USES OF ION EXCHANGE RESINS IN MICROBIOLOGY

BORIS ROTMAN'Instituto de Quimnica Fisiologica, Escuela, de Medicina, Santiago, Chile

The use of synthetic ion exchange resins inmost fields of chemistry has been expandingsteadily for the past two decades. In micro-biology, the use has been practically limited tochemical analysis, biochemical preparations, orpurification of water. More direct applications,such as isolation of microorganisms or removal ofextracellular material by direct treatment of cellsuspensions with resins, are scarce.

This review is concerned with situations inwhich a chemical or physical process was ac-complished by placing microorganisms directlyin contact with ion exchange resins. The aim isto direct the attention of microbiologists towardsion exchange processes which greatly simplifycertain type of operations involving microorgan-isms and which can serve as a powerful tool inphysiological studies.

GENERAL CONSIDERATIONS ABOUT IONEXCHANGE RESINS2

Ion exchange resins may be regarded as in-soluble polymeric electrolytes of high molecularweight composed of an elastic-three-dimensional-hydrocarbon network to which a large number ofionizable groups are bound. Thus, a cationexchange resin is considered as a large non-diffusible anion and a diffusible cation and viceversa for an anion exchange resin. With thisconcept in mind, it becomes easier to predict thebehavior of an ion exchange resin by comparisonwith simple acid-base reactions. In other words,the hydrocarbon network of the resin affects thechemical behavior of the exchanger quantitativelybut not qualitatively. The ideal network shouldconsist of a structure resistant to breakage,mechanical wear, and oxidation or reduction,and insoluble in ordinary solvents. Furthermore,it should be possible to shape it in small sphereswith good hydraulic properties. Polymers ofstyrene and divinylbenzene have most of theseproperties and therefore have been preferred as

I Present address: Veterans AdministrationHospital, Albany, New York.

2 Detailed information can be found in refer-ences (3,8,20,22,25, 29, 34, 35, and 42).

materials for the hydrocarbon network of manyresins. Divinylbenzene links together the straightchains of styrene polymers and therefore itcontributes to the third dimension of the network,makes it insoluble, and determines to what extentthe resin is free to swell and shrink. The term"crosslinkage," in a styrene-divinylbenzene resin,refers to the fraction of divinylbenzene itcontains. A resin of "8 per cent crosslinkage"contains 92 per cent of styrene and 8 per centdivinylbenzene. Crosslinkage is a usefulparameter to describe the porosity of a resinparticle. Particle size and porosity are importantfactors which determine the reaction rate andthe selectivity of the resin for specific ions.Smaller mesh sizes and low crosslinked resinshave relatively faster reaction rates. Low cross-linked resins are less selective for specific ionsbut they can exchange ions of higher molecularweight (24). They are not used routinely incolumn operations because of their low mechan-ical strength and excessive swelling and shrinking.The capacity for exchanging ions is ascribed

to sulfonic, carboxylic, phosphoric, or phenolicgroups for cation exchange resins and to aminegroups for anion exchange resins. In both cases itis possible to account for the capacity of theresin with its entire content of functional groups,indicating that the exchange is not a surfaceeffect but that it takes place throughout theentire structure of the resin (21). The rate ofexchange of ions in most resins is considered tohe controlled by the rate of diffusion of the ionthroughout the structure of the resin (6, 23).Therefore, factors such as particle size, resindensity, temperature, concentration of the ion,and hydration of the resin have a marked effecton the rate of exchange.

Resins exhibit higher exchange rates whentheir functional groups are present in a dissociatedform (table 1). Highly dissociated systems arethe salt forms of weak-basic or weak-acid resinsand all forms of strong-basic or strong-acidresins. The term weak or strong, in ion exchangeresins, refers to the basicity or acidity of theirfunctional group. Thus, -SO3H resins are

251

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

BORIS ROTMAN-

TABLE 1Rates of exchange of weak and strong

cation exchange resins*

Equilibrium Time to Att,Equilibriur

RCOOH + KOH 7 daysRCOONa + CaC12 2 minuteRSO3H + KOH 2 minuteRSO3Na + CaCl2 2 minuite

approximation for ions of the same valence isthat affinity decreases as the size of the hydratedion increases. As an example, for Dowex 50, a

Lain strong catioin exchange resini, the decrealsingLM affinitv series is:

eseses

R represents the hydrocarbon network of theresin.

* Data of Kunin and Barry, Ind. Eng. Chem.,41, 1269 (1949).

strong and -COOH resins are weak. From theseconsiderations it folloNws that the exchange ofweak resins is greatly affected by pH, whereasthat of strong resins is affected to a much lesserextent. A reaction of a strong cation exchangeresin would be:

(RSO3)H + NaCl - (RS03)Na-+ HCl

For a strong anion exchange resin the reactionwould be:

(RR3'N)OH + NaCl -* (RR3')Cl + NaOH

R represents the hydrocarbon network; R', an

organic radical such as a methyl group, which ispart of the ionizable group.

Anion exchange has been limited to the aminetype resin. The exchange in weak resins has beenexplained by adsorption (11):

RNH2 + HCI ---+ RNHC1

However, an exchange mechanism could be inplay if the reaction is visualized as:

RNH2 + H20- RNH30H

RNH30H + HCl - RNH3CI + H20

Even if the strong ion exchange resins willform stable bonds with any ioI, the bonds differin strength. Likewise, all ion exchange resinshave preferences for particular types of ions if a

mixture is presented to them. This preference istermed selectivity of the resin. The selectivity ofa resin is determined by the character of thefunctional groups. In general, at total concen-

trations of less than 0.1 N, trivalent ions havemore affinity for the resin than divalent ions,and these more than monovalent ions. An

Ag+ > T1+ > Cs+ > Rb1 > NH4+ > K+ >Na+ > H+ > Li

The selectivity of a resin also depenids on severalfactors. Among them are ionic strength, tempeira-ture, an(I pressure. Of these three, the mostimportant is ionic strength. For instance, fordivalent ions, the selectivity of the resin increaseswith dilution. Thus, a resin can effectively pickup divalent ions from a dilute solution and theycan be eluted from it with a concentrated solutionof Na+ ions.

ION EXCHANGE TECHNIQUES

Operating Methods

To discuss in quantitative terms the selectionor the performance of an ion exchange columnescapes the scope of this article inasmuch as themicrobiologist is not usually interested in theeconomical or the technical aspect of the process.Therefore, only a few practical rules to manipu-late ion exchange resins will be given here. Moredetailed discussions can be found elsewhere (seefootnote 2).There are two main distinct methods for the

utilizationi of ion exchange resins: batch andcolumnar. The batch method is simpler but atthe same time more inefficient; it is used mainlyfor small scale work. It consists of mixing a fixedvolume of an electrolyte solution with a quantityof exchanger and then separating the two phasesby anv conventional procedure, i.e., centrifuga-tion, decantation, filtration. With the batchmethod the degree of exchange in one operationdepends on the equilibrium constant for thesystem. Therefore, the number of consecutivebatch operations for complete exchange wvillvary from one system to another. For instance,the equilibrium

RSO3-H + NaCl 2 RSO3-Na + HCI

will require a large number of batch treatments,while the equilibrium

RSO3-H + NaOH - RSO3-Na + H20

will require just one. In conclusion, the batchmethod is recommended for svstems in which the

25-2 [VOL. 24

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

ION EXCHANGE RESINS IN MICROBIOLOGY

volume or the time is critical, provided that theequilibrium is quite favorabie to the exchange.The columnar method is the most widely

employed for ion exchange operation. The columntechnique can be visualized as a multiple series ofbatchwise steps. The fresh electrolyte con-tinuously in contact with the resin on the topof the column drives the reaction to completionin that area. Thus, the exchanger bed becomescompletely exhausted in the upper part first andthen gradually downward. The columnar methoddepends less on the selectivity of the resin andthe equilibrium of the reaction. This can beillustrated easily with an oversimplified example.Let us assume that A+ exchanges for B+ andthat the resin has equal preference for each one.The exchange is conducted in a row of batchoperations, each with one equivalent of resin inthe B+ ion form. A volume of solution containingone equivalent of A+ ions is placed in contactwith the first batch of resin. At the completionof the equilibrium the resin will contain 0.5equivalent of A+ and 0.5 equivalent of B+. Thiswould be the non-practical result in a batchoperation. However, if the solution from thefirst batch is added to fresh resin in a secondbatch, the final equilibrium in the solution will be0.75 equivalent of B+ and 0.25 equivalent of A+.After a third batch the solution contains 0.875equivalent of B+ and 0.125 equivalent of A+. Itis easy to visualize that after a great number ofsuch steps we can obtain practically a solutionof pure B+ in spite of the lack of preference ofthe resin.

Choice of ResinThe number of resins available permits the

selection of an optimal type for each field ofapplication. The most important properties ofthe exchangers which need to be considered toselect the optimal type are discussed below.

Crosslinkage. The degree of crosslinkage of aresin is selected according to the molecular sizeof the ion to be exchanged. A lesser number ofcrosslinkages permits exchange with larger ions(24). Resins with 4 to 10 per cent crosslinkage aresatisfactory for most laboratory work involvingcommon organic or inorganic ions.

Particle size. The particle size of resins isgenerally indicated by the range of U. S. Standardscreen sizes. The smaller the particle the fasterthe reaction rate and therefore the less resin

needed per unit time. On the other hand, thesmaller the particle the slower the flow rate of agiven column. Thus, a compromise betweenthese factors has to be accepted. The mesh size20 to 100 has been used to deionize microbialsuspensions; a smaller size, 100 to 200 mesh, iscommonly employed for analytical purposes.

Functional group, weak or strong resin. Capacityand stability are no longer a problem with mostmodern resins. They can be considered practicallyinsoluble in water and in common organicsolvents, e.g., ethanol, butanol, ethyl acetate,xylene, toluene, benzene. Dilute aqueous solu-tions of permanganate (28), chromate (44), andmolybdate (41) attack ion exchangers. Strongalkaline solutions may attack certain types ofexchangers, especially at high temperatures.Complications which may occur with certainchemicals are the exception. For instance, thefree-base form of weak anion exchangers reactswith aldehydes and forms condensation productssimilar to Schiff's bases (15). To circumvent thisparticular difficulty the use of resins in thebicarbonate form was recommended.For cation exchange the most important resin

is the sulfonic type. It belongs to the so-calledstrong type. It remains highly ionized in bothacid and salt form. Whether simple or complex,organic or inorganic, all cations are taken upquantitatively by sulfonic exchangers regardlessof the pH of the initial solution. Ampholyticchemicals, such as amino acids, are also takenup by these resins. Cations of high molecularweight or positively charged colloids are adsorbedslightly or not at all. In most applications thesulfonic resin is used in the hydrogen form. Inthis case, the regeneration is made by an acid,usually hydrochloric.An example of a weak cation exchanger is the

carboxylic acid type. Strong bases have greateraffinity than weak bases for weak resins;therefore, their chief application is for the separa-tion of strong organic bases from weak ones,such as in partition of amino acids. The carboxylicresin is also characterized by its affinity forhydrogen ions; therefore, a small amount ofdilute hydrochloric acid suffices to remove com-pletely the bases from the resin.From these considerations it follows that the

choice of resin will have to depend upon the pHof the solution to be treated and upon the strengthof the base to be removed.

1960] 253

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

BORIS ROTMIAN

The same considerations hold for the anionexchange resins. The strong anion exchange resincan be used in the entire pH range either in thefree-base form or in the salt form, such as chloride,formate, and acetate. The selection of the propersalt permits the separationi of anions of differentstrength from each other. For instanice, neuitral,acid, and some basic amino acids are taken tupby the free-base formn of the exchaniger, w-hereasonly dicarboxylic amino actids are exchanged bythe chloride form (10).Weak anion exchange resinis are higlhly ionized

only below pH 7 wh-11eIn they are in the salt form.Only in that pH range are they useful as ex-changers. This behavior is (cnsistent with thefact that weak anion exchXangers contain primary,secondary, and tertiary aminies as functionalgroups. Their main use is to remove free mineralaci(ls from aqueous or nloIn aqueous solutions andto separate weak from strong acidls.

Preliminar!y Treatmtient of the Resin

Ordinarilv the resinls Imlust he (hemically)pturified and the desired plarticles are separatedto a constant size. These tw-o operations can beperformed simultaneously and improve con-siderably the performance of the resin and thereproducibility of results. A standardl procedureconsists of alternatinig treatments with acid(1 N HCI), water, and alkali (0.5 N NaOH).These treatments are (lone b1 decantation andltherefore they serve at the same time to allowsettling of the larger particles and decantation ofthe fines. The exchangers may be air driedwithout changing their properties. O-en drving isdeleterious to most resins. Some resins are nowcommercially available in the purified form.After these operations the resin is treatedl with

adequate regenerant and then washed carefullywith water until no traces of regenerant can bedetected. For efficiency, it is recommeide(l toregenerate and wash in a column, even if thebatch method is to he uised lateIr foir the exchalnge.

Apparatus and Teclhnique in Conlum OperationiAltlhouighl mniany tyj)es of apparatis hliave l)een

dlesignedol for olumn operation withl ioInexchangers, tlhe most simiiple onIes yield satis-factory results. Essentiall-, a columnlii ((onsists ofa glass tulbe of any kinid witlh (Iradrwni bottomien(d in which a pllug of cottoin, glass vool, oIrsintere(d glass (lisc sustains the resin bed, per-mitting free flow of solutions. The resin bedshould be free of air bubbles because channellinigcauses a considerable decrease in the efficiencN,of the eoluinii. A convenient method to forimi thel)edl is to pour the Iresini in a slurry- and to addmore wvater inlto the column unitil the particlcssettle down-. The resin l)edl should lie kept covere(lwith liqui(d at all timis to prevenit the formationof air)bubhles. This is donie somietimiies by keepinhgthe outlet froimi tlhe column above the top of thceresiin bed. If 1v any chanice air bubbles are

formed, the easiest remedy is to repeat thefilling procedure indicated above. AX burettewi-ithi a pinch clamp can serve effectively aS a

column. The shape of the column does notinfluence the performance of the resin bedwithin certain limits. Usually the length of thecolumni is 10 to 20 times the diameter, but theseproportions caII be v-aried wilely accordling tothe requirements. Extremely narrow columnsare inot recommeni(le(l because of channellingand wa-1ll (effects (6).To o)perate the column, the solutioni to lie

treated is simply placed on top of the resin bed.

TABLE 2Flow rates and particle size of resins colmmonly used in ion exchange chromatography*

Resin Ions Separated Particle Size Flow Rate

Cation exchangersDowex 50..................Zeo-Karb 215

Anion exchangersDowex

De-Acidite B

Dowex 2.........

* From page 107 reference (42).

... Cer ium grotup

Amino acids

... Adenosine polyphosphatesAmino acids

...IAmino acids

[voi,. 24

0.25-0.36

0.25-0.36

<0.0740.14-0.17

0.074-0.14

nilcm?12, 1iin.

0.2-0.42.4

<3.01.20.14

254

Mz III

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

ION EXCHANGE RESINS IN NIICROBIOLOGY

Unless the solution has low ion concentration, itis necessary to ascertain that the amount ofresin will be enough to absorb all the exchangeableions. The flow rate of the column can be adjustedwith a proper outlet, e.g., a stopcock, a pinchclamp, or a capillary tube, although it is moreadvantageous to control it with the particlesize of the resin. Table 2 serves as a guide of theaverage properties of a laboratory ion exchangecolumn. After absorption the residual solutionretained in the resin bed is displaced by washingwith water. To elute or regenerate the columnthe solutions can be passed downwards or up-wards. Theoretically, the latter is more efficient,but in laboratory practice it is not routinelyused because it complicates excessively theapparatus.

ION EXCHANGE IN -MICROBIOLOGY

The factors governing sorption and desorptionof microorganisms from ion exchange resinshave not been the subject of detailed studies ofwhich the reviewer is aware. From the knownchemical composition of the cell wall of certainmicroorganisms it is not difficult to understandthe affinity between cells and ion exchange resinwhich results in adsorption. Cell wall constituentssuch as diaminopimelic acid, amino acids, orhexosamine, could easily provide the necessarycharges to the cell surface. It is much moredifficult to explain why some species of bacteriado not adsorb to ion exchange resins. The cellwall composition and the electrophoretic mobilitypattern of bacteria which do not adsorb to resinsshow no indication that the charges present inthese cells differ strikingly from those present inbacteria which do adsorb. Furthermore, roughand smooth forms of Escherichia coli, whichhave widely different electrophoretic mobility(1), are not adsorbed to mixed beds of strongion exchange resins while "old" cells which havehigher electrophoretic mobility than expo-nentially grown cells (1) are adsorbed to ionexchange resins. The apparent contradictionbetween electrophoretic mobility data andbehavior towards ion exchange resins could beexplained if the charges on the bacterial surfacewhich impart electrophoretic mobility to thecells are hindered by neutral molecules or byposition effects and therefore unable to interactwith the charges on the resin. In old cells,deterioration of the bacterial surface would result

in new charges being formed in more accessibleplaces. The same would occur to bacteria inwhich considerable deterioration of the surfaceis caused by sonic vibrations under conditionsprecluding lysis (39). Like old cells, these sonicallytreated bacteria were found to be adsorbedcompletely by ion exchange resins. The restrictionhas to be considered that bacteria could havelysed in the column and that their constituentsadsorbed thereafter.A hypothesis put forward by Puck and collabo-

rators (36, 37) is that, in the absence of certaincations, viruses have negative charges whichprevent attachment to host cells or to cationicresins. The addition of cations neutralizes therepulsive negative charges of the virus andpromotes its attachment to the host. In line withthis hypothesis, host bacterial cells should havenegative charges; accordingly, Puck and Sagik(37) found that E. coli strain B, the host, wasadsorbed to the phosphate form of an anionexchange resin and did not adsorb to a cationicresin. These results appear in contradiction withthe fact that strain K-12 of the same organismdoes not attach to the OH- form of a similaranionic exchanger (39). The apparent discrepancywas investigated in our laboratory and theconclusion was that it could be attributed to thedifferent resins used. Cells of E. coli were adsorbedcompletely by Dowex 1, 200 mesh, in the chlorideform, while they were not adsorbed by IRA-410,16-50 mesh in the same form. Once more, it hasto be concluded that the presence of charges onbacterial surfaces is not the only factor governingadsorption, but that perhaps the configuration,the relative position of the charges, or otherfactors may play a major role.

Washing of Microorganisms

Even though reports on the use of ion exchangeresin to free microorganisms from extracellularmaterial are scarce, this appears to be one of themajor fields of possible application. A fairevaluation of the ion exchange method would beto compare it with the standard method ofwashing cells by centrifugation. The followingconclusions emerge from such a comparison.The ion exchange method has several ad-

vantages: (a) A far more complete removal ofextracellular ions can be achieved by ion exchangeresin as compared with centrifugation, regardlessof the number of centrifugations which can be

25519601

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

BORISROTMIAN[

considered practical. This is an important factorwhen dealing with radioactive isotopes, vitamins,or growth factors. (b) The removal ofextracellular ions can be achieved in just oneoperation when using resins. The time requiredfor the operation can be less than 30 seconds,whereas at least 20 minutes are necessary forthree centrifugations of bacteria (three is consid-ered an optimal number in many laboratories).(c) Specific removal of certain ions can be accom-plished with ion exchange resins. This operationhas importance when a minimal change in thecell environment is desired. (d) A thoroughwashing with water bY centrifugation cannot bedone with certain cells, like E. coli, which fail topack well after two or three washings withdistilled water (40).

However, the ion exchange method hascertain disadvantages: (a) It does niot removenon-ionic material or high molecular weight

ions. (b) For large amounts of cells the ionexchange method might become more timeconsuming than centrifugation. (c) Althoughthe manipulations required by the ion exchangeresin, prior to its use, are relatively simple,centrifugation would be considered less compli-cated by many laboratory workers.From these considerations the reader might

ponder which method serves him better in anyparticular instance. In some cases a combinationof the two might yield the best result (39).The general desiderata in determiniing whether

an organism can be washed or not by ioni exchangeresin are: (a) The passage through the resinshould not affect the viability of the micro-organism. (b) The microorganism shlould notattach significantly to the exchanger. (c) Theions to be exclude(d should be removed quanitita-tive1v with a minimum number of operations,e.g., one or two passages through the resini bed.

TABLE 3d1(isorption of microorgan ismis to excha,ngers

Organism

Azotobacter vinelandii...........

Bacteriophage ...................

Coxsackie virus.................

Colorado tick virus..............ECHO virus.....................Epidemic typhus rickettsiale..cEscherichia coli.....Escherichia coli B ..............

Escherichia coli K-12 ...........

Influenza virus...............

Neurotropic virus...............Poliomyelitis virus..............

Q fever rickettsiae..............Salmonella schottmnuelleri........Salmonella typhosa..............Shigella dysenteriae..............Staphylococcus aureus...........

Type of Exchanger

Anionic resinCationic resinAtionic resinCationic resinAnionic celluloseAnionic resinCationic resinAnionic cellulose

............. Anioniic celluloseAnionic celluloseAnioniic celluiloseAInioniC resin1

. Anionic resinCationic resinAniioinic resinCationic resinCationic resinAltuminum phosphateCationic resinAnionic resinCationic resinAnionic celluloseAniioinic celltulose

............ iAIionic resinAnionic resinAnionic resinAnionic resin

Per CentRetained in

the Exchanger

0ii 0

10027

0-1000-100

02

0-1000-1000-100<41001900

0-1000-100

00-100

00-1000-100<23<20

<17

Reference

18, 3918, '393737

9, 45191916161616303.73739393314322727

16, 171630303030

256 [ N'OL. 24

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

ION EXCHANGE RESINS IN MICROBIOLOGY

These conditions have been fulfilled for certainorganisms as shown in table 3. For other biologicalsystems, the ideal conditions will have to bedetermined. A few general suggestions can beoffered for this purpose.

The three desiderata given above can beobtained by changing the ionic form of the resin,the medium in which cells are suspended, or both.The work of Puck and Sagik (37) serves well toillustrate the point. These investigators foundthat bacteriophages Ti and T2 attach to anionicexchangers when suspended either in distilledwater or in salt solution. However, they attachto cationic exchangers only in the presence ofsalt.With regard to viability, it might help to use

the resin in the cold in spite of the slower rate ofexchange. Columns with fast flow rate have beenfound to reduce the number of cells lost by attach-ment to the resin. The fast flow rate can beachieved by using large particles of resin, smallheight of resin bed, or a combination of the two.More than one passage through the resin mightbe necessary to remove completely the ionsunder these conditions. Treating the cell suspen-

sion with the resin batchwise also decreases theattachment of cells to the resin, but again thepoor efficiency of the batch method has to beconsidered. The percentage cross-linkage andthe size of the particle are important factors tobe considered (19). In general, low cross linkageand large particle size will result in decreasedadsorption.Mixed bed resins (a mixture of anionic and

cationic exchangers) are particularly useful towash microorganisms (39) because they eliminatein one operation both anions and cations.Analytical grades of mixed bed resins, which are

commercially available and do not requireprevious purification or regeneration, are suitablefor this type of work.

Isolation of Microorganisms by IonExchange Resins

Classical procedures to isolate microorganismsrequire that cells can be grown in vitro. Thisrequisite is not fulfilled by viruses and thereforetheir purification is usually accomplished byphysical or biochemical methods, such as

differential centrifugation and ammonium sulfateor alcohol precipitation. Ion exchange resinshave been used successfully to purify viruses.

The method seems especially suitable for animalviruses because the preparations contain largeamounts of extraneous materials which usuallyinterfere with studies of biochemistry andimmunology.

Mluller's (1950) appears to be the first success-ful purification of a virus using synthetic ionexchange resin (32). He was able to remove upto 90 per cent of the nitrogenous impurities froma neurotropic virus preparation by passing itthrough a bed of cation exchange resin. Foreignmaterial was adsorbed to the resin leaving thevirus in the effluent without significant reductionin the infective titer.

LoGrippo (26) purified poliomyelitis virusfrom human feces by a different technique thanMuller's, using anion exchange resin to treat thecrude virus preparation instead of cation ex-changer. The virus was adsorbed to the resinand, after several washings, it was eluted with10 per cent sodium phosphate. The eluate con-tained the virus with practically its originalinfectivity but only 10 per cent of the initialnitrogen content. Muller and Rose (33) used asimilar technique to purify influenza virus exceptthat the virus was adsorbed to a cation exchanger.More recently cellulose ion exchangers have beenused to purify tobacco mosaic virus (7), bacterio-phage (9, 45), rickettsiae (17), and severalmammalian viruses (16, 17).

Ion exchangers could be used to separate andidentify strains, or cells with different surfaceconstitution, from a culture which appears homo-geneous by usual criteria. Accordingly, Hoyeret al. (16) found definite differences in chromato-graphic behavior among different mammalianviruses and Albertsson (2) reported that differentcell sizes of Chlorella pyrenoidosa can be separatedby step-wise elution from a calcium phosphatecolumn in the manner used in chromatography.

Miscellaneous Applications

Puck and Sagik (36, 37) studied the problemof attachment of viruses to their host cells bymeans of ion exchange resins. Aside from manyinteresting findings connected with their problem,these investigators elucidated the conditionsunder which viruses can attach to anionic orcationic exchangers. For instance, it was foundthat salt is required for attachment of certainbacteriophages to cationic exchangers but not toanionic exchangers. Furthermore, T2 bacterio-

257

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

BORIS ROTMAN

phage was shown to be split into its DNA andprotein fractions shortly after attachment to acation exchanger. The results of Puck and Sagik,together with those mentioned before on viruspurification, give useful principles which mayfind application in the removal of virus frombiological fluids.

Jose and Wilson (18) reported that conversionof Azotobacter vinelandii cells to spheroplasts canbe accomplished by passing the bacteria througha mixed bed ion exchange resin and then treatingwith lysozyme. This method avoids the use ofVersene, which is inhibitory in some systems.

Martin et al. (30) reported attempts to removefrom the intestinal tract organisms associatedwith gastrointestinal disturbances. Several speciesof bacteria were tested for adsorption onto weak-base anion exchange resin and other absorbingmaterial (see table 3). Folsome et al. (13) claimedthat ion exchange resin has antimicrobial activityagainst Trichomonas vaginalis in vivo. Eugere's(12) experiments indicate that this antimicrobialactivity results from lowering the pH or bindingessential growth factors.

Albertsson (2) found that adsorption of dis-integrated cell suspensions of Chlorellapyrenoidosa to a weak anion exchange resin,followed by elution at higher pH, yields a "good"preparation of cell walls.An example which might find similar applica-

tions in microbial physiology is the removal ofcalcium (43) or urea (5) from blood by passagethrough a resin bed. The method presents theunique characteristic that it is possible to removespecific ions, leaving the composition of the fluidpractically unchanged otherwise.

Rothstein (38) removed ions from yeastsuspensions by treatment with a cation exchangeresin in the triethylamine form. In the cellspretreated with the resin, glucose metabolismwas negligible and it was completely dependenton added ions. The resin method was found to bemore effective in the removal of ions than pro-longed washing and starving of the cells. Bair andStannard (4) established that treatment withcation exchange resin does not alter subsequentgrowth of yeast and appears to increase colonyformation of X-ray irradiated yeast.Another instance of physiological interest is

the effect of adenosine triphosphate (ATP) onthe adsorption of rat liver mitochondria tostrong cation exchange resin. Suspensions of

mitochrondria were found to pass through a bedof cation exchange resin (IR-120) in the H+ formwithout noticeable alterations of their oxidativephosphorylating capacity or optical properties(G. Maley and B. Rotman, unpublished results).The addition of 3.6 X 10- M ATP caused theadsorption of 80 per cent of the mitochondria tothe resin. The ATP was added in the cold oneminute before passing the suspension throughthe column. Physical retention of mitochondriain the column due to swelling of the mitochondriain the presence of ATP seems unlikely because nodesorption was observed when the resin wastaken from the column and shaken vigorously.Uridine triphosphate, guanosine triphosphate,and inosine triphosphate caused the same effectas ATP. Adenosine, adenosine mono- anddiphosphate, pyrophosphate, creatine phosphate,thiamin phosphate, and orthophosphate, testedat the same concentration as ATP, did notcause significant adsorption of mitochondria tothe resin. On the other hand, 2 X 10-2 M Versercaused adsorption of the mitochondria to ananion exchange resin (IR-400) which did notadsorb mitochondria even in the presence of ATP,What is the mechanism underlying this specificeffect of ATP and triphosphates? The answerto this question might not be at all simple if thehypothesis is entertained that ATP plays herea role similar to the one it plays in muscle con-traction. A simpler hypothesis, such as specificlysis of ATP-treated mitochondria when incontact with the ion exchange resin, should beruled out before more complicated hypothesesare considered.These experiments illustrate well the unique

kind of information that the use of ion exchangeresins yields.

REFERENCES

1. ABRAMSON, H. A., MOYER, L. S. AND GORIN,M. H. 1942 Electrophoresis of proteins, pp.303-307. Reinhold Publishing Corp., NewYork.

2. ALBERTSSON, P. 1956 Chromatography andpartition of cells and cell fragments. Na-ture, 177, 771-774.

3. ARGERSINGER, W. J. 1958 Ion exchange res-ins and membranes. Ann. Rev. Phys.Chem., 9, 157-178.

4. BAIR, W. J. AND STANNARD, J. N. 1955 Ef-fect of starving and Dowex 50 treatment ongrowth of normal and X-irradiated yeast.J. Gen. Physiol., 38, 505-513.

258 [VOL. 24

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

ION EXCHANGE RESINS IN MICROBIOLOGY

5. BONANOME, A. AND BEGANI, R. 1955 Cat-ion-exchange resin for the use in urea re-moval from blood. Gazz. intern. med. echir., 60, 2005-2010.

6. BOYD, G. E., ADAMSON, A. W., AND MYERS,L. S. 1947 Kinetics of ionic exchange ad-sorption processes. J. Am. Chem. Soc., 69,2836-2848.

7. COMMONER, B., LIPPINCOTT, J. A., SHEARER,G. B., RICHMAN, E. E., AND WuT, J. 1956Reconstitution of tobacco mosaic virus com-ponents. Nature, 178, 767-771.

8. CALMON, C. AND KRESSMAN, T. R. E. 1957Ion exchangers in organic and biochemistry.Interscience Publishers, Inc., New York.

9. CREASER, E. H. AND TAUSSIG, A. 1957 Thepurification and chromatography of bacteri-ophages on anion-exchange cellulose. Vi-rology, 4, 200-208.

10. DAVIES, C. W., HUGHES, R. B., AND PAR-TRIDGE, S. M. 1950 Amino-acid separa-tions by use of a strong-base resin. J.Chem. Soc., 2285-2287.

11. EDWARDS, W. R., SCHWARTZ, M. G., AND Bou-DREAUX, G. 1940 Removal of chloridesand sulfates by synthetic resins. Ind. Eng.Chem. 32, 1462-1466.

12. EUGERE, E. J. 1956 Antimicrobial proper-ties of ion exchange resins. DissertationAbstr., 16, 1697.

13. FOLSOME, C. E., LESLIE, S. A., AND THOMS,R. K. 1955 Control of chronic lower geni-tal tract infections. The use of anion ex-change resin. Obstet. and Gynecol. (U.S.),6, 531-537.

14. FROMMHAGEN, L. H. AND KNIGHT, C. A. 1959Column purification of influenza virus.Virology, 8, 198-208.

15. GABRIELSON, G. AND SAMUELSON, 0. 1952Utilization of ion exchangers in analyticalchemistry. Acta Chem. Scand., 6, 729-737.

16. HOYER, B. H., BOLTON, E. T., ORMSBEE, R. A.,LE BOUVIER, G., RITTER, D. B., AND LAR-SON, C. L. 1958 Mammalian viruses andrickettsiae. Science, 127, 859-863.

17. HOYER, B. H., BOLTON, E. T., RITTER, D. B.,AND RIBI, E. 1959 Simple method forpreparation of purified radioactive poliovirus particles. Virology, 7, 462-464.

18. JOSE, A. G. AND WILSON, P. W. 1959 Meta-bolic activities of subcellular particles fromAzotobacter vinelandii. Proc. Natl. Acad.Sci. U. S., 45, 692-697.

19. KELLY, S. M. 1953 Detection and occur-rence of Coxsackie viruses in sewage. Am.J. Public Health, 43, 1532-1538.

20. KITCHENER, J. A. 1957 Ion exchange resins.John Wiley and Sons, Inc., New York.

21. KUNIN, R. 1949 Ion exchange. Anal.Chem., 21, 87-96.

22. KUNIN, R., MICGARVEY, F. X., AND FARREN,A. 1957 Ion exchange. Ind. Eng. Chem.,49, 507-513.

23. KUNIN, R. AND MYERS, R. J. 1947 The anionexchange equilibria in an anion exchangeresin. J. Am. Chem. Soc., 69, 2874-2878.

24. KUNIN, R. AND NIYERS, R. J. 1949 Exchangeequilibria in anion-exchange resins. Dis-cussions Faraday Soc., 7, 114-118.

25. KUNIN, R. AND MIYERS, R. J. 1950 Ion ex-change resins. John Wiley and Sons, Inc.,New York.

26. LoGRIPPO, G. A. 1950 Partial purificationof viruses with an anion-exchange resin.Proc. Soc. Exptl. Biol. M:Ied., 74, 208-211.

27. LoGRIPPO, G. A. AND BERGER, B. 1952 Useof ion exchange resins in partial purificationand concentration of poliomyelitis virus.J. Lab. Clin. Med., 39, 970-973.

28. LUR'E, JU. JU. AND FILIPPOVA, N. A. 1948Use of organolites in analytical chemistry.Zavodskaya Lab., 14, 159-172.

29. MIARTIN, G. J. 1955 lon exchange and ad-sorption agents in medicine. Little, Brownand Co., Boston.

30. MARTIN, G. J., MIoss, J. N. AND BEILER, J. M.1955 In pages 55-56 of reference 29.

31. MIILLER, H. K. AND SCHLESINGER, R. W. 1955Differentiation and purification of influenzaviruses by adsorption on aluminum phos-phate. J. Immunol., 75, 155-175.

32. MULLER, R. H. 1950 Application of ion ex-change resins to the purification of certainviruses. Proc. Soc. Exptl. Biol. Med., 73,239-241.

33. MULLER, R. H. AND ROSE, H. NI. 1952 Con-centration of influenza virus by a cation ex-change resin. Proc. Soc. Exptl. Biol. Med.,80, 27-29.

34. NACHOD, F. C. 1949 Ion exchange. Aca-demic Press, Inc., New York.

35. OSBORN, G. H. 1956 Synthetic ion exchang-ers. Macmillan Co., New York.

36. PUCK, T. T. 1953 The first steps of virus in-vasion. Cold Spring Harbor SymposiaQuant. Biol., 18, 149-154.

37. PUCK, T. T. AND SAGIK, B. 1953 Virus andcell interaction with ion exchangers. J. Ex-ptl. Med., 97, 807-820.

38. ROTHSTEIN, A. 1954 Enzyme systems of thecell surface involved in the uptake of sugarsby yeast. Symp. Soc. Exptl. Biol., 8, 165-201.

39. ROTMAN, B. 1956 On the mechanism ofsonic lysis of bacteria. J. Bacteriol., 72,827-830.

259

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

BORIS ROTMAN

40. ROTAIAN, B. 1958 Regulation of enzymaticactivity in the intact cell. J. Bacteriol., 76,1-14.

41. RUNEBERG, G. AND SAMIELSON, 0. 1945 Ionexchange substances in analytical chem-istry. Svensk Kem. Tidskr., 57, 250-254.

42. SAMITELSON, 0. 1953 Ion exchangers in ana-

lytical chemistry. John Wiley and Sons,Inc., New York.

43. STEINBERG, A. 1944 A new method for pre-

venting blood coagulation. Proc. Soc. Ex-ptl. Biol. 1Ied., 56, 124-127.

44. SUSSNIAN, S., NACHOD, F. C., AND WOoD, W.1945 Metal recovery by anion exchange.Ind. EiLg. Chem., 37, 618-624.

45. TAItSSIG, A. AND CREASER, E. H. 1957Chromatographic purification of T2r bac-teriophage. Biochim. et Biophys. Acta, 24,448-449.

260 [VOL. 24

on March 11, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from