Novel approach for modifying microporous filters for virus concentration from water

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Novel approach for modifying microporous filters for

virus concentration from water

David R. Preston, Tirucherai V. Vasudevan, Gabriel Bitton, Samuel R.

Farrah, Jean-Louis Morel

To cite this version:

David R. Preston, Tirucherai V. Vasudevan, Gabriel Bitton, Samuel R. Farrah, Jean-Louis Morel. Novel approach for modifying microporous filters for virus concentration from water. Applied and Environmental Microbiology, American Society for Microbiology, 1988, 54 (6), pp.1325-1329. �hal-02726353�

Vol. 54, No. 6 APPLIEDANDENVIRONMENTALMICROBIOLOGY, June 1988,p. 1325-1329

0099-2240/88/061325-05$02.00/0

Copyright © 1988, American Society for Microbiology

Novel Approach

for

Modifying Microporous

Filters for

Virus Concentration

from Watert

DAVID R.

PRESTON,'*

TIRUCHERAI V. VASUDEVAN,2 GABRIEL BITTON,3 SAMUELR. FARRAH,' AND

JEAN-LOUIS MOREL3t

DepartmentofMicrobiology and Cell

Science,'

Departmentof Material Science andEngineering,2 andDepartmentof

Environmental

Engineering,3

The

University

of

Florida,

Gainesville,

Florida 32611

Received 31 August1987/Accepted 2 March 1988

Electronegative microporous filters composed of epoxyfiberglass (Filterite) were treated with cationic

polymers toenhance theirvirus-adsorbing properties. This novel and inexpensive approach tomicroporous filter modificationentails soaking filters inanaqueoussolutionofacationic polymer suchaspolyethyleneimine

(PEI) for2 hatroomtemperatureand then allowing the filterstoair dryovernightonabsorbentpapertowels.

PEI-treatedfilterswereevaluatedfor coliphage(MS2,T2, and 4X174) and enterovirus (poliovirustype1and coxsackievirus type B5) adsorption from buffer at pH 3.5 to 9.0 and for indigenous coliphages from unchlorinatedsecondaryeffluentatambient pH. Adsorbed viruseswererecovered with 3% beefextract(pH 9). Severalothercationic polymerswere used tomodify epoxyfiberglass filters andwere evaluated for their ability to concentrate viruses from water. Zeta potentials of disrupted filter material indicated that electronegativeepoxyfiberglass filtersweremademoreelectropositivewhentreated with cationic polymers. In general, epoxyfiberglass filterstreated with cationic polymers werefound to adsorb a greater percentageof coliphagesandenterovirusesthanwereuntreated filters.

Popular procedures for the detection of viruses in water

samples have taken advantage of the phenomenon of virus

adsorption to and elution from microporous filters to con-centrateviruses fromawidevariety ofwatersources (6,7).

Filtersused in these procedures have been characterized as

electronegativeorelectropositivebased ontheir

electropho-retic mobilities(11, 12). Inaddition, therelative

hydropho-bicities ofthese surfaces have been characterized (9). For viruses to adsorb toelectronegative filters, the water must be pretreated by lowering the pH or by adding salts to enhance virus adsorption to acceptable levels (1, 6). Electropositive

filters,however, canadsorb viruses over a broader pH range without the addition of salts, and so pretreatment of the water is not required prior to virus adsorption (3, 9, 11). Recentadvances in the modification ofsurfaces to enhance

their virus-adsorbing properties (5, 13, 14) have indicated thatsimple modifying proceduresareavailable. In thisstudy

we report asimple andinexpensive procedure similar to that

ofBrownetal. (2) for themodification offiberglassfiltersto enhancetheirvirus-adsorbingpropertiesunderambient wa-terconditions. This modification procedure entailed soaking

afiberglass filterin anaqueoussolutionofcationic polymer

and thenallowingthefilterstoairdryatroomtemperature.

The modification converted the electronegative fiberglass surfaceto anelectropositive surface, asdeterminedbyzeta

potentialmeasurementsofdisruptedfilter materials.

MATERIALSANDMETHODS

Virus and viral assays. Bacteriophages MS2, T2, and ,X174 were determined as PFU by using Escherichia coli

C3000, B, and ATCC 13607, respectively, as hosts by previously described procedures (10). Poliovirus type 1,

*Correspondingauthor.

tPaper no. 8912from the Florida Agricultural Station,

Gaines-ville.

tPresent address: Ecole Nationale Superieure d'Agronomie et

des IndustriesAlimentaires, Vandoeuvre54500,France.

coxsackievirus type B5, and echovirus types 1 and 5 were

determined asPFU by using BGM cells and a methylcellu-loseoverlay, as described previously (8).

Chemicalsand filters. Thefollowing chemicals were used in this study. Glycine, hydrochloric acid, and sodium

hy-droxidewere from FisherScientific Co. (FairLawn, N.J.); imidazole and polyethyleneimine (PEI) were from Sigma Chemical Co. (St. Louis, Mo.); Dellchem cationic polymer

(BASF CF 600) and LCI cationic polymerweregifts from Mike New(KanapahaWastewaterTreatmentPlant, Gaines-ville, Fla.); and Nalco cationic polymer (90% charge, high

molecular weight) was a gift from Kenneth E. DeGarmo

(Leahchem Industries, Inc.,Titusville, Fla.). Thefollowing

filters were used in this study: epoxyfiberglass filters (pore

size, 0.2 to 1.0

ii.m;

from pleated filter cartridges; Duofine

Filterite, Timonium,Md.)andG25fiberglassprefilters(MSI;

through FisherScientific).Allfilterswere25mmindiameter andwere kept in appropriate filter holders.

Modification of epoxyfiberglass filters. Filter sheets or

25-mm-diameter filters were soaked for 2 h at 25°C in an aqueous solution ofa cationic polymer. Filters were then driedat25°Covernightonadsorbent paper towels and stored in paperenvelopes.

Determination ofzetapotentials of filtermaterials. Epoxy-fiberglassfilter material (poresize,0.2,um)was

disrupted

in

deionized water by blending it at

high speeds

and was air dried at room temperature. Next, disrupted filter material

wassoaked for 2 hat roomtemperaturein0.5%PEI,

0.05%

cationic polymer (Nalco), or deionized water and was sub-sequentlycentrifuged at 14,000 x gfor 10 min. The pellets

were collected; air dried at room temperature; and

sus-pendedin3 mMphosphatebufferatpH 3, 5, 7,and10. Zeta

potentials ofdisrupted filter material were determined di-rectly with a meter (model 501; Lazer Zee; Penkem, Inc.,

Bedford Hills,N.Y.).

Virusadsorption-elution studies. (i)Buffer. Buffer

(20

mM

glycineand 20mMimidazole),

adjusted

to

pH

3.5, 5, 7,

or9 which HCl or NaOH as

required,

was seeded to

approxi-1325

1326 PRESTON ET AL.

TABLE 1. Removal ofbacteriophage MS2byepoxyfiberglass filters treated with PEI"

% AqueousPEI %Bacteriophage

used totreatfilters' MS2 removed

0... . . .. 55 10-5... . . . .. . 49 10-4... ...55 lo-,... 55 10-2... 100 lo-'... . ... 100 0.5... 100 1.0... 100 "Buffer (100 ml; pH 7.0) seeded with approximately 105 PFU/ml was

passed throughonefilter layer (poresize,0.2pm)ina25-mm-diameterholder. Theresults are expressed asthepercentage ofvirus in the buffer priorto

filtering. Values indicatethe meanofduplicatedeterminations. The standard error wasless than20% ofthe meanfor allvalues.

" Epoxyfiberglass filtersweretreated with the indicatedpercentageofPEI in deionized waterfor2 h at roomtemperature and allowed todryat room

temperature onadsorbentpaper towels.

mately 105 PFU/ml with bacteriophage orenterovirus. This seeded buffer (100 ml) was passed through one or three layers of filters at approximately 1 ml/s. For recovery of adsorbed viruses, 10 ml of3% beefextract (pH 9.0; Scott Laboratories, Fiskeville, R.I.) or3% beefextract-I MNaCI (pH 9) were passed through the filter at approximately 1 ml/s. The PFU in the eluate and effluent were

determined,

and the results are expressed as the percentage ofvirus in the buffer prior to filtering. Values indicate the mean of

duplicatedeterminations or themeanand standarddeviation of fourdeterminations.

(ii) Indigenous bacteriophage. Unchlorinated secondary

effluent or raw sewage was first prefiltered with a G25

fiberglassfilter to removesuspended solids. Portions of 100 ml of these water samples were then passed through one filterlayer at approximately 1 ml/s. Recovery and determi-nations of adsorption and recovery of viruses were

per-formed as described above.

RESULTS

The removal ofbacteriophage MS2 from buffer at pH 7 was enhanced from 55 to 100% by treating epoxyfiberglass

filters with aqueous PEI, asdescribed above, at a concen-trationof0.01% PEI or greater(Table 1). For later

experi-ments, 0.1 or0.5%PEI was used tomodify epoxyfiberglass

filters. It should be noted that the adsorption of viruses to untreatedepoxyfiberglassfilters wasfound todependon the lot number of thefilter material (data not shown).

The pH influenced virus removal from buffer by three

layers of untreated and PEI-treated epoxyfiberglass filters (Table 2). When allbacteriophages and viruses were

consid-ered, untreated filters removed a greater percentage of

virusesat pH 3.5 (97 + 7) than did PEI-treated filters (71 +

31). However, this condition was reversed at pH 7 and 9. There was little difference in the percent removal of viruses

by treated and untreated filters at pH 5. These results indicate thattreating epoxyfiberglass filters with PEI greatly enhances the removal of both bacteriophages and animal viruses atpH 7 and 9. One very notable exception was the removal of poliovirus type 1 at pH 7; PEI-treated and untreated filters removed less than10%of poliovirus type 1 from the buffer.

In addition to PEI, several othercationic polymers were

investigated for their ability to enhance virus removal by

epoxyfiberglass filters by the simple modification procedure

TABLE 2.

Influence

ofpH onvirusremovalby untreated and PEI-treatedepoxyfiberglassfilters"

%virusremovedfrom:

Virus pH Untreated

PEI-treatedfilters filters

Bacteriophage MS2 3.5 87 98 5 100 57 7 100 28 9 100 24 Bacteriophage T2 3.5 100 85 5 100 92 7 100 20 9 100 60 BacteriophageXX174 3.5 76 100 5 29 97 7 100 28 9 100 41 Poliovirus type 1 3.5 19 100 5 72 100 7 0 7 9 98 0 Coxsackievirustype B5 3.5 72 100 5 100 35 7 100 0 9 100 0 Bacteriophages and 3.5 71 ± 31b 97 ± 7 viruses 5 80 31 76 29 7 80 45 17 13 9 100 11 25 26 Bacteriophages 3.5 88 ± 12 94 ± 8 5 76±41 82±22 7 100 ±0 25 ±5 9 100 ±0 42±18 Viruses 3.5 46 ± 37 100 ± 0 5 86 ±20 68±46 7 50±70 4± 5 9

99±

1 0

"The procedure described in Table 1, footnote a, wasused, exceptthat threelayers of filters (pore size,0.25 pm) wereused. Epoxyfiberglass filters weretreatedwith0.5%PEI,asdescribedinTable 1, footnote b.Theresults areexpressed asthepercentageof virus in thebufferpriortofiltering,and the valuesindicate the meanofduplicatedeterminations.Thestandard errorwas less than 20% ofthe mean for all values.

"Valuesindicatethemean ±standarderrorfortheindicated groups.

described above (Table 3). For the removal ofbacteriophage MS2, the cationic polymers produced by Nalco, Dellchem,

and LCI were found to be as efficient as PEI, with 100% of bacteriophage MS2 being removed from 100 ml ofbuffer at pH 5, 7, and 9. No single polymer-treated filter was able to remove poliovirus type 1 at all pH values tested. However, filters treated with the Nalco cationic polymer were able to remove greater than 99% ofpoliovirus type 1 from buffer at pH 5 and 7, whereas PEI-treated filters were able to remove 97% of poliovirus type 1 at pH 9. These polymer-treated filters were further investigated for their ability to recover adsorbedbacteriophage MS2 andpoliovirus type 1 (Table 4). The recovery of bacteriophage MS2 from PEI and Nalco polymer-treated filters with 3% beef extract was 0 and 5%,

respectively. However, 22 and 18% ofbacteriophage MS2 were recovered from filters treated with Dellchem and LCI

MODIFYING MICROPOROUS FILTERS FOR VIRUS CONCENTRATION

TABLE 3. Effect of pH on the removal of viruses from buffer by epoxyfiberglass filters modified with cationic polymers"

%ofthefollowingremoved: pH Polymer

BacteriophageMS2 Poliovirus type 1

5 None 22 ±6 100 0 PEI 100 ±0 58 15 Nalco 100± 0 100 ± 0 Dellchem 100± 0 84 ± 6 LCI 100 0 96 6 7 None 28 1 21 2 PEI 100 ± 0 3 6 Nalco 100 ± 0 100 ± 1 Dellchem 100 ± 0 74 ± 16 LCI 100 0 89 5 9 None 41 11 12 16 PEI 100 0 97 3 Nalco 100 0 0 0 Dellchem 100 0 0± 0 LCI 100 0 0± 0

"The proceduredescribed in Table 1,footnote a, was used except that 100-ml volumesof seeded buffer were passed through three layers of filters (poresize,0.25purm)in the following order: pH5,7.and 9. Filters were treated with0.1%of the indicated polymer, as described in the text. The results are expressed as the percentage of virus in the buffer prior to filtering. Values indicate the mean± standard deviation of four determinations.

TABLE 4. Adsorption and recovery of viruses to epoxyfiberglass filtersmodified with cationic polymers"

Bacteriophage MS2 Poliovirus type 1 Polymer used to

modifyfiltersb % % % %

Adsorbed Recovered Adsorbed Recovered

None 0 0 0 0

PEI 100 0 100 85

Nalco 100 5 68 42

Dellchem 98 22 34 0

LCI 100 18 39 5

"The procedure describedin Table 1,footnote a, was used, except that threelayersoffilters(poresize, 0.25 ,um) wereused: thebuffer pH was 9.0. Adsorbedviruseswererecovered bypassing10mlof3%beef extractthrough thefilterfollowingpassageofthe seededbuffer. The results areexpressedas the percentage of virus in the buffer prior tofiltering.Valuesindicate the mean ofduplicatedeterminations. The standard errorwasless than20%of themean

for all values.

bEpoxyfiberglassfilters were treated with0.1%of the indicatedpolymerin deionizedwaterfor 2 h at 25°C. Filters were then dried on adsorbent paper towels at25°C. E ow 601 50c 40' 30O 20' 10' 0' o _20 0-IL _n40 N -50 -60 _70 m80 _O90 *Untreated APEI *Nalco N *5T -2 3 4 5 6 8 9 10 pH

FIG. 1. Zeta potentials ofepoxyfiberglass filter material treated withcationicpolymers.

polymers, respectively. Atotallydifferentpatternwasfound

forpoliovirus type 1 recovery, in which PEI-treated filters

gave 85% recovery and Nalco polymer-treated filters gave

42% recovery of poliovirus type 1. We were not able to

recoverpoliovirus type 1 fromDellchem and LCI

polymer-treated filters under the conditions tested.

Untreated, PEI-treated, and Nalco polymer-treated epoxyfiberglassfilterswerethenevaluated for theirabilityto

recover poliovirus type 1, coxsackievirus type B5, and echovirus types 1 and 5 from dechlorinated tap water at

ambientpH (pH 8.1) (Table 5). For the four virusestested, filters treated with cationic polymers adsorbed a greater percentageof viruses(96 7)andallowedagreater percent-ageof virusrecovery(99 + 12)than did untreated filters(20

12%adsorbed and 18 + 25% recovered).

Thezetapotentialsofepoxyfiberglassfilter material mod-ified withcationicpolymers following disruptioninablender

areshown inFig.1. At thepHvaluesinvestigated,untreated

filter material showed a negative charge, PEI-treated filter

TABLE 5. Adsorption and recovery of enteroviruses fromdechlorinated tapwaterusingepoxyfiberglassfilters modified with cationic polymers"

Virus

Polymer" P1 CB5 El E5

% Adsorbed %Recovered % Adsorbed %Recovered % Adsorbed % Recovered % Adsorbed %Recovered

None 37 13 55± 11 8± 13 3±2 20 13 5 1 15 11 7 2

PEI 79 7 75 ±7 99± 1 104 7 99 ± 1 99 ± 99 1 111 5

Nalco 100 1 100 + 0 98±3 112 17 95 ± 1 94 5 98 1 98 7

"Dechlorinated tap water (100ml)atambientpH (8.1)seeded withapproximately105PFU/ml of theindicatedviruswerepassed throughthreefilterlayers(pore size,0.2,um)ina25-mm-diameter filter holder. Adsorbed viruseswererecovered with 10 mlof3%beefextract(certified: DifcoLaboratories,Detroit,Mich.1-l MNaCI(pH 9.0).Theresults areexpressedasthepercentage of virus in the tapwaterpriortofiltering. Valuesindicate themeanand standarddeviations of triplicatedeterminations.

bEpoxyfiberglassfilters were treatedasdescribed in Table 1. footnote h. with0.5% PEIor0.05%cationic

polymner

(Nalco). -=III,& m m F--n a 0 VOL.54, 1988 1327

1328 PRESTON ET AL.APLENRN.Mcoo. material showed a

positive charge,

and Nalco-treated filter

material showed an intermediate

charge.

The

ability

of PEI-treated

epoxyfibergiass

filters to re-cover

indigenous

bacteriophage

from 100 ml of

prefiltered

secondary

unchlorinated effluent andrawsewage atambient

pH

is

presented

in Table 6. A

single layer

of PEI-treated filter materialwasabletoremove100and98% of

indigenous

bacteriophage

from 100 ml of

secondary

unchlorinated ef-fluent and raw sewage,

respectively.

The recovery of ad-sorbed

bacteriophage

with 3% beefextractwas less

impres-sive,

with 18% recovery from

secondary

unchlorinated effluent and 39% recoveryfrom raw sewage.

The

breakthrough

volume ofa

single layer

of PEI-treated

epoxyfiberglass

filter for the

adsorption

of

indigenous phage

from unchlorinated

secondary

effluent at ambient

pH

is shown in Table 7. The virus

breakthrough

volume ofa

single

25-mm-diameter filter under these conditions was found to be between 200 and 300 ml.

DISCUSSION

The

ability

of

electronegative

and

electropositive

filtersto adsorb viruses from water

samples

has been well

docu-mented,and thesefilters have been usedtodetect viruses in environmental water

samples

(1, 6). In

general,

electronega-tive filters do not adsorb viruses well under ambient water

conditions,

whereas

electropositive

filters are more efficient at this task.

Electropositive

filters such as

asbestos-con-taining

filters

(Seitz) (9),

diatomaceous earth and

anion-exchange, resin-containing

filters

(Zeta-plus)

(9, 12), and

charge-modified,

resin-containing

filters (Virasorb 1-MDS

filters)

(9, 11) have been used to concentrate viruses from surface and wastewaters

(9,

11, 12). These

electropositive

filters, however,

have somenoted

disadvantages.

Asbestos-containing

and diatomaceous earth filters have slow flow rates, which

prohibit

the

analysis

of

large

volumesof water, whereas

charge-modified, resin-containing

filtersare expen-sive relativeto otherfiltertypes.

Although

PEI-treated

glass

surfaces have been used to immobilize yeast cells (4), this is the first report on the utilization of this novel

approach

to

modify

microporous

filters for the concentration of viruses from water. The results of this

study

indicate that the

virus-adsorbing

prop-erties of

electronegative

epoxyfiberglass

filters can be

greatly

enhanced

by treating

the filters with an aqueous solution ofacationic

polymer

suchasPEI.This modification results in afilter which adsorbs viruses well under ambient conditions while

retaining

the

high

flow rates

possible

with untreated

epoxyfiberglass

filters. There are

presently,

how-ever, somenoted

disadvantages

tothe useof filters modified with cationic

polymers. Although

modified filters adsorb

TABLE 6. Recoveryofindigenousbacteriophagefrom unchlorinatedsecondary effluent andrawsewage by PEI-treated

epoxyfiberglass filters"

Watertype pH % Adsorbed % Recovered

Secondary unchlorinated 6.4 100 18

effluent

Raw sewage 7.0 98 39

"Theindicated watertype (100ml)prefiltered withaG25fiberglassfilter

werepassedthroughonelayerofPEt-treatedepoxyfiberglassfilter(poresize.

0.25pm).Adsorbed viruswererecovered with 10mlof 3% beefextract(pH 9.0). The resultsareexpressedasthepercentage of viruses in thewaterprior to filtering. Values indicate the mean of duplicate determinations. The standarderrorwasless than20% of themeanfor all values.

TABLE 7. Virusbreakthroughvolume of PEI-treated

epoxyfiberglassfiltersforindigenous phagefrom unchlorinated

secondaryeffluent" Vol(ml) % Adsorbed 100... 100 200... 99 300... 44 400... 27 500... 7

"Five100-mIportionsofG25-prefilteredsecondary unchlorinated effluent (pH 6.5)were passed through asingle layerofPEI-treated epoxyfiberglass

filtermaterial(poresize 0.25pLm).Theseportionswerecollected andassayed separately.The resultsareexpressedasthepercentage of virus in thewater

priortofiltering. Valuesindicate the meanofduplicate determinations. The standarderror wasless than 20%of themeanfor all values.

bacteriophages (MS2,

T2, and (~X174) and enteroviruses

(poliovirus

type 1, coxsackievirus type B5, and echovirus types 1 and 5) well from buffer and tapwater at

pH

values indicative of ambient water conditions

(pH

5, 7,and 9), no

single

polymer

wasabletoadsorb

poliovirus

type 1atall

pH

values tested. The recovery of

bacteriophage

MS2 and

indigenous

bacteriophage

with 3% beef extract

following

adsorption

toPEI-treated

epoxyfiberglass

filters alsowasnot efficient. In

addition,

our

preliminary

results indicated that the

stability

of the modified filters overtime wasless than 2 weeks(datanotshown). This isnotagreat

disadvantage,

as

the modification

procedure

simply

entails

soaking

the filters for2 h in anaqueous solution ofcationic

polymer

and then

allowing

the filterto airdry

overnight.

The type of cationic

polymer

usedto

modify

epoxyfiberglass

filters wasfound to

be

important

for boththe

adsorption

andrecoveryof viruses frombuffer. For

example,

LCI and Dellchem

polymers

were

found to be better suitedfor the recovery of

bacteriophage

MS2, whereas PEI and Nalco polymers were found to be best suited for

poliovirus

type 1 recovery under the same

conditions. All

polymers

tested, however, werefound tobe

equallyeffective for the removal of

bacteriophage

MS2from buffer at

pH

5, 7, and 9, whereas the

adsorption

and recoveryof

poliovirus

type 1werefoundtobe

dependent

on

thetype of

polymer

usedtotreatthefilters,aswellasthe

pH

ofthe buffer.

The resultsof this studyindicate that

fiberglass

filterscan

bemodified with cationic

polymers

simply

and

inexpensively

to enhance their

virus-adsorbing properties

under ambient

waterconditions. We feel that theuseof filters modified with cationic

polymers

is most

promising

for the detection of

indigenous

bacteriophages

and enteroviruses inwaters. Fur-ther work will concentrate on

enhancing

the recovery of

bacteriophage

adsorbedtofilters treated with cationic

poly-mers, as well as the

adsorption

and recovery of

enterovi-ruses under ambient water conditions,

by combining

PEI and Nalco cationic polymers in the filter modification pro-cedures.

ACKNOWLEDGMENTS

This study was supported by the Center for NaturalResources, Institute ofFood andAgricultural Sciences,andbytheEngineering and Experiment Station, University ofFlorida, Gainesville. Jean-Louis Morelwassupported byafellowshipfromthe NorthAtlantic TreatyOrganization.

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