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Applied and Environmental Microbiology, June 2000, p. 2283-2289, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Comparison of Methods for Counting Viruses in
Aquatic Systems
Yvan
Bettarel,1
Telesphore
Sime-Ngando,1,*
Christian
Amblard,1 and
Henri
Laveran2
Laboratoire de Biologie des Protistes, UMR
CNRS 6023, Université Blaise Pascal (Clermont-Ferrand II),
F-63177 Aubière Cedex,1 and
Service Hygiène Hospitalière, Faculté de
Médecine, Université d'Auvergne, F-63000
Clermont-Ferrand,2 France
Received 20 December 1999/Accepted 8 March 2000
 |
ABSTRACT |
In this study, we compared different methods
including
transmission electron microscopyand various nucleic acid labeling methods in which we used the fluorochromes
4',6'-diamidino-2-phenylindole (DAPI),
4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylmethyledene]-1-(3'-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which
can be detected by epifluorescence microscopy (EM), for counting
viruses in samples obtained from freshwater ecosystems whose trophic
status varied and from a culture of T7 phages. From a quantitative and
qualitative viewpoint, our results showed that the greatest efficiency
for all ecosystems was obtained when we used the EM counting protocol
in which YOPRO-1 was the label, as this fluorochrome exhibited strong
and very stable fluorescence. A modification of the original protocol
in which YOPRO-1 was used is recommended, because this modification
makes the protocol faster and allows it to be used for routine analysis
of fixed samples. Because SYBR Green I fades very quickly, the use of
this fluorochrome is not recommended for systems in which the viral
content is very high (>108 particles/ml), such as treated
domestic sewage effluents. Experiments in which we used DNase and RNase
revealed that the number of viruses determined by EM was slightly
overestimated (by approximately 15%) because of interference caused by
the presence of free nucleic acids.
| |
INTRODUCTION |
For a long time it has been thought
that in aquatic ecosystems the bacterioplankton level and
bacterioplankton production are controlled mainly by temperature, the
availability of resources, and predation (6, 19). However,
many attempts at modeling microbial food webs, particularly energy flow
and material flow among dissolved organic matter, bacteria, and
protozoans, have been unsuccessful, undoubtedly because poorly
understood processes are involved in losses that affect pelagic
microbial communities (19, 21, 25, 26). This is one of the
reasons that biologists have recently become interested in the role of
viruses in ecological processes in the pelagic environment. The results
that have been obtained have clearly shown that viruses are abundant,
active, and ubiquitous in aquatic ecosystems, in which they probably
play a dominant role in losses that affect microbial communities
(1, 2, 7-9, 12, 24, 28, 30, 32, 38).
The study of viruses in aquatic environments requires a reliable method
for estimating the levels of these biological entities. In modern
studies of aquatic microbial ecology, the first estimates of viral
levels were obtained by concentrating the viruses by ultracentrifugation (1, 3, 27) or by ultrafiltration
(24, 39) and then counting them by using transmission
electron microscopy (TEM). However, the high costs, the long analysis
time required, and the difficulty of this method led to the development
of alternative, faster, and less expensive methods based on the use of
epifluorescence microscopy (EM); with these methods viruslike particles
(VLPs) can be observed and counted after they have been labeled with fluorochromes that are specific for nucleic acids. DAPI
(4',6'-diamino-2-phenylindole), a fluorochrome specific for
double-stranded DNA (23), was the first fluorochrome used
for labeling and counting of aquatic VLPs by EM (9, 11, 24,
31). Later, Hennes and Suttle (13) developed another
VLP counting protocol in which EM was used; they used a fluorochrome
that was based on cyanine and is specific for DNA and RNA, YOPRO-1
{4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylmethyledene]-1-(3'-trimethyl ammoniumpropyl)-quinilinium diioide}. The long time needed to stain
samples and the requirement for samples that have not been fixed by
aldehydes led Xenopoulos and Bird (40) to propose a modification of the original protocol of Hennes and Suttle
(13). This modification consists of staining samples while
they are hot, which substantially reduces the time needed to stain the samples. Samples fixed with aldehydes can also be analyzed by this
method. More recently, a new fluorochrome that is also specific for
nucleic acids and is intended for counting viral particles by EM (SYBR
Green I) has been tested by Marie et al. (17) and by Noble
and Fuhrman (20).
Some of the methods described above have been compared previously
(10, 11, 13, 20, 35, 40). However, most comparisons have
been made by using plankton samples obtained from oligotrophic marine
environments. To our knowledge, the five protocols described above have
not been simultaneously tested previously with samples of viruses
obtained from aquatic ecosystems that differ in trophic status.
Furthermore, the effects of free nucleic acids (DNA and RNA) on
quantitative estimates of numbers of viruses in aquatic environments
have received very little attention (5, 13) or no attention
(for RNA).
The aim of this study was to compare the five protocols for counting
viral particles. We used TEM and EM coupled with fluorescent staining
with DAPI, YOPRO-1, and SYBR Green I to determine the most efficient
protocol for routine counting of viruses in aquatic ecosystems that
differed in trophic status (oligotrophic to hypereutrophic). We also
tried to estimate the bias related to fading of fluorescently marked
particles and the effects of free DNA and RNA fragments on the total
number of viruses determined by TEM and EM.
Our results revealed that the modified EM-YOPRO protocol was the most
useful method for reliably counting freely occurring viruses in aquatic
ecosystems. Because of the very fast fading time of SYBR Green I, using
this fluorochrome is not recommended for systems in which the viral
concentration is high (>108 particles/ml), such as
domestic sewage treatment plant effluents. Finally, we found that
interference caused by free nucleic acids leads to slight
overestimation of the number of viruses as determined by EM.
| |
MATERIALS AND METHODS |
Sample collection.
Samples were obtained from the following
freshwater ecosystems, which differed in trophic status and were
located in the Massif Central region of France (46°N, 3°E): (i) an
oligotrophic reservoir (the Sep Reservoir), (ii) a moderate-altitude
oligomesotrophic lake (Lake Pavin), (iii) a eutrophic reservoir (the
Villerest Reservoir), and (iv) a domestic sewage works lagoon (the
Roche Blanche plant), which was considered a hypereutrophic
environment. A culture of T7 phages (phages with double-stranded DNA)
that was grown in tryptone-casein-soy broth was also used. Five
replicate water samples were collected at each lake or reservoir site
by using an opaque polyvinyl chloride Van Dorn type of bottle, and five
replicate samples were collected manually with a sterile container at
the entrance to the sewage lagoon. The following two types of samples
(five replicates each) were collected from each aquatic ecosystem that
was tested: samples that were stored at 4°C without a fixative and
were analyzed immediately and samples that were fixed with formaldehyde
(final concentration, 2%) and analyzed within 1 week. Five replicates
for each protocol were also analyzed for the T7 phage culture. Before
analysis, the T7 phage culture was diluted 1:10 with
deionized-distilled water (DDW). All working solutions (i.e., stains,
DDW, mountant, formalin) were filter sterilized immediately before they
were used by using Anotop 10 units (Whatman) equipped with
0.02-µm-pore-size inorganic membranes and sterile syringes.
Virus counts.
For samples examined by TEM, the viruses in 1 ml of each of the five replicates were harvested by ultracentrifugation
onto three TEM grids that previously had been fixed to the platform of
a polymer resin support and placed at the bottom of an
ultracentrifugation tube (27); to do this, we used a
Centrikon TST 41.14 Swing-Out rotor that was centrifuged at
120,000 × g for 2 h at 4°C (4, 27,
31). The electron microscope grids were 400-mesh copper grids
with carbon-coated Formvar films (catalog no. A03; Pelanne Instruments), which provided the best compromise between strength and
electron transparency (31). Immediately before the grids were used, they were soaked for 1 min in 1 drop of 0.1% (wt/vol) poly-L-lysine (catalog no. P8920; Sigma) in order to make
the surfaces of the films evenly hydrophilic (31). Following
ultracentrifugation, each grid was stained for 30 s with uranyl
acetate (2%, wt/wt), and the viruses were counted with a JEOL model
1200EX TEM operated at 80 kV and a magnification of ×40,000. Before
counting, each grid was scanned to make sure that the viruses were
distributed randomly. A minimum of 20 TEM fields were then randomly
selected and the viruses were counted until the total counts exceeded
200 viral particles (31). Taper corrections were used for
the final calculations of viral concentrations by using the formulae
developed by Suttle (31), who provided more details
concerning the protocol used to determine the concentrations of viruses
in aqueous solutions by TEM.
EM was also used to count VLPs, which were stained with DAPI, YOPRO-1,
or SYBR Green I. For the original EM-YOPRO-1 method (13), a
stock solution of YOPRO-1 (Molecular Probes Europe, Leiden, The
Netherlands) was diluted to a concentration of 50 µM by using an
aqueous 2 mM NaCN solution. The viruses in 1-ml test samples were
gently filtered (15-kPa vacuum) onto 0.02-µm-pore-size Al2O3 Anodisc filters (Whatman). While the
filters were still damp, they were placed in a petri dish on 80-µl
drops of YOPRO-1 (final concentration, 50 µM) for 48 h in the
dark. Since fixation with aldehydes interferes with binding of YOPRO,
viruses were filtered and stained immediately after they were
collected. After staining, the filters were rinsed twice by filtering
800-µl portions of DDW through the membranes. The damp membranes were
transferred to glass slides and covered with single drops of a solution
containing 50% glycerol, 50% phosphate-buffered saline (0.05 M
Na2HPO4, 0.85% NaCl; pH 7.5), and 0.1%
p-phenylenediamine (Sigma) (made fresh daily from a frozen
10% aqueous stock solution; Sigma) on 25-mm-square coverslips. This
mountant minimized fading (20). The VLPs were counted by
using an Olympus microscope (model HB 2) equipped with a 100/1.25
Neofluar objective lens and a blue filter set.
The modified EM-YOPRO method described by Xenopoulos and Bird
(
40) can be used to stain fixed samples. For this method,
1-ml samples fixed with formalin (final concentration, 2%) were
filtered onto 0.02-µm-pore-size Anodisc membrane filters and rinsed
three times with 500-µl portions of DDW. The filters were put
sample
side up on 80-µl drops of YOPRO in petri dishes. Each petri
dish was
placed in a cardboard container to protect it from light
and irradiated
in a domestic microwave oven equipped with a turntable
for no more than
4 min at a low to intermediate power level (~400
W). The heated petri
dish was allowed to cool for about 10 min,
and then the filters were
replaced on the filter support and rinsed
three times with 800-µl
portions of DDW. The filters were transferred
to glass slides and VLPs
were counted as described above for the
original EM-YOPRO
method.
For the EM-SYBR Green I method (
20), a stock solution of
SYBR Green I (Molecular Probes Europe) was diluted 1:10 with DDW.
For
each new filter, 2.5 µl of the 10% SYBR Green I working solution
was
added to a 97.5-µl drop of sterile DDW on the bottom of a
petri dish
(final dilution, 2.5 × 10
3). A 1-ml sample fixed
with formalin (final concentration, 2%)
was filtered through a
0.02-µm-pore-size Anodisc membrane filter
(Whatman). The Anodisc
membrane was dried and laid sample side
up on 1 drop of the staining
solution for 15 min in the dark.
The Anodisc filter was mounted on a
glass slide and VLPs were
counted as described above for the EM-YOPRO
methods.
For the EM-DAPI protocol (
31), the viruses in 1-ml samples
that were fixed with formalin (final concentration, 2%) were
stained
with DAPI (final concentration, 1 µg ml
1) in the dark
for 30 min and then filtered onto 0.02-µm-pore-size
filters
(Anodisc). The filters were mounted on microscope slides
as described
above for the other methods, and VLPs were counted
by using a UV filter
set and the Olympus
microscope.
Measuring the fading times of the fluorochromes.
The
intensity of fluorescence of stained biological particles decreases as
the duration of excitation during EM increases. This phenomenon, known
as fading, depends on the nature of the fluorochrome and, therefore,
the excitation and observation wavelengths of the fluorochrome. The
total number of fluorescent particles determined by direct EM counting,
therefore, depends on the rate of fading of the fluorochrome used and
the time taken by the observer to count all of the excited particles
present in the microscope field. In order to estimate the bias
resulting from fading of fluorescently marked viral particles in this
study, we measured the speed of fading of each of the fluorochromes
used. To do this, we measured the time (in seconds) that it took for
the last viral particle to disappear from each microscope field for six
samples that were obtained from the different study sites and had been filtered with 0.2-µm-pore-size filters (to eliminate most of the nonviral particles). Fifty microscope fields were analyzed for each
sample. The shape of the fluorescence decay curve and the related
kinetic parameters might have been more important than the time that it
took for the last viral particle to fade during EM, but technically
these data were difficult to obtain. Our fading data were thus
empirical estimates based on the assumption that the decay of
fluorescence is linear.
Estimate of interference caused by free nucleic acids.
The
fluorochromes used in this study were specific stains for nucleic
acids, which also occur in a free state in aquatic media (34,
37). To estimate the effects of fragments of free DNA and RNA on
the total number of viruses counted, samples were treated with two
enzymes, RNase-free DNase I (catalog no. D7291; Sigma) and RNase A
(catalog no. R4642; Sigma). For DNase I- and RNase A-treated samples,
250 Kunitz units of DNase I per ml and 250 Kunitz units of RNase A per
ml were added, and the preparations were incubated for 30 min
(13). The phages in triplicate samples treated in this way
and in untreated control samples were counted by using both TEM and EM.
These experiments were conducted by using five replicate samples from
each of the study sites.
The supplier (Sigma) acknowledged that the stock solution of RNase A
used in the experiments described above could have contained
between 0 and 10% DNase and that boiling this solution to inactivate
the
residual DNase was not recommended. Therefore, we compared
the
contaminated RNase A with a DNase-free RNase (catalog no.
1119 915;
Boehringer Mannheim) that was obtained from the same
source (bovine
pancreas). Numbers of viruses were determined for
samples collected in
February 2000 from the surface waters of
Lake Pavin and eutrophic Lake
Aydat (also located in the Massif
Central region of France) after the
samples were treated with
the contaminated RNase A and with the
DNase-free RNase (see above).
These tests were performed with five
replicate samples from each
lake, and viral concentrations were
determined by using the modified
EM-YOPRO and TEM methods. As
determined by both protocols, the
samples treated with DNase-free RNase
contained similar to slightly
higher (mean, 1.07-fold; range, 0.96- to
1.19-fold) viral concentrations
than the samples treated with
contaminated RNase A contained.
An analysis of variance performed with
the results showed that
the numbers of viruses in samples treated with
both RNase A and
DNase-free RNase were significantly (
P < 0.05) higher than the
numbers of viruses in untreated samples, but
the values for the
RNase A-treated samples were not significantly
different (
P >
0.05) than the values for the samples
treated with DNase-free
RNase. On the basis of this analysis, we
concluded that comparisons
of the effects of DNase I and RNase A in our
original experiments
were not affected by DNase contamination in the
RNase A stock
solution.
Statistical analyses.
The viral concentrations obtained when
the various protocols were used were compared by performing a one-way
analysis of variance for the differences between treatments. Similar
analyses were also performed for the studies in which we examined the
effects of free nucleic acids and the fading times of the
fluorochromes. Significant differences between the results for
fluorochromes or between the results for different treatments were
tested by using an a posteriori test, the Fisher
least-significant-difference test (29). For all of the
analyses of variance, the null hypothesis was that there was not a
significant difference between the results obtained with the various
treatments or between the results obtained with the different fluorochromes.
| |
RESULTS |
Comparison of methods of counting viruses.
For all of the
methods used and all of the samples analyzed, the coefficients of
variation (coefficient of variation = standard deviation × 100/mean) for the mean concentrations of viral particles, calculated by
using the values for five replicates, were relatively low, between 2.5 and 18.4%. The results which we obtained (Fig. 1) show that the concentrations of VLPs
were up to 50 times higher when EM was used than when TEM was used. The
difference was much more pronounced for the samples obtained from the
lagoon and the T7 phage culture, the samples in which the viral
concentrations were the highest. For all of the samples obtained from
the lagoon and the T7 phage culture, the viral concentrations obtained
by the TEM method were 18 and 3% of the viral concentrations obtained by EM, respectively. For the samples obtained at other sites, at which
the viral particle concentrations were lower, the values were between
27 and 65% (Table 1).
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FIG. 1.
Viral concentrations in the different environments
tested, as determined by TEM and by EM after staining with various
fluorochromes. ND, not determined. The table shows the results obtained
by a one-way analysis of variance in which Fisher's
least-significant-difference test was used. An asterisk indicates
significance at a level of P < 0.05. NS, not
significant. ME(DAPI), EM-DAPI protocol; ME(O-YOPRO), original EM-YOPRO
protocol; ME(M-YOPRO), modified EM-YOPRO protocol; ME(SYBR I), EM-SYBR
Green I protocol; TEM, TEM protocol; O-YOPRO, original YOPRO; M-YOPRO,
modified YOPRO; SYBR I, SYBR Green I.
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Therefore, the TEM method provided lower viral concentrations with a
much higher average coefficient of variation (10.2%)
than the methods
based on epifluorescence provided (EM-DAPI method
coefficient of
variation, 7.7%; original EM-YOPRO method, 5.3%;
modified EM-YOPRO
method, 8.8%; EM-SYBR Green I method, 8.7%).
An analysis of variance
confirmed that the viral concentrations
obtained with the TEM method
were significantly lower (
P < 0.05)
than the
concentrations obtained with the two methods in which
YOPRO
and epifluorescence were used (Fig.
1).
There was, however, considerable variability in the viral
concentrations estimated by the epifluorescence methods depending
on
the fluorochrome used. The original YOPRO staining method resulted
in
concentrations for samples from all of the sites that were
higher than
the concentrations obtained with DAPI and SYBR Green
I. The modified
EM-YOPRO protocol in all cases gave values lower
than the values
obtained with the original EM-YOPRO protocol,
except for the samples
obtained from the T7 phage culture, in
which the viral concentration
was very high. However, the analysis
of variance revealed no
significant difference (
P > 0.05) between
the results
obtained with these two protocols. The values obtained
when DAPI was
used were the lowest values obtained with the EM
methods. There were
significant differences between the results
obtained with this method
and the results obtained with the two
protocols in which YOPRO was
used. The values obtained with the
EM-SYBR Green I protocol were
similar to the values obtained with
the modified EM-YOPRO protocol but
were lower than the values
obtained with the original EM-YOPRO method
for all of the samples
when both YOPRO protocols were used (Fig.
1).
Fading of the fluorochromes.
The fluorochrome fading
experiment clearly showed that the fluorescence of particles that were
stained with the original YOPRO protocol (244 s) and the modified YOPRO
protocol (210 s) lasted the longest. An analysis of variance revealed
no significant difference (P > 0.05) between the
results obtained when these two protocols were used (Fig.
2). DAPI (94 s) and especially SYBR Green
I (49 s) had significantly shorter mean fading times than both the
original YOPRO and the modified YOPRO. For SYBR Green I, the data were equivalent to a fading rate for fluorescent particles that was five
times higher than the fading rate for the original YOPRO. Furthermore,
the coefficients of variation for the fading times when DAPI (32.8%)
and SYBR Green I (45.7%) were used were much higher than the
coefficients of variation obtained when the original EM-YOPRO protocol
(30%) and the modified EM-YOPRO protocol (26.7%) were used.
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FIG. 2.
Comparison of fading times for the various fluorochromes
used with EM. The numbers in parentheses are the coefficients of
variation for the fading times obtained with six samples that
originated from the different bodies of water sampled. The table shows
the results obtained by a one-way analysis of variance in which
Fisher's least-significant-difference test was used. An asterisk
indicates significance at a level of P < 0.05. NS, not
significant. O-YOPRO, original YOPRO; M-YOPRO, modified YOPRO; SYBR I,
SYBR Green I.
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Effects of DNase and RNase.
To evaluate the effects of free
nucleic acids on estimates of viral concentrations, the viruses in
samples obtained from the three lakes which differed in trophic status
were counted by using the modified EM-YOPRO protocol; the samples were
either treated or not treated with RNase-free DNase I (catalog no.
D7291; Sigma) and RNase A (catalog no. R4642; contaminated with 0 to
10% DNase; Sigma). In all cases, the samples treated with DNase I or
RNase A contained significantly lower virus concentrations than the untreated samples (Table 2). On average,
for all of the samples DNase I treatment resulted in an 18.3%
reduction in the total virus level. The reduction after treatment with
RNase A was 26.4%. We observed that the action spectrum of the
DNase-contaminated RNase A used in these experiments was not
significantly different (P > 0.05; analysis of
variance) than the action spectrum of pure RNase (i.e., DNase-free
RNase; catalog no. 1119 915; Boehringer Mannheim). Accordingly, the
cumulative effect of DNase I and RNase A could be estimated by adding
the effects of the two enzymes. The combined effects were equivalent to
reductions in the mean total viral concentration of 39% for the
Villerest Reservoir samples, 53% for the Lake Pavin samples, and 42%
for the Sep Reservoir samples.
After samples were treated with DNase I and RNase A and then TEM counts
were determined, we recorded reductions in mean viral
concentrations of
18.7 and 16.1%, respectively, for Lake Pavin
and the Villerest
Reservoir, the only two bodies of water tested.
When the effects of the
two enzymes were added, the viral concentrations
were reduced by 40%
for the samples obtained from the Villerest
Reservoir and by 30% for
the samples obtained from Lake Pavin.
The analysis of variance
performed with these results showed that
the two enzymes had a
significant effect (
P < 0.05) on total viral
concentration as determined by TEM (Table
2).
| |
DISCUSSION |
Comparative study of the methods of counting viruses.
Our
results show that two microscopic procedures for estimating viral
concentrations in aquatic ecosystems gave different results. With only
one exception, the viral concentrations determined by EM were higher
than the viral concentrations determined by TEM. The values obtained
when TEM was used were comparable to the values obtained when we used
DAPI staining followed by EM but were significantly lower than the
values obtained when the two EM-YOPRO methods were used. The
differences between the values obtained when TEM was used and the
values obtained when EM was used were always more pronounced for the
samples containing higher viral concentrations (i.e., the T7 phage
culture and domestic sewage works lagoon samples) (Table 1). These
findings agree with the results obtained by Hennes and Suttle
(13) and Weinbauer and Suttle (35), who showed
that the difference between estimates of viral concentrations obtained
by EM and TEM increased as the number of viruses in the medium
increased. This is probably due to the high concentrations of
particulate matter that adversely affect TEM counting. The
underestimation of values and the significantly greater variability of
the TEM method (coefficient of variation, 10.2%) than of
epifluorescence methods can also be explained by the high
magnifications needed for viral counting by TEM (13, 31).
Viruses can also be lost during staining of the grids with uranyl
acetate or during the rinsing operations. Due to insufficient dilution
(dilution ratio, 1:10), our TEM data for the level of T7 phages must be
viewed as underestimates (3, 11). However, the
underestimation did not really affect the general observation, based on
the results of this study, in which different aquatic systems were
tested, and the results of previous studies, that the numbers of VLPs
determined by using fluorescence are significantly higher than the
numbers of VLPs obtained when TEM preparations are used (for a review,
see reference 7). Although the reasons for this are
not known (7), it is likely that nonviral fluorescent particles are counted together with viruses by EM. In this study, we
calculated the overestimate of viral concentration due to interference from free nucleic acids when EM was used (see below).
The virus counting protocol in which TEM is used is more expensive and
difficult, requiring highly specialized training. With
the exception of
the original EM-YOPRO method, in which samples
that have not been fixed
are used and a staining time of 2 days
is required, the protocols in
which EM is used are much simpler
and faster than the TEM protocol. Of
the fluorochromes used for
EM, DAPI gave the lowest concentrations.
Similar results have
been reported by several authors (
10,
35), which supports
the hypothesis that VLP concentrations are
underestimated when
DAPI is used; this finding is attributed to the
lower DAPI light
intensity (compared to other fluorochromes), the
affinity of DAPI
for only double-stranded DNA, and a relatively short
fading time
(94 s) (Fig.
2). The concentrations of viruses stained with
SYBR
Green I and examined by EM were similar to the concentrations
obtained when the YOPRO protocol was used; the only exception
was the
value for the sample obtained from the T7 phage culture,
which
contained a very high viral concentration. In the latter
case, the
viral concentration obtained with the EM-SYBR Green
I protocol was much
lower (Fig.
1). Noble and Fuhrman (
20) and
Marie et al.
[
17] have recently tested SYBR Green I with EM
by
using seawater samples. Our results obtained with freshwater
samples
agree with the results of these researchers; the virus
concentrations
determined by the TEM protocol were lower than
the concentrations
determined by the EM-SYBR Green I protocol,
and the concentrations
determined by the EM-DAPI and EM-SYBR Green
I protocols did not differ
significantly from one another. Nevertheless,
despite a fluorescence
intensity that was similar to that of YOPRO,
SYBR Green I had a shorter
fading time (~50 s). In samples treated
with SYBR Green I in which
the viral concentration was less than
5 × 10
7
particles ml
1, the coefficients of variation were lower
at higher viral concentrations.
However, in samples in which the viral
concentration was more
than 5 × 10
7 particles
ml
1, the coefficient of variation increased as the viral
concentration
increased (Fig.
3). Such a
relationship was not found with the
other fluorochromes. These results
suggest that the precision
of the EM-SYBR Green I method decreases with
samples containing
high viral concentrations because of the very short
fading time.
This protocol is therefore not recommended for
quantitative studies
of viruses that do not include an image analyzer
for aquatic systems
containing high viral concentrations, such as
sewage effluents.
It has recently been shown that in a pelagic
oligotrophic environment,
using SYBR Green I with flow cytometry
results in effective counting
of VLPs (
17).
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FIG. 3.
EM-SYBR Green I protocol: relationship between the mean
viral concentrations and the corresponding coefficients of variation
(CV) for the six environments tested.
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In all of the samples with which it was tested, YOPRO staining yielded
viral densities that were higher than the densities
obtained with the
other methods tested. The results of the analysis
of variance showed
that the reduction in the staining time from
48 h (original
EM-YOPRO method) to a few minutes when YOPRO was
hot (modified EM-YOPRO
method) did not have a significant effect
on the quality of staining
(similar intensities and fading times)
and therefore did not have a
significant effect on the estimate
of viral concentration. On the other
hand, the average coefficients
of variation for all of the samples
tested were 5.3% for the original
EM-YOPRO protocol and 8.9% for the
modified EM-YOPRO protocol.
However, the latter value is still
relatively low and represents
an acceptable level of precision and
reproducibility. There has
been only a single study of the use of the
modified EM-YOPRO protocol
(
40), and only a very few samples
were tested in that study.
The authors did not find a significant
difference in viral concentrations
when they compared the modified
EM-YOPRO protocol with the original
protocol of Hennes and Suttle
(
13).
On the basis of our results, we recommend using the modified EM-YOPRO
protocol to count viruses in aquatic systems, since
this protocol is
much faster than the original EM- YOPRO protocol,
can be used with
samples that are fixed with aldehydes, and has
satisfactory precision
and efficiency. Examining TEM preparations
in which phages are
recognizable with much more confidence than
on EM slides is not
precluded when it is possible, at least for
occasional checks. The TEM
method is necessary for describing
the morphological diversity and
functional importance of viruses
in aquatic systems (
27,
28).
Interference from free nucleic acids.
An alternative or
additional explanation for the differences in viral concentrations
observed when EM and TEM are used could be that nonviral fluorescent
particles are counted by EM. These particles could be very small
bacteria (<100 nm), free nucleic acids, or even free mitochondria or
ribosomes that are stained by the nucleic acid-specific fluorochromes.
Since free ribosomes and mitochondria cannot survive for a long time in
the aquatic environment and since the concentration of very small
bacteria in the planktonic environment is negligible (35),
only free nucleic acids can interfere significantly with the viral
concentrations determined by EM. In aquatic ecosystems, free DNA
generally originates from cell lysis (22) and from grazing
activity by consumers (33).
To estimate this interference, we treated samples with DNase and RNase.
The viral concentrations in samples treated separately
with these two
enzymes were significantly lower than the concentrations
in untreated
samples (Table
2). Based on our results, 39 to 53%
of the particles
considered to be viruses by EM were in fact free
nucleic acids. These
results differ from those of Hennes and Suttle
(
13) and
Drake et al. (
5), who observed no significant difference
between marine plankton samples treated with DNase and untreated
samples. In contrast, Hara et al. (
11) reported that about
one-third
of the small VLPs stained by DAPI in the marine environment
are
not in fact viruses. It should be mentioned that to our knowledge,
our study is the first study to consider the effect of RNase on
free
aquatic virus
counts.
However, direct observations by TEM allowed us to demonstrate that
DNase and RNase also break down true viral particles. As
determined by
TEM, a technique that results in certain identification
of phages, the
viral concentrations calculated after samples from
two different
environments were treated with DNase and RNase were
significantly lower
than the viral concentrations in untreated
samples (Table
2). In the
two environments studied, the average
reductions in viral
concentrations were 18.7% after treatment
with DNase and 16.1% after
treatment with RNase. The sensitivity
of phages (viral DNA) to DNase
has been demonstrated previously
by several authors, especially for the
marine environment. For
example, Jiang and Paul (
14) showed
that in a culture containing
T2 phages and plankton samples from the
Gulf of Mexico 33 to 48%
of the encapsulated viral DNA was digested by
DNase. Maruyama
et al. (
18) found that in Tokyo Bay about
10% of the <0.2-µm
fraction of DNA, which could be assumed to be
viruses (i.e., "coated
DNA"), was sensitive to DNase. These authors
used a lower concentration
of DNase (20 Kunitz units/ml) than we used
in this study (250
Kunitz units/ml), which could explain the greater
sensitivity
of viral DNA in our experiments (18.7% reduction as
determined
by TEM and 18.3% reduction as determined by EM, on
average). The
results of this comparison also suggest that DNA is more
likely
to resist the action of DNase in seawater than in freshwater.
The ability of viruses to be adsorbed quickly onto suspended particles
is well known (
36), and this process is undoubtedly more
pronounced
in the marine environment, where the ratio of virus density
to
sensitive host density is lower (
16). It is known that
such
adsorption occurs with nucleic acids in a combined form, which
are
more refractory to the action of DNase (
22). The degradation
of viral RNA by RNase in our experiments led to losses similar
to those
obtained with DNase, and on average, 16.1% and 26.4%
of the
bacteriophages present in our samples were destroyed, as
determined by
TEM and EM, respectively. To our knowledge, these
estimates are the
first data concerning the sensitivity of planktonic
viruses to RNase.
The vertical distribution of RNA concentrations
in the North Atlantic
Ocean has been described by Karl and Bailiff
(
15), but there
is still great uncertainty concerning the origins
of the RNA and its
role in aquatic
ecosystems.
We concluded that the apparent destruction of viruses by DNase and
RNase makes it impossible to use these enzymes routinely
in
virus-counting protocols in which EM is used. However, by comparing
the
viral losses due to the cumulative effects of DNase and RNase
as
determined by EM (ca. 45%) and TEM (ca. 35%), we were able
to
quantify the interference due to free nucleic acids by using
the
following formula: 0.65
A = 0.55(
A+
B), where the sum of
the
fraction of viruses (
A) and the fraction of free nucleic
acids
(
B) in the samples analyzed by EM is equal to 1. By
assuming that
the use of DNase and RNase leads to the destruction of
all of
the free nucleic acids, we were then able to calculate that the
overestimate of the viral concentration determined by EM due to
interference from nucleic acids in all of our samples was 15.4%.
| |
ACKNOWLEDGMENTS |
The field assistance and technical assistance of G. Coffe, C. Doniol, S. Fournet-Fayard, C. Portelli, D. Sargos, and B. Vigues are
greatly appreciated. We are also grateful for valuable comments and
suggestions on an earlier version of the manuscript by two anonymous reviewers.
This study was supported by an MRT graduate fellowship to Y.B.. Funding
by CNRS grant 9507004, ACC-SV 7 (Diversité fonctionnelle des
réseaux trophiques microbiens dans les écosystèmes
aquatiques), also facilitated the investigation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Biologie des Protistes, UMR CNRS 6023, Universite Blaise Pascal
(Clermont-Ferrand II), F-63177 Aubiere Cedex, France. Phone: 33 4 73 40 78 36. Fax: 33 4 73 40 76 70. E-mail:
Telesphore.SIME-NGANDO{at}lbp.univ-bpclermont.fr.
| |
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Top
Abstract
Introduction
