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Round robin investigation of glass wool method for poliovirus recovery from drinking water and sea water
e:>
Pergamon
Wal. Sci. Q
PH: S0273-1223(97)OO301-6
r.ch. Vol. 3S. No. 11-12, pp. 44S-449. 1997. 1997 IAWQ. Published by Elscvier Science Ltd Printed ID Greal Britain. 027J-1223197 S17'00 + 0-00
ROUND ROBIN INVESTIGATION OF GLASS WOOL METHOD FOR POLIOVIRUS RECOVERY FROM DRINKING WATER AND SEA WATER Ph. Vilagines l , B. Sarrette2 , H. Champsaur2 , B. Hugues 2, S. Dubrou 3, J.-C. Joret4, H. Laveran 5, J. Lesne6, J. L. Paquin7, J. M. Delattre8, C. Oger8, J. Alame9, I. GratelouplO, H. Perrollet ll , R. Serceau I2 , F. Sinegre l3 and R. Vilagines l J Centre de Recherche et de Controle des Eaux de Paris 2 Laboratoire d'Hygiene de la ville de Nice
3 Laboratoire d'Hygiene de la ville de Paris Centre de Recherche de la Compagnie Generale des Eaux, Maisons Laffitte S Laboratoire d'Hygiene Hospitaliere, Faculti de Medecine de Clennont-Ferrand 6 Ecole Nationale de la Sante Publique de Rennes 7 Laboratoire d'Hygiene de la ville de Nancy 8 Institut Pasteur de Litle 9 Institut d'Hydrologie de Clennont-Ferrand JO Laboratoire Departemental d'Hygiene et Regional d'Hydrologie / / Institut Pasteur de Lyon J2 Institut Europeen de I'Environnement de Bordeaux /3 Institut Bouisson Bertrand, Montpellier, France 4
ABSTRACf This study was initiated by the AFNOR water microbiology working group to evaluate the perfonnance of the glass wool method for virus recovery. Its reliability was tested with drinking and sea water by respectively nine and thirteen laboratories. In both trials. six were actively involved in water virology research. one was designated as a central laboratory. the others had no experience in virological practices. Analysis of reproducibility and repeatability according to NF-ISO 5725-2 were realized. For drinking waters (24 assays). the average recovery efficiency was 72%. mean standard deviations: repeatability 12.4%; reproducibility 33.6%; inter-laboratories 21 %. For sea waters (39 assays). the average recovery efficiency was 75% and the mean standard deviations 6.9%.17.9% and 11% respectively. CClI997IAWQ. Published by Elsevier Science Ltd
KEYWORDS Virus; glass wool concentration; collaborative study; drinking and sea waters.
INTRODUCfION Adsorption-elution using glass wool (Vilagines et al., 1988) for concentrating virus from water has been widely used in France (Champsaur et al., 1993; Hugues et al., 1993; Menut et al., 1993). Given the need of a 445
446
Ph. Vll.AGINEs et aI.
standard enterovirus detection method with respect to French legislation (Journal Officiel. 1989 and 1991), the AFNOR (Association Franlraise de Normalisation) working group for microbiology initiated "round robin" comparative testing of the glass wool procedure with drinking and sea water. The precision of the procedure. as wen as its repeatability and reproducibility in virus recovery, was examined, rather than its sensitivity. The virus load was intentionally high so that significant amounts of viruses could be measured In the first trial. analyses were performed in triplicate by nine laboratories with their local drinking water, in ~e ~cond trial. thirteen laboratories were involved using the same experimental protocol but with an Identical sea water sample sent by one of the participants. In both trials, only six laboratories having virological equipment went through the whole experimentation including virus enumeration on BGM cens (Baron et aL, 1970). The other laboratories performed only the concentration-elution step. Uniformity was maintained with regard to several aspects: (a) identical protocol for both trials; (b) BGM cells and glass wool were sent two weeks earlier by the central laboratory "CL"; (c) the day before each trial, seed virus and beef extract were sent by express mail; (d) triplicate analyses were performed on the same day and every laboratory immediately posted back a fraction of each water sample and neutralized concentrate to the "CL" for virus titration and retitration within 48h.
MATERIAL AND METHODS Virus and cell culture - poliovirus type I LSc2ab. propagated in BGM cells. infectivity was determined by plaque assay (Vilagin~s et al., 1993). Stock virus was prepared as pools and diluted in MEM medium containing 2% foetal calf serum to approximately 107 PFU/ml. The pools were stored (-70°C) in Sm1 volumes until used. The virus tubes were assigned code letters and dispatched on dry ice to the investigators. G14s, woolfUler - SOg of sodocalcic glass wool Rantigny (R.725, St.Gobain Orgel, France) were packed into a stainless steel holder (Sartorius. ref.SM 16249) as reported earlier (Vilagin~s et aI., 1993).
Concentration - experiments were conducted in triplicate on the same day according to the following protocol: (a) tap water: a 201 dechlorinated tap water sample was spiked with Iml of the virus. mixed thoroughly and an aliquot withdrawn for input determination. The water was filtered at 1001Jh and elution performed with 300m1 0.5% beef extract-SO mM glycine buffer pH 9.5 and immediately neutralised. When qualified, laboratories carried out quantification on the very same day. In addition. samples of the seeded water and of the concentrate were sent to the "CL" for titration within 48h post analysis; (b) sea water: the protocol was identical except that analyses were performed with 11 samples from the same sea water source ftItered at a SO lib flow rate and serum (2%) was added to the mailed water samples. RESULTS Results from participant laboratories were separated into two groups: group A - six laboratories which carried out the whole analytical process (1-6) and group B (7-9 or 7-13) - those who carried out only the concentration. Results in the drinking water trial are reported in Table I. The average recovery efficiencies obtained. after elimination of aberrant values (Cochran test) were 71 % group A. 83% group B and 60% "CL". Comparison of group A data vs "CL" data (group A titration +48h) or group B data (+48h ) vs "CL" data (group A titration +48h), showed no statistical differences (P > 0.05) when t-test, rank test and sign test were performed. Thus. repeatability and reproducibility were calculated from the data provided by the "CL". The mean standard deviation was 12.46% for repeatability and 33.67% for reproducibility (Table 3). Similar results were observed with sea water (Table 2). The average recovery efficiencies were 69%.78% and 72% respectively by group A. "CL" (group A titration +48h) and group B laboratories. No significant difference was observed between "CL" data and those from the laboratories doing their own virus quantifications (P >O.OS). The mean standard deviation was 6.93% for repeatability and 17.94% for reproducibility (Table 3).
Poliovirus recovery from drinking and sea water
447
Table 1. Comparative recovery of viruses from drinking water Group
Lab No.
Laboratory data (t = 0) 6 Recovery Virus pfu. 10 Water Cone· %
*
2
A
3 4
5
6
6.1 5.1 5.1 5.1 5.4
•
5.3
3.8 ~3
84
2.5 2.0
49 39
1.1
20
•
•
•
*
*
14 14 15 12 12 18 9.1
-
62
9.9 8.2 6.4 15
IS
8.4
25 5.2 46
8.4 9.84
7.60
S.7
-
•
71 59 43 125 125 139 57 55 68 71
"CL" data (t = +48h) Recovery Virus pfu.106 % Water Cone 4.8 4.8 100 ~2 3.3 46 SI 36 71 S.4 3.4 63 4.7 3.2 68 4.2 2.7 64 ~8 ~o 26
7.2
9.7 8.1 9.0 7.9
* -
*
8.5 7.4 ~6
6.91 6.7 7.9 7 Not done Not done 5.S 5.6 82 8 Not done Not done 8 7.2 4.6 9 Not done Not done 5.0 53 Mean 6.22 Average from "CL" data (+48h) 6.56 'Cone = concentrate; "CL" - Central Laboratory; -statistically aberrant values average estimation Mean
2.S 35 34 35 6.8 84 6.0 67 S.4 68 14 * 14 15 * 5.0 59 4.1 55 44 67 5.76 60.53 8.91338.4* 1067.1* 1296.1 109 7.1 87 53 74 4.3 93 2.8 56 4.2 79 4.97 83 5.48 72 eliminated by Cochran test in
DISCUSSION AND CONCLUSIONS
Melnick et al. (1984) were the rust to initiate a collaborative study to assess concentration methods to control the virological quality of drinking water. The present study was undertaken to evaluate glass wool repeatability and reproducibility in order to establish a standard method. Among the potential users, the nine and thirteen participant laboratories were geographically scattered throughout France: north (1), north-west (1), south-west (2), south (1), south-east (1), east (1), centre (3) and Paris area (3), seven of which had no experience of virological analysis.
Ph. vn.AGINEs et aI.
448
Table 2. Comparative recovery of viruses from sea water
Group
Lab No
2
A
3
4
5 6 Mean
8
r:aborato~ data (t Virus (pfu. 10 ) Water Cone 496 11.30 3.47 12.10 898 4.35 2.86 5.25 3.05 4.22 2.30 2.11 11.10 22.10 17.50 11.20 20.40 1070 6.47 5.33 5.84 6.62 5.17 396 12.60 9.32 14.70 11.60 1440 13 60 6.63 4.02 7.20 4.90 6.07 5.28 10 21 663
7
Not done
Not done
8
Not done
Not done
9
Not done
Not done
10
Not done
Not done
11
Not done
Not done
12
Not done
Not done
13
Not done
Not done
0) Recovery % 44 29 48 54 72 109 53 64 52 82 88
77
74 79
94
74 68 87 6933
"cLY;
dat! (t - +48h) Virus (pfu 10 ) Recovery Water Cone % 747 532 71 6.69 5.97 89 7.83 682 87 10.80 9.49 88 8.24 6.63 80 9.29 7.22 78 6.82 4.75 70 3.91 63 6.23 6.70 5.23 78 7.18 5.80 81 8.10 6.00 74 640 595 93 4.44 3.47 78 4.97 3.60 72 526 3.79 72 6.27 4.33 69 5.13 86 5.94 605 489 81 6 92 546 7840 4 86' 0748' IS' 4.15' 4.12' 99' 4 67' I 84' 39' 5.83 3.92 67 6.65 3.19 48 8.49 624 73 6.04 4.70 78 5.43 4.87 90 No result No result No result 11.80 6.60 56 10.60 6.66 63 9 69 No result No result 5.52 4.60 83 6.11 4.69 77 594 4.35 73 8.09 542 67 6.88 4.04 59 6.78 408 60
7 57 5.32 72.30 7,2 539 3 oc ran test In average estImatIons Table 3. Results of statistical analysis according to NF-ISO 5725-2 Mean stadard deviation (%) Repeatability (Ur") Reproducibility (UR") Interlaboratories
Drinking water 12.46 33.67 2100
Sea water 6.93 1794 11.01
Their results not only confirmed the simplicity of the virus concentration method by adsorption-elution through glass wool. but demonstrated the ability of non-expert laboratories to carry out water virological analysis limited to the concentration-elution step and within 4gh sending the concentrate to laboratories capable of carrying out virus quantification. This is of major importance because of the scarceness of environmental virological laboratories compared with the number of bacteriological laboratories which are
Poliovirus recovery from drinking and sea water
449
well dispersed throughout the country. Because of cost. only small volumes of sea water were analysed and the better results obtained in this trial may have been due to this smaller sample size and laboratories gaining experience. However. the goal of this study was to assess the repeatability and reproducibility of the glass wool technique for virus isolation from an identical water sample (sea water) or from different quality of waters (drinking water). In that regard. the results show that this method fulf1lled this criterion as it has been proposed and accepted as a French standard for enterovirus evaluation in waters. ACKNOWLEDGEMENTS The authors acknowledge the skillful technical assistance of Sylvie Gosselin and FItderique Retaux for the virological analysis. and Christine Cun for the statistical analysis REFERENCES Norme XP T 90-451 (1996). Recherche des enterovirus: Methode par concentration sur laine de verre et detection par culture cellulaire. AFNOR, Tour Europe, 92049 Paris La Defense Cedex, France, March 1996. Baron, A. L., Olshevesky, C. and Cohen, M. N. (1970). Characteristics of the BGM line of cells from African Green Monkey Kidney. Arch. Virusforsch., 32, 389-392. Champsaur, H., Hugues, B. and Andre. M. (1993). Detection des virus dans les effluents par une nouvelle methode de concentration sur laine de verre. Comparaison avec la methode de concentration sur poudre de verre. J. Fr. Hydrol., 24, 29-40. Hugues. B., Andre. M., Plantat, J. L. and Champsaur, H. (1993). Comparison of glass wool and glass powder methods for concentration of viruses from treated waste waters. Zbl. Hyg., 193,440-449. Journal Officiel (1989). Deeret no. 89-3 du 3 janvier 1989 relatif aux "eaux destinees Ala consommation humaine Al'exclusion des eaux mineraJes naturelles". 4th January 1989. Journal Officiel (1991). Decret no. 91·980 du 20 Septembre fixant les normes d'hygi~ne et de securite applicables aux piscines et aux baignades amenagees. 26th September 1991. Melnick, J. L., Safferman. R., Rao, C., Goyal, S., Berg, G., Dahling, R., Wright, B. A., Akin, E., Stetler, R., Sorher, C., Sobsey, M. D., Moore, R., Lewis, A. and Wellings, F. M. (1984). Round robin investigation of methods for the recovery of poliovirus from drinking water. Appl. Environ. Microbiol.• 47, 144-150. Menut, C., Beril, C. and Schwartzbrod, L. (1993). Poliovirus recovery from tap water after concentration over glass powder and glass wool. War. Sci. Tech., 27(3-4), 291-295. Vilagin~s, Ph., Sarrelte, B. and Vilagin~s, R. (1988). Detection en continu du poliovirus dans des eaux de distribution publique. CR. Mad. Sci. Paris., 307,171-176. Vilagin~s, Ph., Sarrelte, B., Husson, G. and Vilagin~s, R. (1993). Glass wool for virus concentration from water at ambient pH levels. War. Sci. Tech., 27(3-4), 299.306.
Pergamon
Wal. Sci. Q
PH: S0273-1223(97)OO301-6
r.ch. Vol. 3S. No. 11-12, pp. 44S-449. 1997. 1997 IAWQ. Published by Elscvier Science Ltd Printed ID Greal Britain. 027J-1223197 S17'00 + 0-00
ROUND ROBIN INVESTIGATION OF GLASS WOOL METHOD FOR POLIOVIRUS RECOVERY FROM DRINKING WATER AND SEA WATER Ph. Vilagines l , B. Sarrette2 , H. Champsaur2 , B. Hugues 2, S. Dubrou 3, J.-C. Joret4, H. Laveran 5, J. Lesne6, J. L. Paquin7, J. M. Delattre8, C. Oger8, J. Alame9, I. GratelouplO, H. Perrollet ll , R. Serceau I2 , F. Sinegre l3 and R. Vilagines l J Centre de Recherche et de Controle des Eaux de Paris 2 Laboratoire d'Hygiene de la ville de Nice
3 Laboratoire d'Hygiene de la ville de Paris Centre de Recherche de la Compagnie Generale des Eaux, Maisons Laffitte S Laboratoire d'Hygiene Hospitaliere, Faculti de Medecine de Clennont-Ferrand 6 Ecole Nationale de la Sante Publique de Rennes 7 Laboratoire d'Hygiene de la ville de Nancy 8 Institut Pasteur de Litle 9 Institut d'Hydrologie de Clennont-Ferrand JO Laboratoire Departemental d'Hygiene et Regional d'Hydrologie / / Institut Pasteur de Lyon J2 Institut Europeen de I'Environnement de Bordeaux /3 Institut Bouisson Bertrand, Montpellier, France 4
ABSTRACf This study was initiated by the AFNOR water microbiology working group to evaluate the perfonnance of the glass wool method for virus recovery. Its reliability was tested with drinking and sea water by respectively nine and thirteen laboratories. In both trials. six were actively involved in water virology research. one was designated as a central laboratory. the others had no experience in virological practices. Analysis of reproducibility and repeatability according to NF-ISO 5725-2 were realized. For drinking waters (24 assays). the average recovery efficiency was 72%. mean standard deviations: repeatability 12.4%; reproducibility 33.6%; inter-laboratories 21 %. For sea waters (39 assays). the average recovery efficiency was 75% and the mean standard deviations 6.9%.17.9% and 11% respectively. CClI997IAWQ. Published by Elsevier Science Ltd
KEYWORDS Virus; glass wool concentration; collaborative study; drinking and sea waters.
INTRODUCfION Adsorption-elution using glass wool (Vilagines et al., 1988) for concentrating virus from water has been widely used in France (Champsaur et al., 1993; Hugues et al., 1993; Menut et al., 1993). Given the need of a 445
446
Ph. Vll.AGINEs et aI.
standard enterovirus detection method with respect to French legislation (Journal Officiel. 1989 and 1991), the AFNOR (Association Franlraise de Normalisation) working group for microbiology initiated "round robin" comparative testing of the glass wool procedure with drinking and sea water. The precision of the procedure. as wen as its repeatability and reproducibility in virus recovery, was examined, rather than its sensitivity. The virus load was intentionally high so that significant amounts of viruses could be measured In the first trial. analyses were performed in triplicate by nine laboratories with their local drinking water, in ~e ~cond trial. thirteen laboratories were involved using the same experimental protocol but with an Identical sea water sample sent by one of the participants. In both trials, only six laboratories having virological equipment went through the whole experimentation including virus enumeration on BGM cens (Baron et aL, 1970). The other laboratories performed only the concentration-elution step. Uniformity was maintained with regard to several aspects: (a) identical protocol for both trials; (b) BGM cells and glass wool were sent two weeks earlier by the central laboratory "CL"; (c) the day before each trial, seed virus and beef extract were sent by express mail; (d) triplicate analyses were performed on the same day and every laboratory immediately posted back a fraction of each water sample and neutralized concentrate to the "CL" for virus titration and retitration within 48h.
MATERIAL AND METHODS Virus and cell culture - poliovirus type I LSc2ab. propagated in BGM cells. infectivity was determined by plaque assay (Vilagin~s et al., 1993). Stock virus was prepared as pools and diluted in MEM medium containing 2% foetal calf serum to approximately 107 PFU/ml. The pools were stored (-70°C) in Sm1 volumes until used. The virus tubes were assigned code letters and dispatched on dry ice to the investigators. G14s, woolfUler - SOg of sodocalcic glass wool Rantigny (R.725, St.Gobain Orgel, France) were packed into a stainless steel holder (Sartorius. ref.SM 16249) as reported earlier (Vilagin~s et aI., 1993).
Concentration - experiments were conducted in triplicate on the same day according to the following protocol: (a) tap water: a 201 dechlorinated tap water sample was spiked with Iml of the virus. mixed thoroughly and an aliquot withdrawn for input determination. The water was filtered at 1001Jh and elution performed with 300m1 0.5% beef extract-SO mM glycine buffer pH 9.5 and immediately neutralised. When qualified, laboratories carried out quantification on the very same day. In addition. samples of the seeded water and of the concentrate were sent to the "CL" for titration within 48h post analysis; (b) sea water: the protocol was identical except that analyses were performed with 11 samples from the same sea water source ftItered at a SO lib flow rate and serum (2%) was added to the mailed water samples. RESULTS Results from participant laboratories were separated into two groups: group A - six laboratories which carried out the whole analytical process (1-6) and group B (7-9 or 7-13) - those who carried out only the concentration. Results in the drinking water trial are reported in Table I. The average recovery efficiencies obtained. after elimination of aberrant values (Cochran test) were 71 % group A. 83% group B and 60% "CL". Comparison of group A data vs "CL" data (group A titration +48h) or group B data (+48h ) vs "CL" data (group A titration +48h), showed no statistical differences (P > 0.05) when t-test, rank test and sign test were performed. Thus. repeatability and reproducibility were calculated from the data provided by the "CL". The mean standard deviation was 12.46% for repeatability and 33.67% for reproducibility (Table 3). Similar results were observed with sea water (Table 2). The average recovery efficiencies were 69%.78% and 72% respectively by group A. "CL" (group A titration +48h) and group B laboratories. No significant difference was observed between "CL" data and those from the laboratories doing their own virus quantifications (P >O.OS). The mean standard deviation was 6.93% for repeatability and 17.94% for reproducibility (Table 3).
Poliovirus recovery from drinking and sea water
447
Table 1. Comparative recovery of viruses from drinking water Group
Lab No.
Laboratory data (t = 0) 6 Recovery Virus pfu. 10 Water Cone· %
*
2
A
3 4
5
6
6.1 5.1 5.1 5.1 5.4
•
5.3
3.8 ~3
84
2.5 2.0
49 39
1.1
20
•
•
•
*
*
14 14 15 12 12 18 9.1
-
62
9.9 8.2 6.4 15
IS
8.4
25 5.2 46
8.4 9.84
7.60
S.7
-
•
71 59 43 125 125 139 57 55 68 71
"CL" data (t = +48h) Recovery Virus pfu.106 % Water Cone 4.8 4.8 100 ~2 3.3 46 SI 36 71 S.4 3.4 63 4.7 3.2 68 4.2 2.7 64 ~8 ~o 26
7.2
9.7 8.1 9.0 7.9
* -
*
8.5 7.4 ~6
6.91 6.7 7.9 7 Not done Not done 5.S 5.6 82 8 Not done Not done 8 7.2 4.6 9 Not done Not done 5.0 53 Mean 6.22 Average from "CL" data (+48h) 6.56 'Cone = concentrate; "CL" - Central Laboratory; -statistically aberrant values average estimation Mean
2.S 35 34 35 6.8 84 6.0 67 S.4 68 14 * 14 15 * 5.0 59 4.1 55 44 67 5.76 60.53 8.91338.4* 1067.1* 1296.1 109 7.1 87 53 74 4.3 93 2.8 56 4.2 79 4.97 83 5.48 72 eliminated by Cochran test in
DISCUSSION AND CONCLUSIONS
Melnick et al. (1984) were the rust to initiate a collaborative study to assess concentration methods to control the virological quality of drinking water. The present study was undertaken to evaluate glass wool repeatability and reproducibility in order to establish a standard method. Among the potential users, the nine and thirteen participant laboratories were geographically scattered throughout France: north (1), north-west (1), south-west (2), south (1), south-east (1), east (1), centre (3) and Paris area (3), seven of which had no experience of virological analysis.
Ph. vn.AGINEs et aI.
448
Table 2. Comparative recovery of viruses from sea water
Group
Lab No
2
A
3
4
5 6 Mean
8
r:aborato~ data (t Virus (pfu. 10 ) Water Cone 496 11.30 3.47 12.10 898 4.35 2.86 5.25 3.05 4.22 2.30 2.11 11.10 22.10 17.50 11.20 20.40 1070 6.47 5.33 5.84 6.62 5.17 396 12.60 9.32 14.70 11.60 1440 13 60 6.63 4.02 7.20 4.90 6.07 5.28 10 21 663
7
Not done
Not done
8
Not done
Not done
9
Not done
Not done
10
Not done
Not done
11
Not done
Not done
12
Not done
Not done
13
Not done
Not done
0) Recovery % 44 29 48 54 72 109 53 64 52 82 88
77
74 79
94
74 68 87 6933
"cLY;
dat! (t - +48h) Virus (pfu 10 ) Recovery Water Cone % 747 532 71 6.69 5.97 89 7.83 682 87 10.80 9.49 88 8.24 6.63 80 9.29 7.22 78 6.82 4.75 70 3.91 63 6.23 6.70 5.23 78 7.18 5.80 81 8.10 6.00 74 640 595 93 4.44 3.47 78 4.97 3.60 72 526 3.79 72 6.27 4.33 69 5.13 86 5.94 605 489 81 6 92 546 7840 4 86' 0748' IS' 4.15' 4.12' 99' 4 67' I 84' 39' 5.83 3.92 67 6.65 3.19 48 8.49 624 73 6.04 4.70 78 5.43 4.87 90 No result No result No result 11.80 6.60 56 10.60 6.66 63 9 69 No result No result 5.52 4.60 83 6.11 4.69 77 594 4.35 73 8.09 542 67 6.88 4.04 59 6.78 408 60
7 57 5.32 72.30 7,2 539 3 oc ran test In average estImatIons Table 3. Results of statistical analysis according to NF-ISO 5725-2 Mean stadard deviation (%) Repeatability (Ur") Reproducibility (UR") Interlaboratories
Drinking water 12.46 33.67 2100
Sea water 6.93 1794 11.01
Their results not only confirmed the simplicity of the virus concentration method by adsorption-elution through glass wool. but demonstrated the ability of non-expert laboratories to carry out water virological analysis limited to the concentration-elution step and within 4gh sending the concentrate to laboratories capable of carrying out virus quantification. This is of major importance because of the scarceness of environmental virological laboratories compared with the number of bacteriological laboratories which are
Poliovirus recovery from drinking and sea water
449
well dispersed throughout the country. Because of cost. only small volumes of sea water were analysed and the better results obtained in this trial may have been due to this smaller sample size and laboratories gaining experience. However. the goal of this study was to assess the repeatability and reproducibility of the glass wool technique for virus isolation from an identical water sample (sea water) or from different quality of waters (drinking water). In that regard. the results show that this method fulf1lled this criterion as it has been proposed and accepted as a French standard for enterovirus evaluation in waters. ACKNOWLEDGEMENTS The authors acknowledge the skillful technical assistance of Sylvie Gosselin and FItderique Retaux for the virological analysis. and Christine Cun for the statistical analysis REFERENCES Norme XP T 90-451 (1996). Recherche des enterovirus: Methode par concentration sur laine de verre et detection par culture cellulaire. AFNOR, Tour Europe, 92049 Paris La Defense Cedex, France, March 1996. Baron, A. L., Olshevesky, C. and Cohen, M. N. (1970). Characteristics of the BGM line of cells from African Green Monkey Kidney. Arch. Virusforsch., 32, 389-392. Champsaur, H., Hugues, B. and Andre. M. (1993). Detection des virus dans les effluents par une nouvelle methode de concentration sur laine de verre. Comparaison avec la methode de concentration sur poudre de verre. J. Fr. Hydrol., 24, 29-40. Hugues. B., Andre. M., Plantat, J. L. and Champsaur, H. (1993). Comparison of glass wool and glass powder methods for concentration of viruses from treated waste waters. Zbl. Hyg., 193,440-449. Journal Officiel (1989). Deeret no. 89-3 du 3 janvier 1989 relatif aux "eaux destinees Ala consommation humaine Al'exclusion des eaux mineraJes naturelles". 4th January 1989. Journal Officiel (1991). Decret no. 91·980 du 20 Septembre fixant les normes d'hygi~ne et de securite applicables aux piscines et aux baignades amenagees. 26th September 1991. Melnick, J. L., Safferman. R., Rao, C., Goyal, S., Berg, G., Dahling, R., Wright, B. A., Akin, E., Stetler, R., Sorher, C., Sobsey, M. D., Moore, R., Lewis, A. and Wellings, F. M. (1984). Round robin investigation of methods for the recovery of poliovirus from drinking water. Appl. Environ. Microbiol.• 47, 144-150. Menut, C., Beril, C. and Schwartzbrod, L. (1993). Poliovirus recovery from tap water after concentration over glass powder and glass wool. War. Sci. Tech., 27(3-4), 291-295. Vilagin~s, Ph., Sarrelte, B. and Vilagin~s, R. (1988). Detection en continu du poliovirus dans des eaux de distribution publique. CR. Mad. Sci. Paris., 307,171-176. Vilagin~s, Ph., Sarrelte, B., Husson, G. and Vilagin~s, R. (1993). Glass wool for virus concentration from water at ambient pH levels. War. Sci. Tech., 27(3-4), 299.306.