R.B. contributed equally to this work.
This article is contribution number 0015 of the Tara Oceans Expedition
2009 –2012.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AEM.03642-14.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.03642-14
May 2015 Volume 81 Number 9 aem.asm.org 2995Applied and Environmental Microbiology

Suttle and Fuhrman (4). Briefly, samples were filtered onto 0.02-␮m-
pore-size ceramic filters (Whatman Anodisc), stained with SYBR gold
(Invitrogen) for 15 min, and mounted onto a glass slide with an anti-
fade solution (Acros Organics). Viruses were viewed under blue exci-
tation using a Nikon TS100 inversion microscope or a Zeiss Axio Im-
ager epifluorescence microscope at a ⫻1,000 magnification. The viral
concentration was determined by using the average number of fluo-
rescent viruses within a given area of the microscope reticle in 20 fields
of view and the total volume of sample filtered through a measured
area on the filter.
Wet-mount sample preparation and analysis. The wet-mount
method for enumerating viruses involves an optional virus concentration
step followed by combining a known volume of stained sample with a
known volume and concentration of silica beads for relative enumeration
of viruses and beads to calculate the virus concentration in the sample
(Fig. 1). The reagents for assessing viral concentrations using the wet-
mount method are fully described in Table 1, and the protocol is as fol-
lows.
1. If the virus concentration is expected to be ⬍5⫻10
7
viruses ml
⫺1
,
concentrate the viruses by chemical flocculation, as follows. Add 1
␮l iron chloride solution to a 1-ml sample in a microcentrifuge
tube, mix the sample by inversion, and centrifuge the mixture for
20 min at ⬃14,000 ⫻g. Remove the supernatant and resuspend the
pellet in 10 ␮l ascorbate-EDTA buffer. (If a lower concentration
factor is desired, the pellet may be resuspended in a larger volume
of ascorbate-EDTA buffer. In this case, increase the amounts of
SYBR gold, glycerol, and silica beads accordingly in subsequent
steps.)
2. Add 2 ␮l SYBR gold working stock to the concentrated sample,
vortex the mixture, and incubate the mixture for 15 min in the
dark. If the sample was not concentrated with chemical floccula-
tion (i.e., the concentration is expected to exceed 5 ⫻10
7
viruses
ml
⫺1
), mix 10 ␮l unconcentrated sample with 2 ␮l SYBR gold
working stock in a microcentrifuge tube.
3. Add 5 ␮l glycerol, vortex the mixture, and add 2 ␮l working bead
solution (thoroughly vortex the working bead solution before ad-
dition to the sample to ensure accurate pipetting of the beads). If
the sample was not concentrated with chemical flocculation, add 1
␮l ascorbic acid antifade solution as well.
4. Mix the prepared sample thoroughly by pipetting up and down and
then immediately pipette 10 ␮l onto a glass microscope slide and
place a coverslip over the sample (both the glass slide and coverslip
should be cleaned with isopropanol). Avoid trapping air under the
coverslip.
5. Using an epifluorescence microscope, count the number of viruses
in a given area within the microscope reticle under blue (⬃495-
nm) excitation at a ⫻1,000 magnification. Within the same field of
view, count the beads under white light. Continue counting fields
of view until at least 100 each of viruses and beads have been
counted.
6. The virus concentration is calculated as c
virus
⫽n
virus
/n
beads
⫻
v
beads
/v
sample
⫻c
beads
, where c
virus
is the virus concentration in the
sample (viruses ml
⫺1
), n
virus
is the total number of viruses counted,
n
beads
is the total number of beads counted, v
beads
is the volume of
working bead solution added (␮l), v
sample
is the sample volume
FIG 1 Overview of the wet-mount method for enumeration of aquatic viruses.
TABLE 1 Reagent preparation for the wet-mount virus enumeration protocol
Reagent Prepn method
SYBR gold working stock Dilute SYBR gold (Invitrogen) (10,000⫻stock) into PBS to prepare a 1,000⫻solution
Ascorbic acid antifade solution Dissolve ascorbic acid into PBS to create a 10% (wt/vol) solution
a
Working bead solution Thoroughly vortex the stock bead solution (2.34-␮m silica spheres) (catalog no.
SS04N/4186; Bangs Laboratories), dilute it 10-fold into PBS to obtain a concn of
⬃10
8
beads ml
⫺1
, and store it at 4°C
Iron chloride solution Dissolve FeCl
3
·6H
2
O into ultrapure water to form a solution of 10 g Fe liter
⫺1
; the
solution has expired if a cloudy precipitate forms
b
Ascorbate-EDTA buffer Combine equal parts of 0.4 M Mg
2
EDTA and 0.8 M ascorbic acid and adjust with 10
N NaOH to reach a pH of 6–7; prepare fresh within 48 h of use
c
a
See reference 11.
b
See reference 10.
c
An alternative ascorbate-EDTA buffer can be made with MgCl
2
and Na
2
EDTA if Mg
2
EDTA is unavailable (10).
Cunningham et al.
2996 aem.asm.org May 2015 Volume 81 Number 9Applied and Environmental Microbiology

(␮l) (the volume prior to concentration if chemical flocculation is
used), and c
beads
is the bead concentration in the working bead
solution (beads ml
⫺1
).
A full, illustrated protocol describing this method is also available
online for convenience (http://eebweb.arizona.edu/faculty/mbsulli
/protocols/). For analysis of samples with ⬍5⫻10
7
viruses ml
⫺1
, viruses
must be concentrated with chemical flocculation by using a method
adapted from methods described previously by John et al. (10), to obtain
a sufficient concentration of viruses for analysis (Fig. 1A). Samples in this
study with viral concentrations below that threshold were first concen-
trated 100-fold with this chemical flocculation method (step 1 in the list of
procedures above) before being stained with SYBR gold for 15 min and
then combined with silica beads and glycerol (added to create a more
viscous solution and reduce clumping of beads) (steps 2 and 3 in the list of
procedures above) (Fig. 1B). The silica bead size (2.34 ␮m) was selected
due to the ease of visually counting the beads under white light. Due to the
relatively large size of the beads (compared to the size of viruses), the bead
solution must be vortexed thoroughly prior to the addition of beads to the
sample to ensure the addition of an accurate concentration of beads.
These concentrated samples did not require the addition of an antifade
solution since they were resuspended in a buffer containing ascorbic acid,
which reduces fading of the fluorescent signal (11). Samples were then
pipetted directly onto an isopropanol-cleaned glass microscope slide and
covered with a cleaned glass coverslip (step 4 in the list of procedures
above). Viruses and beads were enumerated in multiple fields of view on a
Nikon TS100 inversion microscope or a Zeiss Axio Imager epifluores-
cence microscope at a ⫻1,000 magnification until at least 100 each of
viruses and beads were enumerated to calculate the virus concentration
(steps 5 and 6 in the list of procedures above). For each field of view, the
total number of fluorescent viruses was determined under blue excitation,
after which the total number of beads within the same field of view was
determined under white light (Fig. 2).
For analysis of samples with ⬎5⫻10
7
viruses ml
⫺1
, chemical floccu-
lation of viruses prior to wet-mount sample preparation was not necessary
to obtain a sufficient concentration of viruses for enumeration. These
samples (S-SM1 lysates) were prepared by staining 10 ␮l of sample with
SYBR gold, followed by the addition of an ascorbic acid solution (to act as
an antifade solution), glycerol, and silica beads (steps 2 and 3 in the list of
procedures above) (Fig. 1B). These samples were then wet mounted onto
slides and enumerated exactly as described above for the samples that had
been concentrated with chemical flocculation. To compare the wet-
mount method with the filter mount method for these samples with high
viral concentrations, they were diluted 10-fold in phosphate-buffered sa-
line (PBS) prior to filtering 1 ml of sample for the filter mount method, as
the undiluted sample would have resulted in an excessive viral density on
the filter, preventing analysis. We also note that while p-phenylenedi-
FIG 2 Images of samples prepared by use of the filter mount and wet-mount virus enumeration methods. Shown are epifluorescence images of purified S-SM1
lysate obtained by using the filter mount (A) and wet-mount (B) methods, seawater from 30 m in the Pacific Ocean depth profile by using the filter mount (D)
and wet-mount (E) methods, freshwater from Lake Murray by using the filter mount (F) and wet-mount (G) methods, and unpurified S-SM1 lysate with
Synechococcus cells by using the filter mount (H) and wet-mount (I) methods. These epifluorescence images include arrows pointing to two of the viruses in each
image. Under white light (C), beads are visible in the same field of view as for the wet-mount sample (B), with arrows pointing to two of the beads in the image.
Bar, 10 ␮m.
A Wet-Mount Method for Enumerating Aquatic Viruses
May 2015 Volume 81 Number 9 aem.asm.org 2997Applied and Environmental Microbiology

amine is a popular antifade chemical (4), it reacted with glutaraldehyde to
form a precipitate in these wet-mount samples and thus should not be
used in the wet-mount method with fixed samples.
The minimum number of beads and viruses enumerated per sample is
justified as follows. Counting statistics (also known as shot noise) dictates
that the error in the quantity of viruses or beads counted is given by 1⁄兹n
(12), where nis the number of objects enumerated, and therefore, the total
error in viral abundance is ␴total ⫽兹1⁄nvirus⫹1⁄nbeads, where n
virus
and
n
beads
are the total numbers of viruses and beads counted, respectively.
When at least 100 each of viruses and beads are enumerated, the maxi-
mum error is 14%.
Storage conditions for samples prepared by using the wet-mount
method. Storage conditions were assessed by using two different lysates
concentrated 100⫻using the flocculation method described above. To
assess storage after samples were mounted onto slides, triplicate samples
(S-SM1 lysate) were prepared and analyzed by using the full protocol
described above, with slides being stored vertically at ⫺20°C immediately
after enumeration, and viruses and beads were recounted 7 days later.
Additional triplicate samples (S-WHM1 lysates) were prepared through
step 3 in the list of procedures above, with 10 ␮l of the prepared sample
being analyzed immediately and the remaining sample (⬃10 ␮l) being
stored in a microcentrifuge tube at ⫺20°C until analysis 7 days later.
RESULTS AND DISCUSSION
The wet-mount method resulted in fluorescently stained viruses
with an intensity similar to those of the filter-mount method (Fig.
2). While there was typically a lower density of viruses in the im-
ages derived from samples prepared by using the wet-mount
method, this is favorable because viruses are enumerated in larger
fields of view with the wet-mount than with the filter mount
method. However, images depicting a greater density of viruses
and cells can be obtained with more concentrated samples (Fig.
2I). Viral concentrations obtained by using the wet-mount
method were strongly correlated (Pearson correlation coefficient
of 0.986; P⬍0.001) with those obtained by using the filter mount
method for all sample types tested, including viral lysates and
samples from a variety of oceanic and freshwater regions (Fig. 3).
There was no significant difference in viral concentrations ob-
tained by the use of these methods for the majority of samples (13
of 19 samples; two-tailed ttests) (see Table S1 in the supplemental
material). For the remaining samples with significantly different
viral concentrations, neither method consistently resulted in
higher or lower viral concentrations, nor were these differences
restricted to a specific range of viral concentrations (i.e., high ver-
sus low) or sample type (i.e., freshwater versus marine sample,
natural sample versus lysate, or low versus high chlorophyll con-
centration), indicating stochastic variability inherent to analyses
of samples (Fig. 3; see also Table S1 in the supplemental material).
Furthermore, we consider the low magnitude of the differences in
FIG 3 Viral concentrations in natural samples and lysates obtained by using
the filter mount and wet-mount enumeration methods. Error bars are stan-
dard deviations of the means of data from triplicate samples. Closed symbols
represent samples in which there was no significant difference in virus concen-
trations obtained by using the filter mount and wet-mount methods (P⬎0.05
by two-tailed ttests), while open symbols represent samples in which there was
a significant difference (see Table S1 in the supplemental material). Average
viral concentrations for all samples obtained by using each method were
strongly and positively correlated (Pearson correlation coefficient, 0.986; P⬍
0.001). The solid lines represent a 1:1 relationship, and dashed lines represent
an interval of 70% agreement between methods around the 1:1 relationship to
facilitate visual comparison of results.
Cunningham et al.
2998 aem.asm.org May 2015 Volume 81 Number 9Applied and Environmental Microbiology

average viral concentrations for the few significantly different
samples to be acceptable for studies of aquatic viruses.
It is important to note that there is no available standard used
in aquatic virus enumeration methods. Previous studies compar-
ing aquatic viral concentrations determined by using different
methods (i.e., TEM, filter mount, and flow cytometry) have
shown discrepancies between methods, with one method usually
resulting in consistently higher viral concentrations (13–17).
However, we observed no such consistent differences in our com-
parison of the wet-mount and filter mount methods. Further-
more, the comparison in this study showed that most of the sam-
ples had at least 70% agreement between virus concentrations
obtained by use of the wet-mount method and those obtained by
use of the filter mount method (Fig. 3), which is similar to data
from previously reported comparisons of methods used to enu-
merate viruses (16,17). The wet-mount method also had high
precision; standard deviations of the means for triplicate samples
were 2 to 18% (average, 7% ⫾4%) of the mean virus concentra-
tion and were not significantly different from those obtained by
using the filter mount method (P⫽0.531 by two-tailed ttest).
Thus, the wet-mount method and the filter mount method can be
used with equal confidence.
When enumerating viruses, it is sometimes advantageous to
store prepared samples for enumeration at a later date. For exam-
ple, samples prepared with the filter mount method can be stored
at ⫺20°C for at least 4 months, with no significant change in viral
concentrations (4). For wet-mount samples, we tested storage of
prepared samples both in microcentrifuge tubes and on slides at
⫺20°C (Fig. 4). While the calculated virus concentration was
higher after storage of the prepared samples under both storage
conditions, these differences were not significant (P⫽0.210 and
P⫽0.083, respectively, by two-tailed ttests). Thus, samples pre-
pared by use of the wet-mount method can be stored frozen either
before or after mounting the sample onto slides, with no signifi-
cant change in the calculated virus concentration.
The wet-mount method had one major drawback compared to
the filter mount method, which was the inability to efficiently
enumerate samples with viral concentrations of ⬍1⫻10
6
viruses
ml
⫺1
. Attempted analysis of samples with lower viral concentra-
tions (i.e., samples below 300 m in the Pacific Ocean depth profile)
using the wet-mount method resulted in ⱕ1 virus per field of
view, even after maximum concentration (100-fold) with chemi-
cal flocculation. Thus, the wet-mount method is not recom-
mended for samples with viral concentrations of ⬍1⫻10
6
viruses
ml
⫺1
because the low density of viruses on the slide significantly
extends the time for analysis of a sample. Although this limitation
prevented analysis of the deep-sea samples (⬎300 m) in the Pacific
Ocean depth profile in this study, many deep-sea samples have
viral concentrations above this limit (e.g., see reference 18), and
thus, the wet-mount method should be useful for a wide range of
environmental samples.
The available methods to enumerate aquatic viruses each have
benefits and limitations that are worth considering when planning
research projects. For example, TEM-based analyses of aquatic
samples can generate information about the morphological char-
acteristics of viruses (e.g., see reference 21) in addition to viral
abundance (e.g., see reference 3) but can potentially underesti-
mate the number of viruses because they may be obscured by
debris in the sample (16). Fluorescence-based methods for viral
enumeration (i.e., epifluorescence microscopy and flow cytom-
etry) are significantly faster than TEM but can potentially falsely
include gene transfer agents or cell debris as viruses (reviewed in
reference 19) while excluding single-stranded DNA (ssDNA) vi-
ruses that have very faint fluorescence (20). One additional advan-
tage of fluorescence-based methods is the ability to enumerate
both viruses and bacteria (if present) by using the same prepared
sample (e.g., see reference 16). However, the wet-mount method
presented here has not yet been evaluated for accuracy in counting
of bacterial cells. Among the available epifluorescence-based
methods, the filter mount method also provides an opportunity to
obtain images with a high density of viruses and cells, while the
flow cytometry method does not. The viral density in images ob-
tained by use of the wet-mount method is generally much lower
than that for filter mount samples, although the density of viruses
and cells increases when more concentrated samples are used.
While each of these variables is important when evaluating poten-
tial virus enumeration methods for a given project, we offer the
wet-mount method as a cost-effective alternative to the widely
used filter mount epifluorescence method.
A significant advantage of the wet-mount method over the
filter mount method is the lack of a requirement for costly 0.02-
␮m-pore-size ceramic filters. Currently, these filters are available
from only one supplier and are expensive (⬃$10 each). Instead,
the wet-mount method uses microsphere silica beads that can be
purchased from several suppliers at a ⬃500-fold-lower cost ($0.02
for 20 ␮lofa10
8
-bead ml
⫺1
working solution per sample, calcu-
lated based on $150 for 15 ml of a 10
9
-bead ml
⫺1
stock solution).
Even after accounting for the cost of other reagents and slides, the
per-sample materials cost for the wet-mount method is much
lower (⬃$0.10 per sample). Thus, the wet-mount method is rec-
ommended as an equivalently accurate and precise but cheaper
alternative for enumerating viruses in field and laboratory sam-
ples with viral concentrations of ⬎1⫻10
6
viruses ml
⫺1
.
Conclusion. Enumeration of viruses in field and laboratory
samples is an important tool for investigating the numerous in-
FIG 4 Storage of samples prepared by using the wet-mount method. Concen-
trations of viruses in triplicate samples (S-SM1 lysate for tube storage and
S-WHM1 lysate for slide storage) prepared according to the wet-mount pro-
tocol and stored at ⫺20°C in microcentrifuge tubes (tube storage) or wet
mounted onto slides (slide storage) are shown. Viruses were enumerated im-
mediately after preparation (time [T]⫽0 days) and after 7 days of storage
(T⫽7 days). Error bars are standard deviations of the means for triplicate
samples.
A Wet-Mount Method for Enumerating Aquatic Viruses
May 2015 Volume 81 Number 9 aem.asm.org 2999Applied and Environmental Microbiology

fluences of viruses in aquatic environmental systems. However,
the high cost of enumerating viruses in aquatic samples using the
established filter mount epifluorescence microscopy method can
be a significant burden in conducting aquatic virus research. In
this study, we present a new, less expensive wet-mount method for
aquatic virus enumeration that can be used with accuracy and
precision equivalent to those of the filter mount method for a
variety of environmental and laboratory samples.
ACKNOWLEDGMENTS
We thank Bonnie Poulos from the Tucson Marine Phage Lab at the Uni-
versity of Arizona for her help troubleshooting initial problems with this
method. We also thank the crew and scientists of the R/V New Horizon for
their assistance when sampling in the Eastern Tropical North Pacific
Ocean, as well as the coordinators and members of the Tara Oceans con-
sortium for organizing sampling.
This publication was funded in part by Gordon and Betty Moore
Foundation grant GBMF3305 to S.G.J. and M.B.S. and by grants
GBMF2631 and GBMF3790 to M.B.S. We also acknowledge the following
sponsors for their support in the Tara Oceans Expedition: CNRS, EMBL,
Genoscope/CEA, VIB, Stazione Zoologica Anton Dohrn, UNIMIB,
ANR (projects POSEIDON/ANR-09-BLAN-0348, BIOMARKS/ANR-08-
BDVA-003, PROMETHEUS/ANR-09-GENM-031, and TARA-GIRUS/
ANR-09-PCS-GENM-218), EU FP7 (MicroB3/no. 287589), FWO, BIO5,
Biosphere 2, agnès b., the Veolia Environment Foundation, Region
Bretagne, World Courier, Illumina, Cap L’Orient, the EDF Foundation
EDF Diversiterre, FRB, the Prince Albert II de Monaco Foundation, Eti-
enne Bourgois, and the captain and crew of the Tara schooner. Tara
Oceans would not exist without continuous support from 23 institutes.
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