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Evaluation of procedures for recovery of viruses from water—1 concentration systems
14uler Re.seurch Vol 14. pp. 791 to 793 © Pergamon Pres~ Lid 1980, Printed in Great Britain
0043-1354/80/0701-0791502.00/0
EVALUATION OF PROCEDURES FOR RECOVERY OF VIRUSES FROM WATER--I CONCENTRATION SYSTEMS R. MORRIS* and W. M. W^ITE Regional Laboratory, Severn Trent Water Authority, St Martins Road, Coventry, England Abstract--Three filter media were evaluated for their suitability in recovering seeded poliovirus type 2 from tap and river water. The importance of the presence of aluminium cations was also examined. Fibreglass filters gave best recoveries in this evaluation when used with 0.0005 M AICI36HzO, being up to 4 x more efficient than filters used without AI3+. No such AI3÷ requirement was found for cellulose nitrate membrane filters. A procedure was arrived at suitable for the recovery of enteroviruses from tap and river water. The efficiency of the organic flocculation procedure as a secondary concentration method varied with viral serotype.
INTRODUCTION
A number of workers have used a variety of concentration and detection methods for viruses in natural and potable waters. Concentration techniques such as polyelectrolyte filtration, alum flocculation, alginate filtration and filter adsorption-elution have been reviewed by Hill et al. (1971). Filter adsorption-elution has proved to be the most popular approach to concentration of low levels of virus from water with a wide range of filter media being used. Wallis et al. (1972) examined virus adsorption to cellulose nitrate membranes; Jakubowski et al. (1974) evaluted epoxybound fibreglass filters; Jakubowski et al. (1975) examined a variety of cellulosic and fibreglass media; whilst Farrah et al. (1976) reported on the use of pleated filters. The effects of cations on virus adsorption and subsequent recovery have been examined by Wallis et al. (1972). The filter adsorption-elution procedure allows for the processing of large volumes of water but the resultant eluate volume is normally still too large for direct evaluation. Secondary concentration steps have been introduced to further reduce the sample size to a level suitable for detection procedures to be conducted. Amongst the methods which have been described, the organic flocculation method of Katzenelson et al. (1976) seemed to offer a relatively efficient and easy procedure. Their reported findings of 75% recovery for poliovirus type I, compared with the 35% using the tentative standard method described by Hill et al. (1976) were encouraging Jakubowski (personal communication 1978) failed to recover coxsackie virus type A9 by the organic flocculation method and indicated that the system, whilst being suitable for poliovirus type 1 did not necessarily perform at the same efficiency for other viral types. The present study evaluated two fibreglass filters *To whom all correspondence should be addressed.
and one cellulosic filter for the recovery of poliovirus type 2 seeded into tap and river waters using the organic flocculation technique as a standard secondary concentration procedure, with a view to establishing a reliable method for routine virological surveillance of the aquatic environment. MATERIALS AND METHODS
Viruses
The MEF-I strain of poliovims type 2 (a gift from Dr N. B. Finter. Wellcome Research Laboratories, England) was used as the model virus for all filter evaluations. The virus was propagated in monolayers of Vero cells and virus aggregates removed from the culture fluid by filtration through a 0.05/an cellulose nitrate membrane (Sartorius 11310) prior to storage at -20°C, this preparation being regarded for practical purposes as mono-dispersed. Cell cultures
The continuous cell line derived from African green monkey kidney and designated Veto [CCLSI; Yasumura & Kawakita (1963)] was obtained from Flow Laboratories, Scotland. The line was maintained as roller bottle cultures growing on medium 199 supplemented with foetal calf serum (10%), penicillin (100 units mi- 1) and streptomycin (100/~g ml- i) (Gibco-Biocuit, Scotland). Assay procedure
Plastic flasks (75 crn2, Nunc) seeded with 1.5 x 106 cells in growth medium were used at confluence (3-5 days after planting out). Monolayers were washed with 10 mi serumfree medium 199 and inoculated with the sample. After l½ h adsorption at room temperature, the cultures were washed once and an agar medium added. This overlay consisted of medium 199. foetal calf serum (5%), neutral red (0.0013%, BDH), Noble agar (1.3%, Difco) and antibiotics (penicillin, streptomycin, kanamycin, polymixin B sulphate, neomycin, mycostatin and fungizone used at recommended levels, Gibco-Biocult). Cultures were incubated inverted at 37°C, plaques being counted after 4 days. Waters
Filters were evaluated using both tap and river waters. The tap water used was that produced by conventional water treatment of river water abstracted at Strensham 791
792
R, MORRISand W, M. WAITF Table I. Physico-Chemical Characteristics of test waters [mean + SD)
Electroeonductivity pH Total hardness Alkalinity Total organic carbon Chloride Nitrate Suspended solids Temperature Adsorption pH
~s cm- 1 mgl-~ Ca mgl-I Ca mg 1- ~C mg 1- ~CI ragl -z N rag 1-1 ~C
Treatment Works (Severn Trent Water Authority. 1978}, The second was a river water sampled apwoximately 2 km downstream of Finhara Water Reclamation Works (Severn Trent Water Authority, 1978). Table I details the mean physicocheraicai characteristics of both waters during this study, Water conditionin 0 .
Prior to filtration all waters were adjusted to approximately pH 3.5 by the addition of N/I hydr~hloric acid. Alurainiura cations (AP ÷) were added as required using a 0.5 M solution of aluminiura chloride (AICI~6HzO) to a final 0.0005 M. Filter media
Three filter media were evaluated: (a) cellulose nitrate membrane of 0.45/~ra porosity, 293rata (Milfipore HAWP.293-25) with API5 i0aafibre preflher; (b) epoxybound fibreglass cartridge filter of 8 lain porosity (Bahton type 100-12-C used in a type 92 housing); and (c) epoxybound fibreglass cartridge filter of 8 tan porosity (Whatman grade 80 used in a Gamma:12 honaing). Sample processing
Approximately 450pfu of poliovirus 2 were seeded into 201. samples of tap water and approxinmtely 1750pfu into 51. of river water. After conditioning as above, the treated water was parted through the test filter system under positive prenure (10psi). Adsorbed viruses were eluted with 200 ral volumes of beef extract (3%. pH 9.5) by pressure filtration in the same direction of flow as when adsorbing and further concentrated by the organic fiozeulation procedure of Katzenehon et al. (1976}. Floc formation was induced at pH 3.5 by addition of 5 N HCI and the weopitate deposited by omtrifugation at 2000 0 for 5 rain. The floc was r e ~ m ~ in disodiura hydrogen phosphate solution (0.15 M, pH 9.2), dispersed by sonication (MSE uhrasonicator. 9/an ampfitude. !-2 rain) and adjusted to a final volume of 20 mi. Prior to assay the concentrate was mixed with an equal volume of double strength serum-free medium 199.
Tap water
River water
470 + 62 7.8 + 0.2 171 + 34 103 + 23 2.4 _ 0.4 39 + 7 4.9 + 0.8 Not tested 11.0 4- 1.5 3.4 4- 0.2
1197 + 126 7.4 + 0,1 307 + 33 915 4- 22 9.4 4- 2.3 t 70 + 29 13.9 + 2.7 7.2 + 3,4 14.6 4- 1.8 3.4 4- 0,2 RESULTS AND DISCUSSION
Filter evaluations
The recovery of seeded poliovirus type 2 from both tap water and river water is shown in Table 2. No significant difference was shown between the Millipore system used in the presence or absence of AIa + cations (t-test--independent means, P = 0.2) although it should be noted that whilst the system used without AIa + showed a normal distribution, the results with AI3 + were skew. The two glassfibre systems, however, demonstrated a marked requirement for the presence of AI 3÷ cations (P = 0.001), recoveries then being comparable with those obtained with the Millipore system. This contrasts with previous work where cellulose nitrate membranes used with A! s+ cations gave er.hanced recoveries (Wallis et al., 1972), whilst Jakubowski (personal communication 1978) found that virus recovery was reduced when using the Balston fibreglnss system in the presence of AI a + cations. "" Kessick & Wagner (1978) reported that cellulosic filters at low pH are electrophoretically negative. This is possibly due in part to the presence of ionim~d carboxyl groups on the cellulosic surface. Additionally many nonesterif~d hydroxyl groups contained within the cellulosic material may act as good hydr~wen bonders. As a typical virus surface at pH 3.5 has a nett positive charge, adsorption is likely to be an electrostatic phenomenon. However, when considering epoxybound fibreglass filter systems, they found that the negative charge exhibited at low pH probably arose in part from the ionisation of m S i O H groups on the fibreglass surface. The results presented in Table 2 would seem to sug-
Table 2. Recovery of seeded poliovirus 2 from tap and river water by six concentration methods MiUipore + AI* - AI* Tap River
Mean SD Range Mean SD Range
Balston + Air - AI*
Whatman + Air - AI*
40.8 46.4 45.0 17.7 40.8 10.0 14.1 24,2 16.3 6.4 21.7 5.1 21.3-71.118.3-85.2 14.5--81.3 10.0-26.9 14.9-83.8 2.8-16.8 21.7 32.6 32.2 8.5 35,1 I 1.4 16.0 I 1.6 8.0 3.6 15,4 5.4 7.0-58.2 15.4--48.2 16.8-44.2 3,6--44.2 5.7-56.2 5.3-23.2
* Ten experimental runs. t Twenty experimental runs,
Evaluation of procedures for recovery of viruses from water--I Table 3. Organic floeculation as a concentration step Virus
Source
Polio 1 (Lsc 1) Lab Polio 2 (MEF-I)Lab Polio 2 (t45/77) Raw sludge Polio2 (177/77) River Polio 2 (212/77) River Pofio 2 (216/77) River Cox B2 Lab Cox B3 Lab Cox B4 Lab Cox B5 Lab Echo 1 Lab
Input (pfu) To Recovered 10 117 10 26 105 55 64 57 56 33 41
40; 40 28 _ 9 40; 50 50; 12 45:40 35; 42 22; 6 98; 98 7; 11 9; 3 7; 7
gest that whereas AI a÷ cations are not actively involved in the adsorption of viruses to cellulose nitrate filters, there may be complexing of the cations with components of fibreglass material and the viruses, the cations acting as bridges between the relatively poor virus adsorption sites and the virus partide. The concept of specific ion adsorption to filter surface groups is strongly supported by the work of Davis & Rideal (1961) but the mechanism of the complexing of filter components, virus and cation is not clear. As Kessick and Wagner point out, it is probable that specific interaction of multivalent cations with the viral surface and the filter surface account for a substantial part of the virus-filter adsorption phenomenon. Organic flocculation as a concentration step
Katzenelson et al. (1976) claimed that organic flocculation operated at a mean efficiency of 75% for poliovirus type 1. These workers make no reference to "monodispersion" of the virus suspensions used in their evaluations and the range of recoveries (69-123%) is consistent with the presence of viral aggregates in the initial preparation. Jakubowski (personal communication 1978) found that poliovirus type 1 was more efficiently recovered by the organic flocculation method (mean 46%) than with the glycine method of Hill et al. (1976) (mean 24%). However, he failed to recover coxsackie virus type A9 in three experiments using organic flocculation. Since the organic floeculation procedure had been used as the standard secondary concentration method for the present study, virus recovery efficiency was determined using monodispersed preparations of several viral types and strains. Virus was seeded into 200 ml volumes of beef extract and flocculated as described above. The recovery of poliovirns type 1 (40%) compared favorably with the data of Jakubowski but the mean recovery of 28.2 + 9.2% for poliovirus type 2 (MEF-1 strain), the type virus for the filter evalu-
W.R. 14/7--v
793
ation discussed above, is significantly lower. Additionally, recovery efficiencies varied with viral serotype with some indication that variation may also occur between strains of the same serotype (Table 3). However, because of the reproducibility of the system in recovering poliovirus type 2 in laboratory trials, organic flocculation has been adopted as a standard secondary concentration stage for the routine surveillance of virus levels in the aquatic environment. CONCLUSIONS
Results given in Table 2 show no significant differences in performance between the Millipore system without AI 3+ cations and the two fibreglass systems with AI3+ cations. The Balston system with AP + cations has been adopted as the standard concentration technique by this laboratory on the grounds of ease of handing, cost and apparent reprodudbility. Despite the marked variation in recoveries of different virus types using organic flocculation, this technique has been adopted as the secondary concentration step pending evaluation of alternative procedures. REFERENCES
Davis J. T. & Rideal E. K. (1961) Interracial Phenomena. Academic Press, New York. Farrah S. R., Gerba C. P., Wallis C. & Melnick J. L. (1976) Concentration of viruses from large volumes of tap water using pleated membrane filters. Appl. & Environ. Microbiol. 31, 221-226. Hill W. F. Jr., Akin E. W. & Benton W. H. (1971) Detection of viruses in water: a review of methods and applications. Water Res. .5, 967-995. Hill W. F. Jr., Jakubowski W., Akin E. W. & Clarke N. A. (1976) Detection of virus in water: sensitivity of the tentative standard method for drinking water. Appl. & Environ. Microbiol. 31, 254-261. Jakubowski W., Hill W. F. Jr. & Clarke N. A. (1975) Comparative study of four microporous filters for concentrating viruses from drinking water. Appl. Microbiol. 30, 58-65. Jakubowski W., Hoff J. C., Anthony N. C. & Hill W. F. Jr. (1974) Epoxy-fibreglass adsorbent for concentrating viruses from large volumes of potable water. Appl. Microbiol. 28, 501-502. Katzenelson E., Fattal B. & Hostovesky T. (1976) Organic floeeulation: an efficient second step concentration method for the detection of viruses in tap water. Appl. & Environ. Microbiol. 32, 638-639. Kessick M. A. and Wagner R. A. (1978) Eleetrophoretic mobilities of virus adsorbing filter material. Water Res. 12, 263-268. Severn Trent Water Authority (1978) Water Quality 1977/78. Appendix 3, Avon Division. Wallis C., Henderson M. & Melnick J. L. (1972) Enterovirus concentration on cellulose membranes. Appl. Microbiol. 23, 476--480. Yasumura Y. and Kawakita Y. (1963) Research into SV40 by tissue culture. Nippon Rinsho 21, 1201-1219.
0043-1354/80/0701-0791502.00/0
EVALUATION OF PROCEDURES FOR RECOVERY OF VIRUSES FROM WATER--I CONCENTRATION SYSTEMS R. MORRIS* and W. M. W^ITE Regional Laboratory, Severn Trent Water Authority, St Martins Road, Coventry, England Abstract--Three filter media were evaluated for their suitability in recovering seeded poliovirus type 2 from tap and river water. The importance of the presence of aluminium cations was also examined. Fibreglass filters gave best recoveries in this evaluation when used with 0.0005 M AICI36HzO, being up to 4 x more efficient than filters used without AI3+. No such AI3÷ requirement was found for cellulose nitrate membrane filters. A procedure was arrived at suitable for the recovery of enteroviruses from tap and river water. The efficiency of the organic flocculation procedure as a secondary concentration method varied with viral serotype.
INTRODUCTION
A number of workers have used a variety of concentration and detection methods for viruses in natural and potable waters. Concentration techniques such as polyelectrolyte filtration, alum flocculation, alginate filtration and filter adsorption-elution have been reviewed by Hill et al. (1971). Filter adsorption-elution has proved to be the most popular approach to concentration of low levels of virus from water with a wide range of filter media being used. Wallis et al. (1972) examined virus adsorption to cellulose nitrate membranes; Jakubowski et al. (1974) evaluted epoxybound fibreglass filters; Jakubowski et al. (1975) examined a variety of cellulosic and fibreglass media; whilst Farrah et al. (1976) reported on the use of pleated filters. The effects of cations on virus adsorption and subsequent recovery have been examined by Wallis et al. (1972). The filter adsorption-elution procedure allows for the processing of large volumes of water but the resultant eluate volume is normally still too large for direct evaluation. Secondary concentration steps have been introduced to further reduce the sample size to a level suitable for detection procedures to be conducted. Amongst the methods which have been described, the organic flocculation method of Katzenelson et al. (1976) seemed to offer a relatively efficient and easy procedure. Their reported findings of 75% recovery for poliovirus type I, compared with the 35% using the tentative standard method described by Hill et al. (1976) were encouraging Jakubowski (personal communication 1978) failed to recover coxsackie virus type A9 by the organic flocculation method and indicated that the system, whilst being suitable for poliovirus type 1 did not necessarily perform at the same efficiency for other viral types. The present study evaluated two fibreglass filters *To whom all correspondence should be addressed.
and one cellulosic filter for the recovery of poliovirus type 2 seeded into tap and river waters using the organic flocculation technique as a standard secondary concentration procedure, with a view to establishing a reliable method for routine virological surveillance of the aquatic environment. MATERIALS AND METHODS
Viruses
The MEF-I strain of poliovims type 2 (a gift from Dr N. B. Finter. Wellcome Research Laboratories, England) was used as the model virus for all filter evaluations. The virus was propagated in monolayers of Vero cells and virus aggregates removed from the culture fluid by filtration through a 0.05/an cellulose nitrate membrane (Sartorius 11310) prior to storage at -20°C, this preparation being regarded for practical purposes as mono-dispersed. Cell cultures
The continuous cell line derived from African green monkey kidney and designated Veto [CCLSI; Yasumura & Kawakita (1963)] was obtained from Flow Laboratories, Scotland. The line was maintained as roller bottle cultures growing on medium 199 supplemented with foetal calf serum (10%), penicillin (100 units mi- 1) and streptomycin (100/~g ml- i) (Gibco-Biocuit, Scotland). Assay procedure
Plastic flasks (75 crn2, Nunc) seeded with 1.5 x 106 cells in growth medium were used at confluence (3-5 days after planting out). Monolayers were washed with 10 mi serumfree medium 199 and inoculated with the sample. After l½ h adsorption at room temperature, the cultures were washed once and an agar medium added. This overlay consisted of medium 199. foetal calf serum (5%), neutral red (0.0013%, BDH), Noble agar (1.3%, Difco) and antibiotics (penicillin, streptomycin, kanamycin, polymixin B sulphate, neomycin, mycostatin and fungizone used at recommended levels, Gibco-Biocult). Cultures were incubated inverted at 37°C, plaques being counted after 4 days. Waters
Filters were evaluated using both tap and river waters. The tap water used was that produced by conventional water treatment of river water abstracted at Strensham 791
792
R, MORRISand W, M. WAITF Table I. Physico-Chemical Characteristics of test waters [mean + SD)
Electroeonductivity pH Total hardness Alkalinity Total organic carbon Chloride Nitrate Suspended solids Temperature Adsorption pH
~s cm- 1 mgl-~ Ca mgl-I Ca mg 1- ~C mg 1- ~CI ragl -z N rag 1-1 ~C
Treatment Works (Severn Trent Water Authority. 1978}, The second was a river water sampled apwoximately 2 km downstream of Finhara Water Reclamation Works (Severn Trent Water Authority, 1978). Table I details the mean physicocheraicai characteristics of both waters during this study, Water conditionin 0 .
Prior to filtration all waters were adjusted to approximately pH 3.5 by the addition of N/I hydr~hloric acid. Alurainiura cations (AP ÷) were added as required using a 0.5 M solution of aluminiura chloride (AICI~6HzO) to a final 0.0005 M. Filter media
Three filter media were evaluated: (a) cellulose nitrate membrane of 0.45/~ra porosity, 293rata (Milfipore HAWP.293-25) with API5 i0aafibre preflher; (b) epoxybound fibreglass cartridge filter of 8 lain porosity (Bahton type 100-12-C used in a type 92 housing); and (c) epoxybound fibreglass cartridge filter of 8 tan porosity (Whatman grade 80 used in a Gamma:12 honaing). Sample processing
Approximately 450pfu of poliovirus 2 were seeded into 201. samples of tap water and approxinmtely 1750pfu into 51. of river water. After conditioning as above, the treated water was parted through the test filter system under positive prenure (10psi). Adsorbed viruses were eluted with 200 ral volumes of beef extract (3%. pH 9.5) by pressure filtration in the same direction of flow as when adsorbing and further concentrated by the organic fiozeulation procedure of Katzenehon et al. (1976}. Floc formation was induced at pH 3.5 by addition of 5 N HCI and the weopitate deposited by omtrifugation at 2000 0 for 5 rain. The floc was r e ~ m ~ in disodiura hydrogen phosphate solution (0.15 M, pH 9.2), dispersed by sonication (MSE uhrasonicator. 9/an ampfitude. !-2 rain) and adjusted to a final volume of 20 mi. Prior to assay the concentrate was mixed with an equal volume of double strength serum-free medium 199.
Tap water
River water
470 + 62 7.8 + 0.2 171 + 34 103 + 23 2.4 _ 0.4 39 + 7 4.9 + 0.8 Not tested 11.0 4- 1.5 3.4 4- 0.2
1197 + 126 7.4 + 0,1 307 + 33 915 4- 22 9.4 4- 2.3 t 70 + 29 13.9 + 2.7 7.2 + 3,4 14.6 4- 1.8 3.4 4- 0,2 RESULTS AND DISCUSSION
Filter evaluations
The recovery of seeded poliovirus type 2 from both tap water and river water is shown in Table 2. No significant difference was shown between the Millipore system used in the presence or absence of AIa + cations (t-test--independent means, P = 0.2) although it should be noted that whilst the system used without AIa + showed a normal distribution, the results with AI3 + were skew. The two glassfibre systems, however, demonstrated a marked requirement for the presence of AI 3÷ cations (P = 0.001), recoveries then being comparable with those obtained with the Millipore system. This contrasts with previous work where cellulose nitrate membranes used with A! s+ cations gave er.hanced recoveries (Wallis et al., 1972), whilst Jakubowski (personal communication 1978) found that virus recovery was reduced when using the Balston fibreglnss system in the presence of AI a + cations. "" Kessick & Wagner (1978) reported that cellulosic filters at low pH are electrophoretically negative. This is possibly due in part to the presence of ionim~d carboxyl groups on the cellulosic surface. Additionally many nonesterif~d hydroxyl groups contained within the cellulosic material may act as good hydr~wen bonders. As a typical virus surface at pH 3.5 has a nett positive charge, adsorption is likely to be an electrostatic phenomenon. However, when considering epoxybound fibreglass filter systems, they found that the negative charge exhibited at low pH probably arose in part from the ionisation of m S i O H groups on the fibreglass surface. The results presented in Table 2 would seem to sug-
Table 2. Recovery of seeded poliovirus 2 from tap and river water by six concentration methods MiUipore + AI* - AI* Tap River
Mean SD Range Mean SD Range
Balston + Air - AI*
Whatman + Air - AI*
40.8 46.4 45.0 17.7 40.8 10.0 14.1 24,2 16.3 6.4 21.7 5.1 21.3-71.118.3-85.2 14.5--81.3 10.0-26.9 14.9-83.8 2.8-16.8 21.7 32.6 32.2 8.5 35,1 I 1.4 16.0 I 1.6 8.0 3.6 15,4 5.4 7.0-58.2 15.4--48.2 16.8-44.2 3,6--44.2 5.7-56.2 5.3-23.2
* Ten experimental runs. t Twenty experimental runs,
Evaluation of procedures for recovery of viruses from water--I Table 3. Organic floeculation as a concentration step Virus
Source
Polio 1 (Lsc 1) Lab Polio 2 (MEF-I)Lab Polio 2 (t45/77) Raw sludge Polio2 (177/77) River Polio 2 (212/77) River Pofio 2 (216/77) River Cox B2 Lab Cox B3 Lab Cox B4 Lab Cox B5 Lab Echo 1 Lab
Input (pfu) To Recovered 10 117 10 26 105 55 64 57 56 33 41
40; 40 28 _ 9 40; 50 50; 12 45:40 35; 42 22; 6 98; 98 7; 11 9; 3 7; 7
gest that whereas AI a÷ cations are not actively involved in the adsorption of viruses to cellulose nitrate filters, there may be complexing of the cations with components of fibreglass material and the viruses, the cations acting as bridges between the relatively poor virus adsorption sites and the virus partide. The concept of specific ion adsorption to filter surface groups is strongly supported by the work of Davis & Rideal (1961) but the mechanism of the complexing of filter components, virus and cation is not clear. As Kessick and Wagner point out, it is probable that specific interaction of multivalent cations with the viral surface and the filter surface account for a substantial part of the virus-filter adsorption phenomenon. Organic flocculation as a concentration step
Katzenelson et al. (1976) claimed that organic flocculation operated at a mean efficiency of 75% for poliovirus type 1. These workers make no reference to "monodispersion" of the virus suspensions used in their evaluations and the range of recoveries (69-123%) is consistent with the presence of viral aggregates in the initial preparation. Jakubowski (personal communication 1978) found that poliovirus type 1 was more efficiently recovered by the organic flocculation method (mean 46%) than with the glycine method of Hill et al. (1976) (mean 24%). However, he failed to recover coxsackie virus type A9 in three experiments using organic flocculation. Since the organic floeculation procedure had been used as the standard secondary concentration method for the present study, virus recovery efficiency was determined using monodispersed preparations of several viral types and strains. Virus was seeded into 200 ml volumes of beef extract and flocculated as described above. The recovery of poliovirns type 1 (40%) compared favorably with the data of Jakubowski but the mean recovery of 28.2 + 9.2% for poliovirus type 2 (MEF-1 strain), the type virus for the filter evalu-
W.R. 14/7--v
793
ation discussed above, is significantly lower. Additionally, recovery efficiencies varied with viral serotype with some indication that variation may also occur between strains of the same serotype (Table 3). However, because of the reproducibility of the system in recovering poliovirus type 2 in laboratory trials, organic flocculation has been adopted as a standard secondary concentration stage for the routine surveillance of virus levels in the aquatic environment. CONCLUSIONS
Results given in Table 2 show no significant differences in performance between the Millipore system without AI 3+ cations and the two fibreglass systems with AI3+ cations. The Balston system with AP + cations has been adopted as the standard concentration technique by this laboratory on the grounds of ease of handing, cost and apparent reprodudbility. Despite the marked variation in recoveries of different virus types using organic flocculation, this technique has been adopted as the secondary concentration step pending evaluation of alternative procedures. REFERENCES
Davis J. T. & Rideal E. K. (1961) Interracial Phenomena. Academic Press, New York. Farrah S. R., Gerba C. P., Wallis C. & Melnick J. L. (1976) Concentration of viruses from large volumes of tap water using pleated membrane filters. Appl. & Environ. Microbiol. 31, 221-226. Hill W. F. Jr., Akin E. W. & Benton W. H. (1971) Detection of viruses in water: a review of methods and applications. Water Res. .5, 967-995. Hill W. F. Jr., Jakubowski W., Akin E. W. & Clarke N. A. (1976) Detection of virus in water: sensitivity of the tentative standard method for drinking water. Appl. & Environ. Microbiol. 31, 254-261. Jakubowski W., Hill W. F. Jr. & Clarke N. A. (1975) Comparative study of four microporous filters for concentrating viruses from drinking water. Appl. Microbiol. 30, 58-65. Jakubowski W., Hoff J. C., Anthony N. C. & Hill W. F. Jr. (1974) Epoxy-fibreglass adsorbent for concentrating viruses from large volumes of potable water. Appl. Microbiol. 28, 501-502. Katzenelson E., Fattal B. & Hostovesky T. (1976) Organic floeeulation: an efficient second step concentration method for the detection of viruses in tap water. Appl. & Environ. Microbiol. 32, 638-639. Kessick M. A. and Wagner R. A. (1978) Eleetrophoretic mobilities of virus adsorbing filter material. Water Res. 12, 263-268. Severn Trent Water Authority (1978) Water Quality 1977/78. Appendix 3, Avon Division. Wallis C., Henderson M. & Melnick J. L. (1972) Enterovirus concentration on cellulose membranes. Appl. Microbiol. 23, 476--480. Yasumura Y. and Kawakita Y. (1963) Research into SV40 by tissue culture. Nippon Rinsho 21, 1201-1219.