Electrophoretic mobilities of virus adsorbing filter materials

B'alt,r Re.~,'arch Vol. 12. pp. 263 to 268 ~) Pergamon Press Lid. 1~78. Printed in Grcal Britain

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ELECTROPHORETIC MOBILITIES OF VIRUS ADSORBING FILTER MATERIALS M. A. KESSICK* and R. A. WAGNER'[" Department of Environmental Science and Engineering, Rice University. Houston. TX 77001 U.S.A. (Received in final form 10 September 1977)

Abstract--Surface charge characteristics of virus adsorbing filters were studied by electrophoretic measurements of particulate suspensions under conditions commonly encountered in the adsorption technique for virus concentration. A mechanism for virus interaction with filter surfaces, involving cross-complexation by multivalent cations, is discussed.

CTION

be to study the surface charge characteristics of the filter surfaces compared to those of viruses under a variety of conditions. Electrophoretic mobilities of filter material particles in aqueous suspension were therefore measured as a function of pH, ionic strength, salt-type, and protein concentrations. The results were considered indicative of the surface charge of the filters under operating conditions. Surface charge properties of particles in suspension may be experimentally determined by the microscope method of electrophoresis (microelectrophoresis). The technique depends on the direct observation of microscopicaUy visible particles as they migrate in an electric field. The particles are suspended in a suitable electrolyte and placed in a glass (or plastic) cell through which an electric current is passed. Since particle surfaces are in thermodynamic equilibrium with ions in solution, the surface charge of a particle actually fluctuates as a function of time. However. these fluctuations are shorter than the time necessary to measure a particle's mobility, with the result that a statistical time average of surface charge is given by electrophoresis (Brinton & Lauffer, 1959). Electrophoresis yields no information on local surface charges of particles. Although the examination of the surfacc charge properties of viruses themselves was not undertaken during this study, some information on this topic is available in the literature. A virus is known, for instance, to consist predominantly of a nucleic acid core surrounded by a protein or a protein-lipid shell (Goodheart, 1968). The physical and chemical characteristics of viruses are in many ways similar to those of proteins (Hill et al., 1971). Mandel (19711 has characterized poliovirus type I, an enterovirus, by direct electrophoretic analysis. By examining this virus over a broad pH range, he was able to construct a curve relating electrophoretic mobility rate (mm min-~) to pH. The curve reveals that at pH 3.5 (at which virus * Present addresses: Alberta Research Council, Edmonadsorption to filter surfaces seems optimuml, the ton, Alberta, T6G 2C2, Canada t Department of Environmental Science and Engineer- polio virion is net positively charged, and at pH 7 ing. University of North Carolina. Chapel Hill, NC. U.S.A. or above it is negatively charged. INTROD

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For many years it has been known that under certain conditions viruses can be removed from aqueous systems by adsorption to filters which have nominal pore sizes many times the viruses' own diameter (see, for instance, Cliver, 1965). In particular a virus concentrator has recently been developed (Wallis et al., 1972a, b; Homma et aL, 1973) which consists basically of such a filter serving as a virus adsorbent in a continuous flow system. Optimum conditions for operation of the concentrator are reached after adjustment of the pH to 3.5 and after addition of a small quantity of a multivalent metal salt, usually AICI3. Adsorbed viruses can then be eluted from the filter with small volumes of high pH buffer, effectively concentrating them from many liters of sampled water into volumes of one liter or less. Several mechanisms for virus adsorption to surfaces other than filters have been proposed. Valentine & Allison (1959) described adsorption rates of viruses to non-biological surfaces in terms of diffusional forces (Brownian motion) operating in the aqueous system. They noted that multivalent cations increased the adsorption rates. Cookson (1969); Gerba et al. (1974) studied virus adsorption to activated carbon and concluded electrostatic interaction between the two surfaces mediated adsorption. Carlson et al. (1968); Schaub et al. (1974) studied virus adsorption to clays, and observed that adsorption was dependent upon concentration and type of cation present in the suspending medium. They explained adsorption in terms of chemical and ,~an der Waals forces operating in their systems. Little discussion, however, appears in the literature on the mechanisms of adsorption of viruses to filter materials. In light of the above, it was felt that a promising avenue for investigation in this area would

263

264

M.A. KESSI('K and R. A. WAGNER MATERIALS AND METHODS

The microelectrophoresis apparatus

The microelectrophoresis apparatus employed in this research was modified after an electrophoresis cell originally described by Briggs (1940). This cell was purchased from the Ace Glass Company of Rockville, Maryland. Palladium electrodes as described by Neihof (1969) were constructed and used successfully. Microelectrophoresis of filter suspensions was performed according to procedures described by Black & Smith 0962). Electrophoretic mobilitics of the filter particles were determined by substituting experimental values into the equation: = (dX/tIRs)

AICI3, were prepared by dissolving the various salts in deionized water. Protein solutions were prepared by dissolving various amounts of purified bovine serum albumin in 0.02 M KCI. Tapwater was obtained from laboratory faucets. Based on recent analysis it was calculated to have an ionic strength value of 0.011. Secondary sewage effluent was obtained from a local trickling-filter treatment plant. The effluent was centrifuged at 3000 rpm for 15 rain to remove particulate matter, and total organic carbon analysis of the centrifuged effluent yielded a value of 20mgl-~, As no analysis for ions present in the secondary sewage effluent was available, the ionic strength of the effluent was estimated at 0.019. This estimate was calculated by considering the average concentrations of ions added during water use (American Chemical Society. 1969).

where ~ is elcctrophoretic mobility (tim s - i per V cm - i), d is dislancc of particle migration (itm), X is cross-sectional Filter particle suspensions area of the electrophoresis cell (cm2). t is time of particle Particle suspensions suitable for electrophoretic analysis migration is). I is current (amps), and R, is specific resist- were prepared by blending the various filter materials in ance of the particle suspension (ohm-cm). If particles 150ml of suspending medium in a one speed Waring migrated toward the positive pole in the electrophoresis blender for two min. The following amounts of filter materapparatus, a net negative surface charge was indicated, and ials were used: (11 Millipore--three 47 mm diameter memhence a negative electrophoretic mobility (and vice versa). branes/150 ml; (2) Commercial Filters-- 15 cm yarn/150 ml; For each determination, six particles were timed with the (3) Filterite = 10cm 2 membrane material/150ml. These polarity in one direction, and a further six with the polarity quantities were blended and filtered through two gauze reversed, t was thus an average of 12 values. pads to yield particle suspensions sufficiently concentrated The microelectrophoresis apparatus was standardized by to produce a readily visible particle density in the microthe determination of the electrophoretic mobility of human scope viewing field. The particles were generally irregularly red blood cells (Black & Smith, 1962). Abramson (1964) shaped, except in the case of the Commercial Filters materreported the mean value for the mobility of human RBC ial which yielded fiber-like particles. Only particles whose suspended in KHzPO,~(0.067 M),/Na2HPO4 (0.067 M) at diameters [or length) fell in the approximate range of pH 7.40 to be +l.31,ums - t per Vcm -~ at 25°C. This 2-10/~m were timed as they migrated in the electric field. value has been verified by other investigators (Neihoff, 1969). Using the Briggs cell with palladium electrodes, the RESCLTS AND DtSCtJSStON electrophoretic mobility of human RBC was determined to be + 1.47 + 0.13 [mean and standard deviation of five p H effects values) at room temperature (25 + 2 C). The value did not appear to vary with temperature within this range. Because The electrophoretic mobilities of filter particles susthis microelectrophoresis apparatus yielded an electropended in KC1 (0.02 N)/HCI(0.02 N) solutions were phoretic mobility for human RBC that was higher than published values, the electrophoretic mobilities for filter measured as a function of pH over the range 2-7, materials reported in this study may be slightly higher than The choice of suspension medium was such that the real values. In most cases, however, a relative change in ionic strength was maintained at 0.02. The use of a mobility was the significant result (i.e. change in mobility . symmetrical monovalent salt in these studies also as a function of pH or ionic strength). served to minimize specific ion effects resulting from adsorption or interaction with the filter surface. Filter materials Although the buffering capacity of a KCI/HCI system Three lilter materials capable of adsorbing viruses were is known to be poor a r o u n d neutral pH, the maxistudied in this research. (1) Millipore membrane filter: type MF HATF, 47 mm diameter, 0.45/am porosity, manufacm u m change in pH measured during these experitured by Millipore Corp.. Bedford, Mass. These fiat memments was 0.34 units at a mean value of 6.59. This brane filters are made from mixed esters of cellulose with constituted a range of 5.2,°/o. This c o m p a r e d to a maxicellulose nitrate predominating (Mix, 1973), and had not m u m 9 5 ~ confidence interval of 5.7~ in electrobeen treated with non-ionic detergents by the manufacphoretic mobility measurement. For the sake of clarturer. {2) Commercial Filters depth wound filters: type Fulflo WIOA-7, 10 inch tubes, I ltm porosity, manufactured ity p H ranges a n d confidence intervals are not by Commercial filters Division, Carborundum Corp., Niadepicted in the Figures. gara, N.Y. These filters are made from cellulose acetate The experimental results d e m o n s t r a t e that filter yarn which is wound around a perforated tube (Wallis et materials capable of a d s o r b i n g viruses exhibit net al., 1972a). (3) Filterite pleated membrane filter: type Duoline, 10 inch tubes. 0.45 ,urn porosity, manufactured by Filnegative surface charges in the presence of KCI/HCI terite Corp., Timonium, MD. These filters consist of a fiat over the entire pH range studied (Fig. 1). In all cases rectangular membrane which has been pleated and these materials were least negative at low pH. The wrapped around a perforated tube. The membrane is made charge characteristics of cellulose nitrate and cellulose of glass fibers bonded together by epoxy resins. Melamine acetate appear very similar. The net negative charge impregnated paper is layered on the outer filter surface. This paper was removed prior to preparation of filter parexhibited by these cellulosic filters may be due in part ticle suspensions (Farrah et al., 1976l. to the presence of ionized carboxyl groups on the cellulosic surface. Such carboxyl groups are known Particle .~u.spen.sion me,lia Several filter particle suspension media were used in this to be present in purified natural cellulose (Sookne study. Salt solutions, including KCI, MgCI2, CaCI2, and & Harris, 1954). It is also possible that many nones-

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medium. Under elution conditions (pH 11.5) the viral and filter surfaces would have net negative charges, and hence electrostatically repel one another.

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materials. terified hydroxyl groups contained within the cellulosic materials may, like phenolic hydroxyl groups (Edsall & Wyman, 1958), serve as good hydrogen bonders, and perhaps form hydrogen bonds with hydroxide ions from solution. The fiberglass/epoxy filter material exhibited slightly different charge characteristics in that the net negative charge appears to be greatest at pH 5.0 within the range studied, although it would be expected to increase again at high pH. The composite nature of this material may explain the occurrence of this negative peak in the curve relating electrophoretic mobility to pH (Fig. 1). The negative charge exhibited by the fiberglass/epoxy material probably arises in part from ionization of - S i O H groups on the fiberglass surface. The effect of the epoxy binding agent in unknown. The dependence of filter charge on pH would seem to support an electrostatic theory of adsorption. It is known that at pH 3.5, a typical viral surface is positively charged (Mandel, 1971), and hence would be attracted to the negatively charged filter surfaces. Indeed, Sobsey et al., (1973) demonstrated that by simply acidifying tapwater (pH 3.5) viruses could be efficiently adsorbed to filters from the aqueous 2.01

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Protein effects Electrophoretic mobility of Millipore cellulose nitrate filter material as a function of protein concentration (purified bovine serum albumin) was studied at pH 3.5 and pH 7.0. In both cases protein concentration varied from 0.001 m g l - | to 10mg-~. The protein was first dissolved in 0.02 N KCI; this protein solution was then adjusted (0.02 N HCI or 0.02 N NaOH) to either pH 3.5 or 7.0 and stirred 20rain prior to electrophoresis. The results are shown graphically in Fig. 2. The equations of the regression lines that best fit the data were calculated for pH 3.5 and 7.0, and the correlation coefficients were found to be 0.97 and 0.99 respectively. The studies using protein were carried out to help support the concept of electrostatic adsorption since the viral surface is proteinaceous in nature and generally may be considered to exhibit charge properties similar to those of proteins. The protein used, purified bovine serum albumin, is isoelectric at pH 4.75 (Longsworth & Jacobson, 1949y From Fig. 2 it may be seen that at pH 3.5 in KCI/HC1 (ionic strength 0.02) the cellulose nitrate surface exhibits a positive character at high protein concentration (_>0.1 mg I-~). This may be attributed to the fact that the positively charged protein molecules are adsorbing to the negative filter surfacc, effectively masking the negative charge of the filter material. At pH 7.0 ........

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in KCI/NaOH (ionic strength 0.02), the net negative charge of the cellulose nitrate did not change as protcin concentration varied from 0.001 to 10mg/l. At this pH one would not expect adsorption of the negatively charged protein to the negative filter surface, and the data arc consistent with this concept.

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The effects of four salts (KCI, MgCI2, CaCI2, AICI3) on the surface charge properties of Millipore cellulose nitrate lilter material were studied at both pH 3.5 i and pH 7.0. In addition the effects of AICI 3 on surface charge properties of Filterite epoxy-fiberglass were studied at both pH levels. The data describing electrophoretic mobility as a function of ionic strength of the various salt solutions are presented graphically in Figs. 3 and 4. The equations of the regression lines that best fit the data and correlation coefficients were calculated for each salt study (except AICIs at pH 7.0). In all cases correlation coefficients were greater than or equal to 0.98. Several observations indicate that any interpretation of virus adsorption to filter surfaces must include discussion of the role of salts in modifying surface charges since (1) virus adsorption is known to occur at neutral pH in the presence of salts despite the fact that both virus and filter are negatively charged, (Wallis et al., 1972c) and (2) the requirements for salt enhancement of virus adsorption at acidic pH levels has been demonstrated in attempts to concentrate viruses from tapwaters at high flowrates (Farrah et al., 1976) and polluted waters (Homma et al., 1973). The effects of the four different salts (KCI, MgCI2, CaCI2, AICI3) on the surface charge properties of cellulose nitrate at both pH 3.5 and 7.0 clearly demonstrate that the action of certain salts on the filter charge must be explained by phenomena other than simple ionic strength effects. This is apparent from the significant differences in cellulose nitrate electrophoretic mobilities in the presence of the various salts for a given ionic strength value (at both pH 3.5 and 7.0). In general monovalent cations are less effective than divalent cations which are in turn, lcss effective than trivalent cations at decreasing the net negative charge characteristics of cellulose nitrate. Indeed, just as Nordin et al. (1967) observed with glass surfaces and Carlson et al. (1968) with clay surfaces, thc trivalent cation (AI3÷) was capable of actually reversing the filter surface charge from negative to positive, at both 3.5 and 7.0. The AI 3+ cation was similarly capable of reversing the surface charge of the fiberglass/epoxy material also at both pH values (Figs. 3 and 4). This is strong evidence for the concept of specific ion adsorption to filter surface groups (Davis & Rideal, 1961). It should be noted that at pH 7.0, increasing AIC!3 concentration beyond a certain level did not appreciably change the electrophoretic mobility of cellulose nitrate or fiberglass/epoxy filter particles. This may

indicate that saturation of filter surface groups with Ai 3÷ or its charged hydrolysis products has occurred. Tapwater and effluent effects

Microelectrophoresis of cellulose nitrate filter particles in tapwater and secondary sewage effluent at pH 3.5 (Fig. 5) revealed that the net surface charge of the filter materials remained the same whether the particles were suspended in tapwater, effluent, or tapwater/effluent mixtures. This implies that proteinaceous organic material in particular was not present in the elttuent in significant amounts since this has been shown to adsorb strongly to filter materials (sec Fig. 2 and Homma et al., 1973). The ionic strength of both water types is approximately the same (tapwater--0.011: effluentS.019), and the effect of this parameter would be expected to be similar in each case.

The effect of addition of AICI3 on the cellulose nitrate surface in tapwater and effluent at pH 3.5 is also shown. It was discovered that at high AICI 3 concentration (0.005 M) the filter materials became positively charged in tapwater, but remained negatively charged in effluent. This was attributed to the presence of soluble organics in the effluent with which the AI3÷ cations may have preferentially interacted. resulting in the formation of soluble complexes (Perdue et aL, 1976).

Virus interactions with filters

Conditions necessary for the adsorption and elution of viruses can be summarized as follows. It is known that viruses in natural waters will absorb to filter surfaces: (l) At acidic pH levels at low flow rates (Sobsey et al., 1973); (2) At acidic pH levels at high flow rates in the presence of salts (Homma et al., 1973; Farrah et al., 1976); (3) At neutral pH levels in the presence of salts (Wallis et al., 1972c). Viruses can be desorbed or eluted from the filters: (l) In buffered systems (0.05M) glycine) at high pH (ll.5) (Wallis et al., 1972c); (2) At neutral pH in the presence

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Electrophoretic mobilities of virus of protein (Wallis & Melnick, 1967). High pH buffers seem to be the best eluents, although virus inactivation occurs at pH 11.5 as a function of time, (Farrah et al., 1976). Although the effect of cations on the surface charge properties of viruses does not appear to have been studied, it is known that both cations and anions may adsorb to protein molecules, and by inference may change the net surface charge of a virus (Mix, 1973). More specifically, Smith (1936) demonstrated that divalent cations shifted the isoelectric pH of ovalbumin to higher pH values. Thus, while it is most probable that specific interaction of multivalent cations with filter surfaces is of primary importance in the adsorption of viruses to these surfaces, it is also probable that specific interaction of the multivalent cations with the viral surface is of comparable importance. It is believed that virus adsorption to filters at neutral pH may be described in terms of a more generalized theory of interactions between surfaces of like charge (such as clays and organics in natural waters) in the presence of multivalent counterions (Stumm & Morgan, 1970). According to such theory, at pH 7.0, although both viral and filter surfaces exhibit net negative surface charges, these are reduced by specific adsorption of multivalent cations to both surfaces. This, coupled with a possible ionic strength effect on the configuration of the double layers, facilitates the approach of the two surfaces, allowing van der Waals forces to have significant effect. When multivalent cations are present we believe there is the further distinct possibility of cross-complexation between the viral and filter surfaces. That is, a multivalent cation may complex with groups on the two separate surfaces simultaneously. The trivalent cation (AI 3÷) would be expected to serve as an excellent cross-complexing agent owing to its high charge. The divalent cations (Ca 2÷ or Mg 2+) would not be expected to cross-c0mplex as effectively. This could explain the requirement for higher concentrations of these cations to effect adsorption (Wallis et al., 1972c). Monovalent cations would be expected to be poor complex formers, incapable of cross-complexation. At the high concentrations of these ions necessary to enhance virus adsorption, it is very likely that adsorption in this case is due solely to van der Waals attractive forces, operating by virtue of a short distance of closest approach at high ionic strength, as described in the VODL theory of coagulation (Stumm & Morgan, 1970). It is suggested that crosscomplexation by multivalent cations is more important than van der Waals forces in promoting attachment of surfaces in trivalent and possibly divalent systems, and that the action of these cations might be described in terms of a "cationic adhesive" effect. This "adhesive" effect seems to be demonstrated best by adsorption characteristics at pH 3.5 when the filter surface is net negatively charged and the viral surface positive, and by conditions required for subsequent elution of the viruses. At pH 3.5, electrostatic

267

attractive forces appear sufficient at low flow rates to promote adsorption from tap water, (Sobsey, 1973). although some cross-complexation involving Mg 2+ and Ca 2+, which were present to a total concentration of 0.003 M, might be occurring to some small degree. At high flow rates, however, the presence of an efficient cationic "adhesive", such as AI3÷, seems necessary to maintain adsorption after the initial impaction step. Although the viral surface exhibits a net positive charge at this pH it is likely that there are sufficient residual negatively charged surface groups to enter into the cross-complexation process. At pH 3.5 and at the concentrations involved, thermodynamic data (Hayden, 1974) indicate that AI 3+ is the only aluminum species present in significant concentration. The fact that the concentration of AICI3 (0.0005 M) reported necessary to enhance virus adsorption (Wallis et al., 1972c) is not capable of reversing thc surface charge on cellulose nitrate in tapwater or secondary sewage effluent at pH 3.5 (Fig. 5) also indicates that negative groups are available for crosscomplexion on this surface under these conditions. In cases where secondary effluent is present, an equilibrium might be established as follows: [AI3+--filter] ~ [AI 3+ ] ~ [Al3+--organics] In heavily polluted water samples this equilibrium would be shifted to the right, and fewer AI3÷ would be available for complexing with the filter or viral surfaces. On the other hand, it is conceivable that organic-Al 3+ complexes under some circumstances may accumulate on the filter surface and actually serve as additional virus adsorption sites. The most efficient desorption, or elution, or viruses from filters is carried out under basic conditions (i.e. 0.05 M glycine buffer at pH 11.5) (Wallis et al., 1972c). Equilibrium considerations of the AI 3÷ cation and its hydrolysis products (Morgan, 1967) show that aluminum is amphoteric and will form the soluble species AI(OH)~ at pH 11.5. Under these conditions the "'adhesive" would be dissolved out, allowing electrostatic repulsive forces to dominate the system and cause desorption of the viruses from the filter. As calcium or magnesium are not amphoteric and will not form soluble species at high pH, breakdown' of any adhesive effect from their cations would not occur by pH elevation alone, and elution would be more difficult. Data supporting this concept havc been reported by Stagg (1976). He found that bacteriophage MS2 could be adsorbed efficiently to cellulose nitrate surfaces in the presence of MgCI2 (0.05 M) at pH 7.0. However, satisfactory elution of viruses from the filter was not achieved using the conventional high pH eluent. Stagg observed, however, that the addition of EDTA. a chelating agent capable of strongly binding divalent cations, to the high pH eluent greatly facilitated the desorption of viruses from the filter surface. In this case, then, the EDTA can be considered to have dissolved out the "'cationic adhesive".

268

M.A. KEg.SICK and R. A. WAGNER REFERENCES

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Mix T. (1973) The physical chemistry of membrane-virus interaction. Devs. Ind. Microbiol. 15, 136-142. Morgan J. J. (1967) Applications and limitations of chemical thermodynamics in natural water systems. In: Equilibrium Concepts in Natural Water Systems, Advances in Chemistry Series No. 67, (Edited by Stumm W.) Am. Chem. Soc. Publications, Washington. Neihof R. (1969) Microelectrophoresis apparatus employing palladium electrodes. J. Colloid Interface Sci. 30, 128-133. Nordin J. S.. Tsuchiya H. M. & Fredrickson A. G. (1967) lnterfacial phenomena governing adhesion of chlorella to glass surfaces. Biotech. Bioen.qnq. 9, 545-558. Perdue E. M., Beck K. C. and Reuter J. H. (1976) Organic complexes of iron and aluminum in natural water. Nature, Lond. 260, 418-420. Schaub S. A., Sorber C. A. & Taylor G. W. (1974) The association of enteric viruses with natural turbidity in the aquatic environment. In: Virus Survival in Water and Wastewater Systems (Edited by Malina J. and Sagik B.). Univ. Texas Press, Austin, Texas. Smith E. R. B. (19361 The influence of method of preparation and of cations on the isoelectric point of ovalbumin. J. hiol. Chem. 109, 473-478, Sobsey M. D., Henderson M., Wallis C. & Melnick J. L. (1973) Concentration of enteroviruses from large volumes of water. Appl. Microbiol. 26, 529-534. Sookne A. M. & Harris M. (1954) Chemical nature of cellulose and its derivitives--base exchange properties. In: Cellulose and Cellulose Derivitires, Vol. 1 (Edited by Ott E., Supurlin H. M. & Grafflin M. W.) Interscience. New York. Stagg C. H. (19761 Inactivation of solids-associated virus by hypochlorous acid. Doctoral thesis, Rice Univ., Houston, Texas. Stumm W. & Morgan J. J. (1970) Aquatic Chemistry, pp. 458-465, 285-287. 496--497. Wiley-lnterscience. New York. Valentine R. C. & Allison H. C. (1959) Virus particle adsorption I. Theory of adsorption and experiments on the attachment of particles to non-biological surfaces. Biochem. Biophys. Acta 34, 10-23. Wallis C., Henderson M. & Melnick J. L. (1972c) Enterovirus concentration on cellulose membranes. Appl. Microbiol. 38, 476-480. WaUis C.. Homma A. & Melnick J. L. (1972a) Apparatus for concentrating viruses from large volumes. J. Am. War. Wks Ass. 64, 189-196. Wallis C.. Homma A. & Melnick J. L. (1972b) A portable virus concentration for testing water in the field. Water Res. 6, 1249-1256. Wallis C. & Melnick J. L. (1967) Concentration of viruses from sewage by adsorption on millipore membranes. Bull. WId HIth Or9. 36, 219-225.