Journal of Virological Methods 117 (2004) 19–25

Standardised evaluation of the performance of a simple membrane

filtration-elution method to concentrate bacteriophages
from drinking water
Javier Méndez a , Ana Audicana b , Ana Isern c , Julián Llaneza d , Belén Moreno e ,
Marı́a Luisa Tarancón f , Juan Jofre a , Francisco Lucena a,∗
a Departamento de Microbiologı́a, Universidad de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain
b Dirección de Salud Pública del Gobierno Vasco, C/ Marı́a Dı́az de Haro, 60, 48010 Bilbao, Spain
c Laboratori Municipal de Barcelona, Institut Municipal de Salut Pública, Avda. de les Drassanes, 13-15, 08001 Barcelona, Spain
d Laboratorio de Salud Pública de Asturias, Finca de la Cedellada, Camino de Rubı́n s/n, 33011 Oviedo, Spain
e Laboratorio de Salud Pública de Gipuzkoa, Avda. Navarra, 4, 20013 Donostia-San Sebastián, Spain
f Conselleria de Salut i Consum, Plaça Hospital 3, 07012 Palma de Mallorca, Spain

Received 7 July 2003; received in revised form 17 November 2003; accepted 18 November 2003

Abstract

The bacteriophage elution procedure described further after adsorption to acetate–nitrate cellulose membrane filters allows better recovery
of phages concentrated from 1 l of water than elution proceduresm used previously. The improvement is due to the combined effect of the
eluent (3% (w/v) beef extract, 3% (v/v) Tween 80, 0.5 M NaCl, pH 9.0) and the application of ultrasound instead of agitation or swirling.
Average recovery of somatic coliphages, 82 ± 7%, was the greatest, and that of phages infecting Bacteroides fragilis, 56 ± 8%, the lowest,
with intermediate values for F-specific and F-specific RNA bacteriophages. Thus, the method allowed recovery of over 56% for all the phages
suggested as surrogate indicators. The method was then validated according to an International Standardisation Organisation validation
standard procedure and implemented in routine laboratories, which obtained reproducible results.
© 2003 Elsevier B.V. All rights reserved.

Keywords: Bacteriophages; Concentration; Membrane filter; Elution; Validation

1. Introduction approaches: the first is a presence/absence method, which

Bacteriophages have been investigated as potential model
organisms for water quality assessment (IAWPRC, 1991;
Grabow, 2001; Jofre, 2002). Phages proposed for water
quality assessment are somatic coliphages (Kott and Ben
Ari, 1968), F-specific RNA bacteriophages (Havelaar et al.,
1986) and bacteriophages infecting Bacteroides fragilis
(Tartera and Jofre, 1987). Although phages are present in
surface waters at concentrations high enough to allow di-
rect enumeration in small volumes of sample, this is not
the case with groundwater or drinking water. In this case it
is necessary to test phages in larger, at least 1 l, volumes.
Detecting phages in 1 l volumes can be achieved by two
∗ Corresponding author. Tel.: +34-934-021-484;
fax: +34-934-110-592.
E-mail address: fluena@ub.edu (F. Lucena).
is only qualitative and the second is through concentration
and plaque assay, which is quantitative. Many methods
for concentrating phages from water samples had been
described (Logan et al., 1980; Schulze and Lenk, 1983;
Shields and Farrah, 1986; Sobsey et al., 1990; Borrego
et al., 1991; Lucena et al., 1995; Sinton et al., 1996). Most
of the methods have only been described for one type of
bacteriophages and generally validated with a laboratory
strain. Among these, we find very feasible a method based
on adsorption to acetate–nitrate cellulose ester membrane
filters described by Sobsey et al. (1990) that was modified
further by Sinton et al. (1996). The method is very efficient
for concentrating coliphages from 100 ml, but results for
1 l volumes are below 50% recovery (Hsu et al., 1998). We
tested the method as modified by Sinton et al. (1996) for
concentrating somatic coliphages from 1 l of drinking water
and the recovery was also below 50% (data not shown).

0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jviromet.2003.11.013
20 J. Méndez et al. / Journal of Virological Methods 117 (2004) 19–25
It is well known that concentration efficiencies obtained
when testing natural samples for viruses are frequently sig-
nificantly lower than those reported in the publications de-
scribing the method as developed with reference laboratory
bacteriophages. Also, Round Robin tests among different
laboratories show significant differences in concentration ef-
ficiencies between the different laboratories (Melnick et al.,
1984; Vilaginés et al., 1997). Many factors other than the
concentration method itself seem to influence the recovery:
the characteristics of the water sample, the amount of sam-
ple, the numbers of bacteriophages spiked and the bacterio-
phages groups themselves. Because of the many sources of
uncertainty it is important to establish “standardised” pro-
cedures for evaluating concentration procedures, based on
both reliability and efficiency. Recently, a standard proce-
dure for validation of methods for concentration of bacte-
riophages from water has been adopted by the International
Standardisation Organisation (Anonymous, 2002). This pro-
cedure does not give specific details of concentration meth-
ods, but provides a general framework for the evaluation
of the suitability of a particular method for a given type
and volume of water. Secondly, it also discusses how to use
this information in establishing the actual values of bacte-
riophages in a given sample taken into account the numbers
recovered after concentration.
We describe modifications introduced to the method first
reported by Sobsey et al. (1990) and then modified by Sinton
et al. (1996). The modifications introduced were examined
for their impact upon the concentration of phages belonging
to the three groups reported above as potential indicators
from 1 l of drinking water. The final modified method was
then validated according to the international validation stan-
dard mentioned above. Finally, the implementation of the
method in routine microbiology laboratories and the repro-
ducibility of the results with sewage derived bacteriophages
in these laboratories were assessed.
2. Materials and methods
2.1. Phage assay
Enumeration of bacteriophages was done by determin-
ing the number of plaque forming units (pfu) by the dou-
ble agar layer technique according to standards ISO10705-1
(Anonymous, 1995), ISO10705-2 (Anonymous, 2000) and
ISO10705-4 (Anonymous, 2002), which describe methods
for the detection and enumeration of F-specific (F-TOTPH)
and F-specific RNA bacteriophages (F-RNAPH), somatic
coliphages (SOMCPH) and phages infecting strains HSP40
(BFBHSP40) and RYC2056 (BFBRYC2056) of B. fragilis,
respectively.
2.2. Bacteriophages
Phages X174, MS2 and B40-8, recommended by the
Standard ISO methods indicated above as reference phages
for somatic coliphages, F-specific RNA bacteriophages
and B. fragilis phages, respectively were used as con-
trols in enumeration tests and for spiking of water in the
method development experiments indicated further. As well,
sewage derived phage stocks for SOMCPH, F-TOTPH,
F-RNAPH, BFRPHHSP40 and BFRPHRYC2056 were
prepared as indicated further for method validation
studies.
2.3. Spiking phage suspensions
Two kinds of phage suspensions were used.
2.3.1. For developing the modified method
Volumes of suspensions of phages X174, MS2 and
B40-8 containing between 1.5 × 104 and 5.0 × 105 pfu per
ml were added to the water sample to be submitted to con-
centration to reach a final concentration of 60–200 plaque
forming units per litre. After vigorous agitation, phages
in the sample were enumerated, either directly or after
dilution.
2.3.2. For validating the modified method
Sewage derived phage stocks were prepared as indicated
in the ISO 10705-3 Standard (Anonymous, 2003). Briefly,
primary or secondary sewage effluent was either centrifuged
for 10 min at 2000 × g or filtered through a 12 ␮m pore
size membrane filter as described in Mendez et al. (2002).
Target bacteriophages were enumerated for initial titre. The
phage suspensions were stored at 4 ◦ C until the enumeration
results were available. If necessary, the suspensions were
diluted to obtain concentrations of 60–200 plaque forming
units per ml of each of the phage groups. Since their numbers
in sewage are different, these means that three sets of phage
spike-stocks were prepared. Glycerol was added to a final
concentration of 10% (v/v). Then, these suspensions were
distributed in 10 ml volumes in plastic containers, and frozen
at −20 or −70 ◦ C.
To ensure that sewage derived phages were distributed at
random within and between the containers, the within and
between containers homogeneity was tested. Two contain-
ers for each phage group suspension (somatic coliphages,
F-specific/F-specific RNA phages and phages infecting B.
fragilis infecting strains HSP40 and RYC2056) were thawed
at room temperature, and from each container two 0.5 ml
aliquots for the target bacteriophages were assayed. The
counts were then analysed for within (T1 ) and between con-
tainers (T2 ) homogeneity.
T1 is a measure for the variation within one container of
spiking material (replicate variation). T2 is a measure for
the variation between different vials of one batch of spiking
material.
J
I

 

(zij − zi+ /J) 2
T1 =
(zi+ /J)
i=1 j=1
 J. Méndez et al. / Journal of Virological Methods 117 (2004) 19–25
where zij is the number of pfu per analytical portion (j) of
vial i, and
J

were assayed with the eluting solution 1 that was confirmed
zi+ = zij
j=1
being the sum of numbers of pfu in all containers counted
(I, that in this case is 2) and J (in this case 2) the number
of analytical portions per container. Degrees of freedom for
T1 are I(J − 1).
J   2.5. Concentration method proposed and validated

(zi+ − z++ /J)
T2 =
z++ /I
j=1
where
I 



z++ = zij
i=1
is the total count of plaques over all vials and duplicates.
Degrees of freedom for T2 are I − 1.
As indicated in the ISO Standard the phage suspensions
were considered as suitable for the validation of the method
if 0.01 < T1 < 5.99 and T2 < 3.84.
2.4. Modifications assayed for the phage concentration
method
The experiments to test the effect of changes of protocol
in concentration efficiency were done spiking bottled wa-
ter samples with suspensions of laboratory reference phages
followed by enumeration of phages before and after con-
centration. The protocol was applied to the assays described
further.
2.4.1. Sample amendment
The effect on virus adsorption of different MgCl2 concen-
trations (0.005, 0.05 and 0.5 M, the last one being the con-
centration proposed by Sinton et al. (1996)) in the sample
was assayed. For these tests a 4.14 M MgCl2 ·6H2 O solution
was prepared and the volumes necessary to reach the above
indicated final concentrations were added to the reference
phage spiked water sample.
2.4.2. Eluting solutions
The following eluting solutions were tested: (1) 3% (w/v)
beef extract, 3% (v/v) Tween 80, 0.5 M NaCl, pH 9.0; (2)
1% (w/v) beef extract, 0.5 M NaCl, pH 9.0; (3) 3% (w/v)
beef extract, 0.5 M NaCl, pH 9.0; (4) 3% (w/v) beef extract,
0.5 M NaCl, 0.4 M urea, pH 9.0; (5) 1% (w/v) beef extract,
0.5 M NaCl, 0.4 M urea, pH 9.0; (6) 1% (w/v) beef extract,
0.5 M NaCl, 0.25 M glycine, pH 9.0; (7) 0.5 M NaCl, 0.25 M
glycine, pH 9.0; (8) 1% (w/v) beef extract, 0.25 M glycine,
pH 9.5.
21
2.4.3. Eluting conditions
The effect on elution efficiency of swirling agitation and
ultrasound was compared as follows. The eluting conditions
be the most efficient as indicated in the results section. The
swirling agitation of the membrane filter submerged in elut-
ing solution was applied as indicated by Sinton et al. (1996).
Ultrasound was applied by submerging the flask or tube con-
taining the membrane filter into an ultrasound cleaning bath
for 4 min.
As indicated above, the method came up as a modification
of a membrane filter phage concentration method previously
described (Sobsey et al., 1990; Sinton et al., 1996) accord-
ing to different changes tested herein. The method is recom-
mended for volumes ranging from 100 ml to 1 l of waters
with turbidity <2.0 NTU. For concentrating bacteriophages
from 1 l of water proceed as follows. First, add MgCl2 to the
water sample to reach a final concentration 0.05 M. Then,
filter the sample through an acetate–nitrate cellulose ester
membrane filter, 0.22 ␮m pore size, 47 mm diameter. Filter
at a flow rate of approximately 1 l in 30 min. Cut the mem-
brane filter in eight fragments and place into a glass flask
containing 5 ml of eluting solution (1% beef extract, 0.5 M
NaCl and 3% Tween 80). Place the flask into an ultrasound
cleaning bath for 4 min. Count the eluted bacteriophages
by the double layer agar method. Count the bacteriophages
retained in the membrane fragments placing the fragments
face down onto a host monolayer.
2.6. Evaluation and validation of the method with sewage
derived phages
This was carried out as indicated in the standard
ISO10705-3 (Anonymous, 2003).
Briefly, five samples of bottled water or tap water
dechlorinated by adding sodium thiosulphate (Anonymous,
1998) were tested. Turbidity was measured after spiking
and ranged from 0.1 to 2.0 nephelometric turbidity units
(NTUs). Since 1 l is the maximum volume proposed for
this method, containers containing 125, 250, 500, 1000 ml
were prepared. One millilitre of sewage derived spiking
material prewarmed at room temperature was added to each
container, and the remainder of the spiking material was
kept on melting ice for enumeration.
2.6.1. Calculation of recovery
Let Nc be the total number of plaques recovered from a
concentrate, and Ns be the number of plaques from 1 ml of
the spiking material. The percentage recovery was calculated
as R(%) = Nc /Ns × 100.
The arithmetic average recovery, the standard deviation
and the confidence interval were calculated for all assayed
volumes. The R(%) plus the confidence intervals were
22 J. Méndez et al. / Journal of Virological Methods 117 (2004) 19–25

plotted against the volume of sample and examined for Table 1

linearity. If confidence intervals of R(%) for the different Comparison of elution efficiencies using different eluents
volumes overlapped, it was considered that there were no Eluent
significant volume effects. 1a 2 3 4 5 6 7 8
The results of all volumes tested were combined and the
arithmetic mean of R(%), the standard deviation and the rela- X174 100b 56.9 60.0 63.1 73.8 79.7c 23.4 73.5
MS2 100 52.0 68.1 63.4 50.7 41.4 2.9 64.4
tive standard deviation (standard deviation/arithmetic mean) B40-8 100 25.2 42.5 42.5 38.1 44.1 4.0 77.3c
were calculated. According to the ISO standard the method
Results are the average of nine independent experiments.
can be considered reliable if the relative standard deviation a Indicated in the Section 2.
is <0.5. b Recovery by eluent 1 has been assigned as 100%. All other recoveries

are expressed as relative (%) to recovery by eluent 1.

2.7. Concentration in different laboratories
Five routine laboratories, which in a previous collabora-
tive study aimed to ensure the correct implementation in the
laboratory of the methods for enumerating bacteriophages
succeeded in the implementation of the methods, partici-
pated in the trial.
Each laboratory prepared its own spiking material from
sewage as indicated above.
They assayed (4/5 assays for each group of bacterio-
phages per laboratory) the concentration of somatic col-
iphages, F-specific bacteriophages and phages infecting B.
fragilis RYC2056 from 1 l of spiked water.
2.8. Statistical tests
c The values were not significantly different from data in the first
column.
3.1.2. Eluting solution
The eluting solution number 1 (3% (w/v) beef ex-
tract, 3% (v/v) Tween 80, 0.5 M NaCl, pH 9.0), which
was established as 100% recovery (Table 1) gave re-
coveries significantly higher (ANOVA, P < 0.05) than
any other of the eluting solutions tested for the three
bacteriophages, with the exception of solution number
6 for phage X174 and solution number 8 for phage
B40-8. Consequently it was decided to elute the ad-
sorbed phages with the following solution: 3% beef ex-
tract 3% Tween 80, 0.5 M NaCl, pH 9.0 in the modified
method.

Confidence intervals based

√ on Student’s
√ t-tests were
calculated as {X̄ − t × s/ n; X̄ + t × s/ n} where X̄ is
the arithmetic average recovery; s the standard deviation,
n the numbers of R(%) values and t values of a Student’s
t-distribution (Anonymous, 1999).
Statistical tests (ANOVA, Student’s t-test, Multiple
range test analysis using least significant differences be-
tween means using Student’s t-test) were performed using
the Statistical Package for Social Sciences (Anonymous,
3.1.3. Eluting conditions
Ultrasound gave significantly higher elutions (ANOVA,
P < 0.05), but for phage X174, than swirling elution
and vigorous shaking (Fig. 1). Consequently it was de-
cided to include elution by ultrasound in the final modified
method.
100

1999). Differences were considered significant when 90

P < 0.05.
80

70
3. Results 60
% Recovery

50
3.1. Modifications assayed for the phage concentration
method 40

30
3.1.1. Amendment of the sample
The three reference bacteriophages studied (X174, MS2 20

and B40-8) adsorbed over 93% in the presence of 0.5 and 10

0.05 M MgCl2 . No significant differences (ANOVA, P > 0
0.05) were observed between the numbers of phages ad- N= 6 6 17 18 6 8

ØX174 (SE) ØX174 MS2 (SE) MS2 B40-8 (SE) B40-8

sorbed in the presence of the two concentrations of MgCl2 .
In contrast, the lower concentration, 0.005 M, of MgCl2
tested showed significantly (ANOVA, P < 0.05) lower ad-
sorption of bacteriophages ÔX174 and MS2. Consequently
it was decided to amend the samples with 0.05 M MgCl2
before the concentration step.
Phage
Fig. 1. Comparison of swirling elution method (SE) and ultrasound for
the elution of adsorbed laboratory reference phages. (䊐) Indicates the
arithmetic mean and bars indicate the 95% confidence intervals of six
independent experiments.
 J. Méndez et al. / Journal of Virological Methods 117 (2004) 19–25
3.1.4. Bacteriophages remaining in the filters
Even applying the best eluting solution and the best elut-
ing conditions, non-negligible numbers of phages, ranging
from 1 to 28% depending on the experiments and phages,
remained in the membrane filter. Consequently, it was rec-
ommended in the proposed method to include the count of
phages retained in the filter as part of the total number of
phages recovered as was recommended in the original meth-
ods (Sobsey et al., 1990; Sinton et al., 1996).
As a consequence of the results reported in this section,
the final modified method was established as indicated in
the Section 2.
3.2. Evaluation and validation of the method
The recovery efficiency of the method was evaluated for
natural populations of phage, using phages partially purified
from sewage as indicated in the material and methods sec-
tion. Results expressed as the arithmetic mean of the recover-
ies for all volumes tested, since as indicated below there was
no volume effect, are shown in Fig. 2. Recovery of somatic
coliphages was significantly higher (multiple range test anal-
ysis using least significant differences between means using
Student’s t-test, P < 0.05) than the recoveries of F-specific,
F-RNA bacteriophages and bacteriophages infecting B.
fragilis. In contrast, the recoveries of F-specific bacterio-
phages, F-specific RNA phages and phages infecting B.
fragilis were similar (multiple range test analysis using least
significant differences between means using Student’s t-test,
P > 0.05).
100
90
80
70
60
% Recovery
50
40
30
20
10
0
N= 20 20 20 20 20
SOMCPH FTOTPH FRNAPH BFRPHHSP40 BFRPHRYC2056
Phage Group
Fig. 2. Average recovery of different groups of sewage derived phage from
1 l of water. (䊐) Indicates the arithmetic mean and bars indicate the 95%
confidence intervals. The arithmetic means correspond to the recoveries
obtained in five tests performed at each of the four volumes. (SOMCPH),
somatic coliphages; (F-TOTPH) F-specific bacteriophages; (F-RNAPH),
F-specific RNA bacteriophages; (BFRPHHSP40) bacteriophages infecting
B. fragilis strain HSP40 and (BFRPHRYC2056) bacteriophages infecting
of B. fragilis strain RYC2056.
23
The percentages of phages counted by platting the mem-
brane filters varied from 13.5 to 21.6% for somatic col-
iphages, 1.8–9.8% for F-specific bacteriophages, 0.8–8.8%
for F-specific RNA phages, 11.6–19.6 for phages infecting
B. fragilis HSP40 and from 19 to 26.9% for phages infecting
strain RYC2056 of B. fragilis.
The ISO validation procedure was applied to all phages
reported in Fig. 1 and results indicated neither volume ef-
fect, nor unacceptable relative standard deviation >0.5 for the
five groups of phages studied. The relative standard devia-
tions for the concentration efficiencies of the different phages
were: 0.19 for somatic coliphages, 0.23 for F-specific bacte-
riophages, 0.24 for F-specific RNA bacteriophages, 0.34 for
bacteriophages infecting B. fragilis HSP40 and 0.28 for bac-
teriophages infecting B. fragilis RYC2056. Therefore, it was
concluded that the method is reliable for volumes ranging
from 100 ml to 1 l for somatic coliphages, F-specific RNA
bacteriophages and bacteriophages infecting B. fragilis.
For brevity, only the results corresponding to the
F-specific RNA bacteriophage are reported in detail below.
The sewage derived spiking material showed an excellent
homogeneity both within and between vial since values of
T1 and T2 were 0.034 and 0.41 well below 5.99 and 3.84
indicated in the ISO validation standard.
Fig. 3 shows the plotted arithmetic average of the recover-
ies (R(%)) plus the confidence intervals obtained by concen-
trating phages from 125, 250, 500 and 1000 ml. No signifi-
cant differences (ANOVA, P > 0.05) were detected. There
was not volume effect in the percentage of phages detected
by platting the membrane filter.
The calculation of the combined (all volumes) recoveries
(R(%)) gave an arithmetic mean average of value of 63.7 with
a standard deviation of 14.8. The relative standard deviation
100
90
80
70
60
% Recovery
50
40
30
20
10
0
N= 5 5 5 5
125 250 ml 500 ml 1000 ml
Volume
Fig. 3. Average recovery of sewage derived F-specific RNA phages from
four different volumes. (䊐) Indicates the arithmetic mean and bars indicate
the 95% confidence intervals. Each value corresponds to five independent
experiments.
24 J. Méndez et al. / Journal of Virological Methods 117 (2004) 19–25
100
90
80
70
60
% Recovery
50
40
30
20
10
0 The results of the standardised validation procedure in-
N= 20 21 20 22 20
SOMCPHc SOMCPH FRNAPHc FRNAPH BFRPHRYC2056c
Phage Group
Fig. 4. Average recoveries obtained by the laboratories participating in
the interlaboratory exercise utilizing sewage derived phage. (䊐) Indicates
the arithmetic mean and bars indicate the 95% confidence intervals.
For each phage group the first value (c) is the one obtained by the
central laboratory and the second, the average value obtained by the all
participating laboratories. (SOMCPH) somatic coliphages; (F-RNAPH)
F-specific RNA bacteriophages and (BFRPHRYC2056) bacteriophages
infecting of B. fragilis strain RYC2056.
gave a value of 0.23 well below 0.5 as recommended in the
ISO standard.
3.3. Results of the collaborative study
Results expressed as the arithmetic mean of the
recoveries ± the confidence intervals obtained by all the
participating laboratories are shown in Fig. 4, with the
central laboratory recoveries isolated for comparison In the
figure are also reported the values obtained. Though not
significantly different (Student’s t-test for of each group of
bacteriophages, P > 0.05), the average arithmetic mean re-
coveries of somatic coliphages and F-specific phages were
lower than those of the validation study, whereas recoveries
of phages infecting Bacteroides were identical.
As well, the relative standard deviations were 0.27 for
somatic coliphages, 0.40 for F-specific RNA bacteriophages
and 0.36 for bacteriophages infecting B. fragilis RYC2056,
all well below 0.5.
4. Discussion
The method reported above introduces some modification
to the methods based in adsorption-elution to acetate–nitrate
cellulose ester membrane filters described first by Sobsey
et al. (1990) and modified further by Sinton et al. (1996). The
modified method demonstrated good recoveries of the three
groups of phages suggested as surrogate indicators from 1 l
of drinking water. Somatic coliphages were recovered more
efficiently that the others; however, average recoveries of all
phage groups were over 55%. Average recovery of somatic
coliphages was slightly over 80%, which is clearly supe-
rior to the original method method/s for 1 l of water which
is slightly below 50% (Hsu et al., 1998). The improvement
is due to the combined effect of the new eluent and the
ultrasound treatment. Results referring to elution of phage
X174 and the overall results for somatic coliphages may
seem contradictory, but it should be considered that the so-
matic coliphages are a very heterogeneous group (Ackerman
and Nguyen, 1983; Pedroso and Martins, 1995; Muniesa
et al., 1999) and that in municipal sewage the phages of the
family of X174 are not the most abundant (Muniesa, et al.,
1999).
32
BFRPHRYC2056 dicate that the method is reproducible and that it can be
easily implemented in routine laboratories with highly re-
producible results. The validation method used is simple
and allows one to work with naturally occurring phages at
concentrations approaching those found in nature. Some
experiments performed with un-homogeneous phage sus-
pensions gave very variable recoveries (data not shown)
and consequently indicated that when working with low
numbers of phage, the homogeneity of spiking material is
essential for calculating recovery efficiencies of concen-
tration methods. This may explain some extreme results
reported in Round Robin exercises when concentrations of
low numbers of phages were tested (Melnick et al., 1984).
In our opinion it will be worthwhile to assay the suit-
ability of similar approaches for validating concentration
methods for animal viruses and oocysts of protozoa as
Cryptosporidium.
Acknowledgements
This study was partially supported by REN2002-04035-
C03-03/HID from CICYT, Ministerio de Ciencia y Tec-
nologı́a, Spain; and by grant 1999SGR00023 and CeRBa
(Biotechnology Center Reference) from the Generalitat de
Catalunya.
References
Ackerman, H.W., Nguyen, T.M., 1983. Sewage coliphages studied by
electronic microscopy. Appl. Environ. Microbiol. 45, 1049–1059.
Anonymous, 1995. ISO 10705-1: Water Quality. Detection and Enu-
meration of Bacteriophages. Part 1. Enumeration of F-specific RNA
Bacteriophages. International Standardisation Organisation. Geneva,
Switzerland.
Anonymous, 1998. Standard methods for the examination of water and
wastewater, 20th. ed. American Public Health Association, Washing-
ton, D.C.
Anonymous, 1999. Statistics program. SPSS for Windows, Version 10.0.6,
1989–1999. Statistical Package for Social Sciences Inc. Chicago, IL.
Anonymous, 2000. ISO 10705-2: Water Quality. Detection and Enumer-
ation of Bacteriophages. Part 2. Enumeration of Somatic Coliphages.
International Standardisation Organisation, Geneva, Switzerland.
 J. Méndez et al. / Journal of Virological Methods 117 (2004) 19–25
Anonymous, 2002. ISO 10705-3/FD: Water Quality. Detection and Enu-
meration of Bacteriophages. Part 3. Validation of Methods for Con-
centration of Bacteriophages from Water. International Standardisation
Organisation. Geneva, Switzerland.
Anonymous, 2003. ISO 10705-4: Water Quality. Detection and Enumera-
tion of Bacteriophages. Part 4. Enumeration of Bacteriophages Infect-
ing Bacteroides fragilis. International Standardisation Organisation.
Geneva, Switzerland.
Borrego, J.J., Cornax, R., Preston, D.R., Farrah, S.R., McElhaney, B., Bit-
ton, G., 1991. Development and application on new positively charged
filters for recovery of bacteriophages from water. Appl. Environ. Mi-
crobiol. 57, 1218–1222.
Grabow, W.O.K., 2001. Bacteriophages: update on application as model
for viruses in water. Water SA 27, 251–268.
Havelaar, A.H., Furuse, K., Hogeboom, E.H., 1986. Bacteriophages and
indicators bacteria in human and animal faeces. J. Appl. Bacteriol.
60, 255–262.
Hsu, F.C., Handzel, T.R., Lovelance, G., Stewart, J.R., Sobsey, M.D.,
1998. Improved methods to detect low levels of coliphages in wa-
ter by enrichment presence-absence and membrane filter methods.
In: Proceedings of the 1998 Water Quality Technology Conference,
American Water Works Association. Denver, CO.
IAWPRC Study Group on Health Related Water Microbiology, 1991.
Bacteriophages as model viruses in water quality control. Water Res.
25, 529–545.
Jofre, J., 2002. Bacteriophages as indicators. In: Bitton, G. (Eds.), Encyclo-
pedia of Environmental Microbiology. Willey, New York, pp. 354–363.
Kott, Y., Ben Ari, H., 1968. Bacteriophages as marine pollution indicators.
Rev. Int. Océanogr. Méd. ix, 207–217.
Logan, K.B., Rees, G.E., Primrose, S.B., 1980. Rapid filtration of bacte-
riophages from large volumes of freshwater: evaluation of positively
charged % microporous filters. J. Virol. Methods 1, 87–97.
Lucena, F., Muniesa, M., Araujo, R., Jofre, J., 1995. Simple concentration
methods for bacteriophages of Bacteroides fragilis in drinking water.
J. Virol. Methods 45, 121–130.
25
Melnick, J.L., Safferman, R., Rao, V.C., Goyal, S., Berg, G., Dahling,
D.R., Wright, B.A., Akin, E., Stetler, R., Sorber, C., Moore, B.,
Sobsey, M.D., Moore, R., Lewis, A.L., Wellings, F.M., 1984. Round
Robin investigation of methods for the recovery of poliovirus from
drinking water. Appl. Environ. Microbiol. 47, 144–150.
Mendez, J., Jofre, J., Lucena, F., Contreras, N., Mooijman, K., Araujo, R.,
2002. Conservation of phage reference materials and water simples
containing bacteriophages of enteric bacteria. J. Virol. Methods 106,
215–224.
Muniesa, M., Lucena, F., Jofre, J., 1999. Study of the potential relationship
between the morphology of infectious somatic coliphages and their
presence in the environment. Lett. Appl. Microbiol. 87, 402–409.
Pedroso, D.M.M., Martins, M.T., 1995. Ultra-morphology of coliphages
isolated from water. Water Res. 29, 1190–1202.
Schulze, E., Lenk, J., 1983. Concentration of coliphages from drinking
water by Mg(OH)2 flocculation. Naturwissenschaften 7, S: 612.
Shields, P.A., Farrah, S., 1986. Concentration of viruses in beef extract
by flocculation with ammonium sulphate. Appl. Environ. Microbiol.
51, 211–213.
Sinton, L.W., Finlay, R.K., Reid, J.A., 1996. A simple membrane
filtration-elution method for the enumeration of F-RNA, F-DNA and
somatic coliphages in 100 ml water samples. J. Microbiol. Methods
25, 257–269.
Sobsey, M.D., Schwab, K.J., Handzel, T.R., 1990. A simple membrane
filter method to concentrate and enumerate male-specific RNA col-
iphages. J. Am. Water Works Assoc. 82, 52–59.
Tartera, C., Jofre, J., 1987. Bacteriophages active against Bacteroides
fragilis in sewage polluted waters. Appl. Environ. Microbiol. 53,
1632–1637.
Vilaginés, P., Sarrette, B., Champsaur, H., Hughes, B., Dubrou, S., Joret,
J.C., Laveran, H., Lesne, J., Paquin, J.L., Delattre, J.M., Oger, C.,
Alame, J., Grateloup, I., Perrollet, H., Serceau, R., Sinegre, F., Vi-
laginés, R., 1997. Round Robin investigation of glass wool method
for poliovirus recovery from drinking water and sea water. Water Sci.
Technol. 35, 445–449.