Botanica Lithuanica

2011, 17(23): 127133

COMPARATIVE ANALYSIS OF METHODS FOR QUANTITATIVE ASSESSMENT OF

VIRUS-LIKE PARTICLES IN EUTROPHICATED AQUATIC ENVIRONMENTS
Sigitas ulius1, Juozas Staniulis2, Riardas Pakauskas1,2
Klaipda University, Coastal Research and Planning Institute, H. Manto Str. 84, LT-92294 Klaipda,
Lithuania; e-mail: sigas@corpi.ku.lt
2
Nature Research Centre, Institute of Botany, alij Eer Str. 49, LT-08406 Vilnius, Lithuania;
e-mail: juozas.staniulis@botanika.lt, ricardas.paskauskas@botanika.lt

Abstract
ulius S., Staniulis J., Pakauskas R., 2011: Comparative analysis of methods for quantitative
assessment of virus-like particles in eutrophicated aquatic environments [Metod, skirt virusini
daleli kiekiui eutrofikuotose hidroekosistemose vertinti, lyginamoji analiz]. Bot. Lith., 17(2
3): 127133.
The applicability of two microscopy-based methods for virus enumeration was evaluated for the
eutrophicated Curonian Lagoon. Total abundance of viruses determined by means of electron and
epifluorescence microscopy ranged from 1.91 107 to 5.06 107 viruses ml-1 and from 72.81 107
to 141.88 107 viruses ml-1, respectively. T-test for dependent samples showed the significant (p <
0.001) differences in counts observed by the two different methods from the same site of the studied
environment. Based on method precision, effectiveness of visualization, time consumption and
detection capacity, we suggest that the use of SYBR Green I in parallel with image analysis makes
the application of epifluorescence microscopy more accurate and less time consuming for overall
estimation of phages abundance in the eutrophicated aquatic environments.
Keywords: virioplankton, transmission electron microscopy, epifluorescence microscopy, Curonian
Lagoon.

INTRODUCTION
The importance of viruses is based on their significant contribution to biogeochemical cycles (Fuhrman,
1999) and plankton communities regulation (Jardillier et al., 2005). Viruses exhibit a high infection rates
(Winter et al., 2004) and significantly contribute to
phytoplankton (Baudoux et al., 2006) and bacteria
(Jacquet et al., 2005) mortality. Moreover, viruses
are maintaining the microbial diversity (Weinbauer
& Rassoulzadegan, 2004) and activity (BonillaFindji et al., 2008). They are numerically abundant,
globally distributed and are found in all aquatic environments.

Quantitative evaluation of study object is an essential initial step in many ecological investigations of
targeted environmental system. The first studies of free
viruses without isolation or host cultivation procedures
were performed three decades ago (Sieburth, 1979;
Torrella & Morita, 1979). It has been shown
that bacteriophages extremely dominate the aquatic
environments (Bergh et al., 1989; Suttle, 2005). The
abundance and high variation of free floating viruses
seems to depend on host community (Murray &
Jackson, 1992), trophic gradient (Weinbauer et al.,
1993; Noble & Fuhrman, 2000), depth (Hara et al.,
1996), physical factors (Suttle & Chen, 1992) and the
type of system (marine or freshwater) (Maranger &
127

Bird, 1995). These observations implicate not only

the importance of enumeration of viruses, but also
the application of quantitative methods for particular
environment, assuming that different water or trophic
conditions (sample type) can significantly affect the
efficiency and accuracy of countings and introduce
biases that might lead to misunderstanding of the results
(Wen et al., 2004).
Recently three culture-independent methods have
been proposed for virus enumeration from natural
aquatic environments (Suttle, 2007), including flow
cytometry as most relevant and accurate approach
nowadays. However, two microscopy-based methods,
transmission electron microscopy (TEM) and epifluorescence microscopy (EFM), are already widely used
for virus counting. Both approaches rely on different
operating principles and provide numerical index of
free virus particles. The eutrophic conditions, including
the detritus and alloctonous organic substances, are
known to interfere with quality of the data produced
by both approaches (Proctor, 1997). The aim of our
study was to evaluate suitability of application of the
two different methods of virus enumeration for the
eutrophicated Curonian Lagoon, in terms of precision,
effectiveness of visualization, costs of analysis, time
consumption and detection capacity.
St.

Coordinates (WGS 84)

1
2
3
4
5
6
7
8
9
10
11
12
13

550 41,200' N, / 210 07,400' E

550 37,571' N, / 210 08,867' E
550 34,886' N, / 210 08,867' E
550 32,367' N, / 210 07,393' E
550 30,041' N, / 210 07,643' E
550 27,857' N, / 210 06,995' E
550 25,898' N, / 210 06,714' E
550 23,571' N, / 210 08,200' E
550 22,249' N, / 210 08,933' E
550 20,939' N, / 210 09,692' E
550 19,620' N, / 210 10,500' E
550 24,735' N, / 210 06,002' E
550 18,097' N, / 210 01,002' E

MATERIALS AND METHODS

Sampling
The Curonian Lagoon lies along the Baltic coast of
Lithuania and Kaliningrad Region of the Russian Federation. It is a shallow eutrophicated freshwater body
typical of the south-eastern coast of the Baltic Sea. The
irregularly recurrent seawater intrusion in the northern
part of the lagoon creates the north-south salinity gradient. The study was performed at 13 stations (Fig.1).
The samples were taken from surface water layer
(0.5m) along Lithuanian part of the Curonian Lagoon
during cruise at the end of July 2005.
Transmission electron microscopy
Material for TEM analysis (1000 ml) was collected
into polyethylene bottles and kept in cold (+4 C) until further processing. In the lab samples were filtered
through 0.45-m-pore-size membrane filter to remove
larger particles. Virus-like particles (VLP) were concentrated 200 times by filtration onto Pragopor 11 nitrocellulose filters and preserved with glutaraldehyde to final 1 % concentration and stored at +4 C until analysis.

The Curonian Lagoon

Fig. 1. Scheme and coordinates of the 13 sampling sites in the Curonian Lagoon
128

Three microlitres of a high-titer viral stock preparation

were applied onto a Formvar-carbon-coated 400-mesh
palladium grid and allowed to adsorb to the grid and
desiccate. The grid was then stained with 1 or 2 drops of
a uranyl acetate solution (2 %) for 30 s. The phages on
the grid were counted with a JEOL JEM-100S transmission electron microscope at instrumental magnification
of 15.000 and 25.000, an accelerating voltage 60 kV.
At least 10 randomly taken micrographs (Fig. 2 a) were
analysed.
Epifluorescence microscopy
Samples (50 ml) for the estimation of virus abundance were collected into PE bottles and immediately
fixed with 0.2-m-pore-size pre-filtered 37 % formaldehyde (to final 1 % concentration) and kept at -20 C
until processing. Direct counts of viruses were obtained
according to Noble & Fuhrman (1998) after staining
with SYBR Green I (Fig. 2 b). The analysis was made by
epifluorescence microscope Olympus IX70 at blue light
excitation (488 nm) to take a close-up at 1000magnifications by taking the pictures from at least 10 randomly
selected fields per slide. The pictures were examined by
Image ProPlus 4.5 software. In parallel the total number
of bacteria was identified following the same approach.
Statistical analysis

RESULTS
Total abundance of viruses in water samples from the
Curonian Lagoon ranged from 1.91 107 to 5.06107
viruses ml-1 and from 72.81 107 to 141.88 107
viruses ml-1, determined by means of electron and epifluorescence microscopy, respectively. There was no
significant (p > 0.05) decrease (b = -0.001, r2 = 0.002 for
both methods) along the north-south gradient (Table1),
whereas salinity was observed for stations 1, 2 and 4
(not shown). The total abundance of viruses determined
by both methods showed no significant differences
(p> 0.05; df 11) between freshwater and saline zones
of the lagoon. Total bacteria abundance determined by
EFM varied from 0.64 106 bacteria ml-1 to 1.87 106
bacteria ml-1, and did not correlate with total counts of
VLP derived by both methods.
Variation in virus abundance (up to 2.6) as well as
standard deviation (0.92) obtained by TEM method
was higher than the variation and standard deviation of
EFM (up to 2.0 and 0.26, respectively) for the Curonian
Lagoon (Table 2). EFM counts were higher from 19 to
71 times than TEM counts in all cases. TEM averaged
2.6 % of the total EFM counts. T-test for dependent
samples (Table 2) showed the significant (p < 0.001)
differences in counts observed by the two different
methods from the same site of the studied environment.

T-test for dependent samples and correlation analysis

was applied in order to compare the results obtained by
the two different methods, where a probability of < 0.05
was considered as significant.

DISCUSSION

a)

b)

The methodological advancements or the development of new methods, which allow obtaining more
accurate information on size and amount of microbes

Fig. 2. Electron micrographs (a) and pictures (b) taken by TEM and EFM, respectively. In both pictures dotted
arrows indicate virus-like particles, whereas solid arrows indicate bacteria cells
129

still have many limitations, particularly when comparing the results obtained by different approaches. First
studies aimed to compare EFM and TEM counts yielded inconsistent results (Hara et al., 1991; Proctor &
Fuhrman, 1992). The significant differences between
counts obtained in the Curonian Lagoon led to the
considerations of possible uncertainties of both methods
applying them for eutrophicated water bodies. Moreover, it is critical to infer which method is more suitable
when the number of viruses is particularly of interest.
It has been shown that detritus, particulate and organic allochthonous matter, which is most relevant in
eutrophic environments, could strongly and negatively
influence TEM counts (Proctor, 1997). In addition, it
has been suggested that detritus food chain could constitute a significant part of the functioning of the Curonian Lagoon (Lesutiene et al., 2008). Thus, the lower
number of virus-like particles yielded by TEM might
result from high concentrations of organic matter or detritus. The similar effects of detritus and organic matter could result in an unacceptable level of background
fluorescence in EFM and virus counts (Bettarel et al.,
2000). The choice of SYBR Green I, instead of other
fluorochromes such as SYBR Gold or Yo-Pro for EFM
in eutrophicated aquatic environment partly compen-

sates the miscounting, since humic content or detritus

is not stained or emit yellow colour instead of green of
virus particles (Noble & Fuhrman, 1998). Thus the use
of EFM over TEM can significantly eliminate the negative effect of increased allochthonous organic matter
concentration and result in higher numbers of viruses in
the Curonian Lagoon.
It was suggested that in the presence of high virus
abundance, which is characteristic of eutrophic aquatic
environments, the decrease in precision occurs due to
short fading time (SYBR Green in our case), which in
turn increases variations in virus numbers. However, the
variation in virus number yielded by EFM in general is
lower than observed by TEM, suggesting that the use
of image analysis provides steady and sufficient counts.
On the other hand, Hara et al. (1991) concluded that
low ratio between EFM and TEM indicates that EFM
observed particles are viruses. However, application
of the TEM makes difficult to identify filamentous and
pleomorphic or RNA viruses, which could be stained
and observed using EFM (Patel et al., 2007), even if
produced signal of these viruses is quite weak. Thus,
it could at least partially explain the higher numbers of
virus-like particles given by EFM in the Curonian Lagoon.

Table 1. Abundance of virus-like particles observed by transmission electron (VLP-TEM) and epifluorescence
microscopy (VLP-EFM). VBR virus to bacterium ratio obtained from EFM counts
Station

VLP-EFM, 107 ml-1

VLP-TEM, 107 ml-1

EFM/TEM

Bacteria, 106 ml-1

VBR

1
2
3
4
5
6
7
8
9
10
11
12
13

140.12
112.97
140.05
87.67
141.88
118.13
85.75
72.81
141.49
84.09
119.36
134.64
139.23

2.36
2.03
2.55
1.91
2.88
5.06
3.61
3.80
2.59
3.03
2.03
2.28
1.95

59
56
55
46
49
23
24
19
55
28
59
59
71

0.89
0.94
1.09

0.92
1.87
0.96
0.78
1.66
0.64
0.93
1.21
1.07

27
22
23

31
27
38
49
16
48
22
19
18

Table 2. Analysis of differences between the TEM and EFM counts of each pair of a sample (Student test for
dependent samples)
VLP-EFM, 10 ml
VLP-TEM, 107 ml-1
9

130

df

13

12

-1

Mean
1.168462
2.775385

SD
0.257612
0.916793

-5.61670

0.000113

The drawback of EFM is that other organisms and

particles containing genetic material could also be
stained. Ultramicrobacteria, free mitochondria or ribosomes as well as free oligonucleotides originated
from cell lysis or grazing activity are stained by the
nucleic acid-specific fluorochromes. Although the first
two have a minor effect on total virus counts (Hennes
& Suttle, 1995; Weinbauer & Suttle, 1997), the
free DNA of non-viral origin can constitute from a few
up to 99 % for samples from estuaries as well as other
environments (Paul et al., 1991). So far there are no data
on the concentration of free DNA in the water column
of the Curonian Lagoon. In the samples taken from the
Curonian Lagoon the TEM numbers constituted from
1.40 % to 5.22 % and averaged 2.6 % of the total EFM
counts. However, in case of high amount of free DNA,
the differences between TEM and EFM counts might
be less than observed or TEM counts could be even
higher as it has been shown by Paul et al. (1991). The
use of nuclease enzymes (such as DNase and RNase)
to degrade and remove unprotected (non-virus) DNA
from samples seems to be insufficient, since it could
damage the true viruses (Bettarel et al., 2000).
As an alternative to short fading time of SYBR Green
I, other fluorochromes such as Yo-Pro may be used.
Although Yo-Pro demonstrates a very long fading over
SYBR Green, the staining protocol requires 48 h and
is not applicable for fixed samples (Hennes & Suttle,
1995), making this approach ineffective in terms of time
consumption. In addition, the long incubation with dyes
could result in fast decrease of viruses and thus the final
counts. Assuming that decrease rate of viruses is equal
to their production (to keep the standing stock), the
application of Yo-Pro is not very suitable for eutrophic
environments such as the Curonian Lagoon as well as for
high number of samples. Moreover, the advancements
in technology and instrumentation such as cameras and
image analysis software enable recording of almost all
emitted signals as well as more accurate signal analysis
to distinguish e.g. detritus, making the SYBR Green I
more suitable for use in eutrophic waters than Yo-Pro.
The possible ecological role of viruses may be inferred from what is known about viruses themselves.

Thus, not just identification of the present viruses is

critical, but as high precision as possible as well. It has
been suggested that TEM underestimates viral abundance due to technical problems of samples preparation
(unequal collection and staining), observation (low detection of non-typical viruses) and recently due to introduction of digital technology (Ackermann & Heldal,
2010). The time consumption, procedure difficulties
and service charges, which may differ for other environments and purposes, are important features of the
analysis (Table 3). In addition, the use of different fixatives, sample storage time or pre-treatment procedures
may have a significant effect on counts produced by
both methods, which were not examined in the present
study. However, the evaluation of these biases still remains difficult, since relationship of particle decay rates
over storage time is non-linear and can change dramatically either using the same or different fixatives (Wen
et al., 2004).
The use of SYBR Green I in parallel with image
analysis make the application of EFM more accurate
and less time consuming for overall estimation on
phages abundance. However, TEM method provides
not just the number of viruses, but it is surpassing
so far in morphological characterization and sizing
of phages, burst size measurements and at least can
provide an excellent contrast of phage-like particles if
microscopists skill is proper.
ACKNOWLEDGEMENTS
This study was funded by a grant (No. T-66/05) from
the Lithuanian State Sciences and Studies Foundation.
The authors also express their appreciation to the staff
of the Department of Immunology and Cell Biology
(Institute of Biotechnology, Vilnius) for a valuable
support with epifluorescence microscopy technique.
The authors thank the anonymous reviewer whose
comments and suggestions improved the manuscript.
Ms. Violeta Ptaekien is acknowledged for her
contribution to improve the language of the manuscript.

Table 3. Comparison of TEM and EFM approaches

Time consumption Price of analysis Precision

Effectiveness of visualisation

Detection capacity
Moderate (Phages)
High (unusual/RNR
viruses)

TEM

High

High

Moderate

High

EFM

Moderate

Low

High

Low

131

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Metod, skirt virusini daleli kiekiui eutrofikuotose hidroekosistemose

vertinti, lyginamoji analiz
Sigitas ulius, Juozas Staniulis, Riardas Pakauskas
Santrauka
Atliekant iuos tyrimus buvo vertintas dviej mikroskopijos metod tinkamumas planktono virusini
daleli kiekiui eutrofikuotame Kuri mari vandenyje nustatyti. Virioplanktono gausumas, vertintas
pervieiamosios elektronins mikroskopijos (TEM)
bdu, svyravo nuo 1,91107 ml-1 iki 5,06107 ml-1, o
epifluorescencins mikroskopijos metodu (EFM) nuo
72,81107 ml-1 iki 141,88107 ml-1. Porini imi statistin (T-test) analiz atskleid, kad kiekybiniai veriai, gauti TEM ir EFM, reikmingai tarpusavyje ski-

riasi. Remiantis abiej tyrim bd tikslumo, objekto

vizualizacijos efektyvumo ir kokybs, laiko snaud
(apimani visas procedras nuo mginio paruoimo
iki galutinio rezultato) bei objekto aptikimo ir identifikavimo tikslumo kriterijais, galima teigti, jog epifluorescencins mikroskopijos, naudojant SYBR Green I
fluorochrom ir nuotrauk analizs programin rang,
taikymas yra tinkamesnis bdas siekiant vertinti bendr virioplanktono gausum eutrofikuotuose vandens
telkiniuose.

133

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