Conservation of phage reference materials and water samplescontaining bacteriophages of enteric bacteria

J. Mendez a, J. Jofre a, F. Lucena a, N. Contreras a, K. Mooijman b,R. Araujo a,*

a Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, Barcelona 08028, Spainb National Institute of Public Health and the Environment Bilthoven, Bilthoven, The Netherlands

Received 25 April 2002; received in revised form 26 July 2002; accepted 29 July 2002

Abstract

The survival was determined in different conservation conditions of: somatic coliphages, F-specific RNA

bacteriophages and phages infecting Bacteroides fragilis proposed as model micro-organisms for water quality control.

Titres of phages of all groups either in pure culture phage suspensions or in naturally occurring phage suspensions were

stable at (�/709/10) 8C and at (�/209/5) 8C when protected with glycerol. Moreover, phage analysis of stored

suspensions demonstrated that their numbers were homogeneous, both between vials and within vials, and

consequently they can be used as reference materials. Furthermore, changes in the storage temperature of the vials

cause unpredictable changes in the numbers of bacteriophages. Consequently, phage reference materials and samples

containing a quantitative number of phages must be maintained and dispatched at a constant temperature.

Consequently, the results indicate that bacteriophages should be packed in dry ice during transport and storage.

Finally, the number of phages in water samples stored at (59/3) 8C in the dark does not decrease significantly during the

first 72 h of storage. In addition, phage concentrates from natural samples obtained by adsorption�/elution to cellulose

nitrate filters and mixed with 10% glycerol were stable at least for 2 months at (�/709/10) 8C and at (�/209/5) 8C.

# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Reference materials; Sample conservation; Temperature; Somatic coliphages; F-specific RNA bacteriophages; Phages of

Bacteroides fragilis

1. Introduction

Somatic coliphages (Kott et al., 1974), F-specific

RNA bacteriophages (Havelaar et al., 1984) and

bacteriophages infecting Bacteroides fragilis (Jofre

et al., 1986) have been suggested as model micro-

organisms for water quality assessment (Kott et

al., 1974; Havelaar et al., 1984; Jofre et al., 1986;

IAWPRC, 1991). Bacteriophages have several

advantages as model organisms. Firstly, the meth-

ods are cheap, simple to perform and rapid. For

example, results for somatic coliphages are avail-

able after 4�/6 h. Secondly, phenomena like

‘‘stress’’, ‘‘injury’’, or ‘‘reactivation’’, which lead

* Corresponding author. Tel.: �/34-3-402-1491; fax: �/34-3-

411-0592

E-mail address: araujo@porthos.bio.ub.es (R. Araujo).

Journal of Virological Methods 106 (2002) 215�/224

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0166-0934/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 6 - 0 9 3 4 ( 0 2 ) 0 0 1 6 3 - 5

frequently to misinterpretation of environmentaldata on bacterial indicators, are greatly reduced.

And thirdly, sample analysis may be delayed after

collection, as the reduction in the number of

bacteriophages is presumed to be low when the

sample is stored at (59/3) 8C according to data on

the persistence of bacteriophages in the environ-

ment (Lasobras et al., 1999; Duran et al., 2002).

Standard methods for the detection and enu-meration of bacteriophages (Anonymous, 1995,

2000, 2001a,b) are now available, and protocols

for quality assurance have been developed. Some

of the tools used for quality assurance are refer-

ence materials (RMs). They were first implemen-

ted in chemistry, where they are used widely, but in

microbiology the process of implementation is

slower and more complex, especially because ofthe need to conserve the microorganisms alive and

capable of multiplying without long latency peri-

ods, when tested in the quality assurance working

schemes. So, the media, cryoprotectors and tem-

perature of conservation are decisive factors in the

development of robust reference materials. In

recent years bacterial preparations have been

introduced as reference materials (In’t Veld andNotermans, 1992; Mooijman et al., 1992). But

there are very few studies about bacteriophages

and viruses prepared as reference materials. In the

literature there is only one example, the bacter-

iophage MS2 suspension developed by RIVM

(Schijven et al., 1995; Mooijman et al., 1999).

The generalised use of reference materials requires

transport. Consequently, studies on the conditionsfor conservation and transport of reference sus-

pensions of all groups of phages are needed.

Knowing for how long the samples can be

conserved before testing for bacteriophages with-

out losing phage titre has a dual interest: (i) for

conserving natural samples; and (ii) for conserving

phages concentrated from natural samples.

Consequently, the conditions for the conserva-tion of bacteriophages suspended in aqueous

solutions need to be determined. To this end, the

survival in different conservation conditions of

somatic coliphages, F-specific RNA bacterio-

phages and phages infecting B. fragilis proposed

as model micro-organisms for water quality con-

trol was determined.

2. Materials and methods

2.1. Bacteriophages and bacterial strains

Bacteriophages B40-8 (ATCC 51477-B1),

FX174 (ATCC 13706-B1) and MS2 (NCTC

12487, ATTC 15597 B1) were used as reference

pure cultures of bacteriophages infecting B. fragi-

lis , somatic coliphages and F-specific RNA bac-

teriophages, respectively. B. fragilis HSP40

(ATCC 51477), Escherichia coli strain WG5

(ATTC 700078) and Salmonella typhimurium

strain WG49 (NCTC 12484) were used as host

strains for those phages. These three bacterial

strains and B. fragilis RYC2056 (ATTC 700786)

were used as bacterial hosts for naturally occurringbacteriophages.

2.2. Phage assays

Phages were enumerated using the double agar

layer plaque assay (Adams, 1959) following the

standardised methods described below. Phagesinfecting Bacteroides were enumerated with B.

fragilis strains onto BPRM-agar and incubated

in anaerobic jars according to ISO 10705-4 (Tar-

tera et al., 1992; Anonymous, 2001b; Araujo et al.,

2001). Somatic coliphages were enumerated with

E. coli WG5 onto Modified Scholtens Agar

according ISO 10705-2 (Havelaar and Hogeboom,

1983; Anonymous, 2000) and F-specific RNAbacteriophages were enumerated with S. typhimur-

ium WG49 onto TYGB-agar according to ISO

10705-1 (Havelaar et al., 1984; Anonymous, 1995).

2.3. Reference materials

Two types of reference materials were prepared:

(i) suspensions of pure phage cultures, and (ii)suspensions of naturally occurring phages partially

purified from sewage. In both cases glycerol was

added to a final concentration of 5 or 10% (v/v), as

indicated, and the suspensions were distributed

into vials of 1.5 or 2.5 ml and stored at (�/709/

10) 8C.

J. Mendez et al. / Journal of Virological Methods 106 (2002) 215�/224216

2.3.1. Pure culture phage suspension

Each phage, B40-8, MS2 and FX174, was

cultured as indicated in the relevant ISO-method

(ISO10705-4, -1, -2, respectively), and diluted with

peptone saline solution (ISO 10705-1) to a final

phage concentration between 30 and 100 pfu/ml.

2.3.2. Suspensions of naturally occurring phages

Samples from influent raw sewage from awastewater treatment plant in the city of Barce-

lona were filtered through a membrane of 12 mm

pore size or centrifuged for 10 min at 2000�/g and

the filtrate or the supernatant used as the source of

suspensions of naturally occurring phages. The

three types of phages, somatic coliphages, F-

specific RNA bacteriophages and phages infecting

strains HSP40 and RYC2056 of B. fragilis, werequantified and when necessary diluted to a final

concentration ranging from 30 to 100 pfu/ml.

2.4. Storage at changing temperatures

Some experiments were performed to evaluate

the effect of changes of temperature on the

stability of bacteriophages. For it, 20 vials ofeach batch of phage suspension stored at (�/709/

10) 8C were placed at (59/3) 8C for 24 h. Then five

vials were enumerated. The remaining 15 vials

were stored at (�/209/5) 8C for another 24 h and

five vials were analysed. The remaining ten were

stored at (59/3) 8C for 24 h and five vials were

analysed. Finally the last five vials were stored at

(�/209/5) 8C and were enumerated after 24 h.

2.5. Conservation of water samples

Three different water types were used: raw

sewage water from a wastewater treatment plant

of Barcelona, fresh water from the Llobregat river

and seawater from Badalona on the Mediterra-

nean coast. The samples were transported to the

laboratory in a cool box at (59/3) 8C and within 3h stored in a refrigerator at (59/3) 8C in the dark.

River water and sea water were then spiked with

an aliquot of filtered sewage (Nylon Membrane of

11 mm of pore), at a dilution 1:25 v/v, and mixed

by magnetic stirring for 2�/3 h at (59/3) 8C.

Afterwards the three samples were dispensed in

duplicate in volumes of 150 ml into screw cappedbottles that were stored at (59/3) 8C in the dark.

The bacteriophages in the different samples were

counted, in triplicate, in aliquots removed from the

bottles at the times indicated in Section 3.

2.6. Phage concentration by adsorption�/elution

The phages were concentrated by adsorption tonitrate�/acetate membrane filters followed by elu-

tion according to the method first described by

Sobsey et al. (1990). Briefly, MgCl2 was added to

the water sample to a 0.05 M final concentration.

Next it was filtered through acetate�/nitrate cellu-

lose ester membrane of 0.22 mm pore size and 47

mm diameter. The membrane was cut into frag-

ments, placed in a glass flask containing 5 ml ofeluting solution (3% of Tween 80 (v/v), 1% beef

extract (w/v) and 0.5 M of NaCl) at pH 9. The

flask was placed in an ultrasonic cleaning bath for

4 min. Finally, the suspension was adjusted to pH

7, and the content was mixed with glycerol up to

10%, frozen and stored at (�/209/5) 8C and (�/

709/10) 8C.

2.7. Data computing and statistical analysis

To make the descriptive statistics, U-Mann

Whitney test, which was performed in order to

determine the significance of the differences be-

tween the series of results, the Statistical Package

for Social Science (Anonymous, 1999) was used.

Differences were considered significant at P B/

0.05.Some data were plotted as boxes and whiskers.

This plotting provides a summary statistics using

five numbers: the minimum, the maximum, the

median, the 25th percentile and the 75th percentile.

The homogeneity of the phage suspensions was

calculated according to T1 (a measure for homo-

geneity within vials) and T2 (a measure for

homogeneity between vials) tests according toHeisterkamp et al. (1993). Suspension with T1B/

5.99 and T2B/3.84 were considered to fulfil the

homogeneity criteria.

Long time stability of the phage suspensions or

reference materials was determined by the pre-

paration of control charts according to van

J. Mendez et al. / Journal of Virological Methods 106 (2002) 215�/224 217

Dommelen (1995). For this purpose, 20 vials ofeach batch were enumerated on different days.

From these results the mean (x) and the standard

deviation (s) were calculated. Next, the warning

limits (x9/2s) and the action limits (x9/3s) were

calculated. The control chart was used for quality

control and every time a new experiment was

performed one vial of the batch was analysed and

the result was indicated in the chart. Results wereconsidered out of control in the case of a single

violation of the action limit (x9/3s ) and two out of

three observations in a row exceeded the same

warning limit (x9/2s ).

Inactivation values were calculated as the log10

of Nt /N0, with N0 being the initial value of the

phage (pfu/ml) and Nt the values at the indicated

time.

3. Results

3.1. Conservation of pure culture phage suspensions

Suspensions of bacteriophages B40-8, FX174

and MS2 were prepared to test their capacity to be

conserved at a given temperature without losingtitre. The stability during 30 days of the number of

infectious bacteriophages in the suspension kept at

different temperatures was evaluated. For it, each

suspension of the pure phage cultures of FX174,

MS2 and B40-8, was distributed into vials with 5%

of glycerol and stored at (�/709/10) 8C and at (�/

209/5) 8C. Then, aliquots of the suspensions were

quantified at different time intervals up to 30 days.The results are shown in Fig. 1. During the total

assay time the phage numbers of the vials stored at

(�/709/10) 8C did not decrease significantly (U

Mann Whitney test, P B/0.05) for any of the three

phages. However, when the vials were stored at (�/

209/5) 8C, B40-8 showed a decrease in the mean

number of PFU per ml, mainly in the first 7 days

of storage. This drop in numbers was prevented infurther experiments by increasing the concentra-

tion of glycerol to 10% (v/v) (data not shown).

In order to determine stability for longer periods

of time, new batches of vials were prepared with

each pure culture phage suspension mixed with

glycerol until a final concentration of 5% (v/v) and

stored at (�/709/10) 8C. Firstly it was determinedthat the variation in numbers of pfu per ml

between vials as well as within vials fulfilled the

homogeneity criteria indicated above (data not

shown). Then, control charts were prepared with

each phage suspension and the mean, the warning

limits (x9/2s ) and the actions limits determined

(Fig. 2). Stored vials were then analysed at regular

intervals for almost 1 year. The results areindicated in the control charts and values were

always within the limits of the chart (Fig. 2). These

data showed the stability of the phage batches for

almost 1 year in these conditions.

3.2. Conservation of suspensions of naturally

occurring phages

The stability at �/70 8C and at �/20 8C of the

naturally occurring phages infecting B. fragilis ,somatic coliphages and F-specific RNA bacterio-

phages phages was tested. For it, filtered samples

of sewage prepared as indicated in Section 2 were

used. The samples were cryoprotected with 10%

glycerol, dispensed into vials and stored at (�/709/

10) 8C and at (�/209/5) 8C. The vials were ana-

Fig. 1. Results of the quantification of vials of pure culture

phage suspensions: FX174 (somatic coliphage), MS2 (F-specific

RNA bacteriophage) and B40-8 (phage of B. fragilis ) stored

during 1 month with 5% of glycerol (a) at (�/709/10) 8C and (b)

at (�/209/5) 8C. (�/"�/, �/174; �/j�/, MS2; �/'�/, B40-8).

J. Mendez et al. / Journal of Virological Methods 106 (2002) 215�/224218

Fig. 2. Control charts of phage suspensions stored at (�/709/10) 8C with 5% of glycerol: (a) FX174 (somatic coliphage), (b) MS2

(F-specific RNA bacteriophage) and (c) B40-8 (phage of B. fragilis ).

J. Mendez et al. / Journal of Virological Methods 106 (2002) 215�/224 219

lysed periodically for quantitation of somatic

coliphages, F-specific RNA bacteriophages and

phages of B. fragilis (Fig. 3). The numbers of the

three types of phages of suspensions of naturally

occurring phages were stable for more than 1

month at (�/709/10) 8C and at (�/209/5) 8C.

In order to determine stability for longer periods

of time, vials containing suspensions of naturally

occurring bacteriophages of the three groups with

10% glycerol were tested for homogeneity after 15

days of storage at (�/709/10) 8C and at (�/209/

5) 8C. Suspensions prepared by centrifugation for

10 min at 2000�/g were more homogeneous

regarding the fulfilment of the homogeneity cri-

teria indicated in Section 2 than those filtered

through a membrane of 12 mm pore size. Homo-

geneity was maintained at both temperatures, both

within and between vials (data not shown). Then,

Fig. 3. Results of the quantification during 45 days of vials containing suspensions of naturally occurring phages: somatic coliphages,

F-specific RNA bacteriophage and phages of B. fragilis . (a) at (�/709/10) 8C and (b) at (�/209/5) 8C. (�/"�/, SOM; �/j�/, F-RNA;

�/'�/, BfHSP40).

J. Mendez et al. / Journal of Virological Methods 106 (2002) 215�/224220

control charts were performed and during 1 yearthe phages of the three groups were quantified at 3

weeks intervals. The numbers obtained were al-

ways within the control limits of the control chart

showing that the three types of phages were stable

during at least 1 year at both temperatures (data

not shown) similar to the pure culture suspensions.

3.3. Effect of changing temperatures

Fig. 4 shows the results of quantifying somaticcoliphages from suspensions of naturally occurring

phages stored at (59/3), (�/209/5), (59/3), and

finally to (�/209/5) 8C as indicated in Section 2.

Changes in storage temperature resulted in

changes in the numbers of bacteriophages that

do not follow any trend. Changes were also

observed in the numbers of the other two groups

of bacteriophages, though the patterns of thechanges in numbers of bacteriophages were differ-

ent from group to group and as in the case of

somatic coliphages no trend could be observed

(data not shown). The trends in the changes of

phages numbers were always unpredictable.

3.4. Conservation of water samples

Numbers of bacteriophages in sewage, and river

water and seawater spiked with filtered sewagewere determined after 4 h, 24 h, 48 h, 72 h and 7

days storage at (59/3) 8C and the log10 reductions

(log10 of Nt /N0) plotted in Fig. 5. These data show

that the numbers of bacteriophages of the three

groups remained stable for 3 days at 5 8C, since

numbers did not change significantly (U MannWhitney test, P �/0.05) whereas a certain decrease

in numbers were observed after 7 days of storage

at (59/3) 8C.

3.5. Conservation of concentrated samples

There were no significant (U Mann Whitney

test, P �/0.05) changes in the numbers of any ofthe three groups of bacteriophages obtained by

concentration of natural water samples after 2

months of storage at �/20 8C or at �/70 8C (Fig.

6).

4. Discussion

Available microbiological reference materialsare prepared with pure cultures of microorganisms

(In’t Veld and Notermans, 1992; Mooijman et al.,

1992). Phages B40-8, FX174 and MS2, which are

the reference phages in the standardised methods

for the detection of, respectively, B. fragilis phages

(Anonymous, 2001b), somatic coliphages (Anon-

ymous, 2000, 2001a) and F-specific RNA phages

(Anonymous, 1995), were chosen to preparereference materials with pure cultures of bacter-

iophages. Firstly, 5% glycerol was chosen as the

cryoprotector to conserve the bacteriophages at �/

70 8C because these conditions are optimal for

MS2 conservation (Schijven et al., 1995). Our

results also showed that these conditions were

suitable for B40-8 and FX174. However, we tested

conservation at �/20 8C, since many routinelaboratories do not have �/70 8C freezers. At this

temperature, 5% glycerol was not suitable for B40-

8 conservation, whereas 10% was.

Phages detected by the standard methods may

be diverse. Thus, somatic coliphages constitute a

heterogeneous group of DNA phages which in-

cludes phages from the following families: Myo-

viridae, Siphoviridae, Podoviridae andMicroviridae (Muniesa et al., 1999). F-specific

RNA bacteriophages belong to the Leviviridae,

but there are several subgroups (Furuse et al.,

1979). The most abundant bacteriophages infect-

ing B. fragilis belong to the Siphoviridae (Laso-

bras et al., 1997). This diversity when counting

Fig. 4. Box plots of the phage numbers of somatic coliphages

from samples naturally polluted stored at different tempera-

tures successively.

J. Mendez et al. / Journal of Virological Methods 106 (2002) 215�/224 221

Fig. 5. Bacteriophages inactivation of water samples from different origins: sewage, seawater and river water kept in the dark at

(59/3) 8C. (�/"�/, SOM; �/j�/, F-RNA; �/'�/, Bact. fragilis phage detected by strain RCY2056).

J. Mendez et al. / Journal of Virological Methods 106 (2002) 215�/224222

phages in natural samples by the double agar layer

method gives a variety of plaques that differ in

morphology and size. We therefore sought refer-

ence material containing a variety of phages.

According to available data (Lasobras et al.,

1997; Muniesa et al., 1999), raw sewage is a good

source of phages to prepare reference material

with natural occurring bacteriophages, because the

variety and the numbers are high enough. Because

of the results with B40-8 we decided to conserve

these suspensions of naturally occurring bacterio-

phages with 10% glycerol. Results showed that it is

possible to preserve naturally occurring phages

with 10% glycerol at �/20 and �/70 8C for up to 1

year. An additional result of these experiments is

that centrifuging sewage at 2000�/g for 10 min

provides a suspension of naturally occurring

bacteriophages that, once distributed in vials,

fulfils the homogeneity criteria required for the

RM. It is concluded that it is possible to prepare

stable reference material of both pure cultures and

naturally occurring bacteriophages using glycerol,

at 5 or 10% depending on the material, both at �/

20 and �/70 8C. According to the results obtained

here it seems that 10% will cover all phages and

temperatures.

The extended use of reference materials requires

their transport, which may entail temperature

changes, even when precautions are taken. Results

presented herein indicated that changes in tem-

perature produced unpredictable changes inphages numbers. The reasons are not evident,

but they may include aggregation, desegregation

and inactivation. However, from the practical

point of view, changes in temperature seem to

produce changes in the numbers. To avoid this,

and taking into consideration that at �/20 8C the

reference materials are stable, we advise dispatch-

ers to transport reference materials or naturalsamples with glycerol and packed in dry ice.

Finally, the results described above show that

natural samples can be kept in a 5 8C refrigerator

for at least 3 days before testing without any

significant loss in the number of phages detected.

It is also possible to keep concentrated bacterio-

phages frozen in the presence of glycerol for at

least 50 days without a significant loss in thenumber of infectious phages. No doubt these are

important advantages of bacteriophages if they are

to be used as model microorganisms for water

quality assessment.

Acknowledgements

This work was supported by European Com-

munity Contract No. SMT4-CT95-1603 (DG 12-

RSMT).

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