Pohl - Biological Assay of Fresh Waters

8/13/2019 Pohl - Biological Assay of Fresh Waters

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RESEARCH PROJECT COMPLETION REPORTOWRR PROJECT NO A 056 0KLA

BIOLOGICAL ASSAY OF FRESH WATERS Y A N W METHODDIELECTROPHORESIS

Submitted toThe Oklahoma Water Resources Research Inst i tuteOklahoma State UniversityStillwater Oklahoma

Prepared byHerbert A PohlaMKent PollockDepartment of PhysicsOklahoma State University

The work upon which this report is based was supported in part byfunds provided by the United States Department of the Interior Officeof Water Resources Research as authorized under the Water ResourcesResearch Act of 1964


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TABLE OF CONTENTS

Chapter RESEARCH OBJECTIVES.

Page

INTRODUCTORY OMMENTS ON NUFE.Nonuniform Fie ld Eff ec ts NUFE

SPECIFIC OBJECTIVES.IV EXPERIMENTAL SECTION

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Biological Materials 3The Dielectrophoretic Collect ion Spectrum of an Alga 4The Development of Continuous Separators 4The Development of Stream centered Continuous Dielectrophoresis 4V SUMM RY AND CONCLUSIONS.

VI GOALS FOR FURTHER STUDY.VII REFERENCES

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LIST OF FIGURES

Figure1 Lielectrophoretic Collection Rate Spect rum o f Chl or el la Vulgari s

2 Cell Concentration vs Optical Density 3 Cylindrical Electrode Chamber 4 Multiple Wire Wire Chamber 5 Stream Centered Continuous Dielectrophoresis6A Stream Centered Continuous Dielectrophoresis Chamber6B S Chamber 7 Agar Filled S Chamber

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BIOLOGICAL ASSAY OF FRESH WATERS BY A W METHOD, DIELECTROPHORESIS

I RESEARCH OBJECTIVESRapid and selective methods for assaying cellular organisms in freshwater are being sought through the applicat ion of nonuniform electr ic fieldeffects NUFE .The original objectives i t will be recalled were twofold. Firs t adirect attempt was to be made to develop a rapid but selective assay usingNUFE, and Chlorella sp. as th e model o rgani sm . Secondly the developmentof a continuous rather than a batch type of dielectrophoretic separatorusing NUFE was to be done. The l a t ter would have wide applicat ion in study-in g f re sh wat er biota .

I I INTRODUCTORY COMMENTS O NUFESince the NUFE techniques are re la t ively new, the next few paragraphs

will present a br ie f i nt roduct ion to the basic principles of i t s operationbefore going on into a discussion of t he r es ea rch procedures.1. Nonuniform F ie ld Eff ec ts NUFEA gentle force is exerted upon a l l matter by a nonuniform electr icf ie ld . Recently i t has been shown to be usefully applicable in thestudy of single cel l organisms. The nonuniform field effect die lect-rophoresis is the t ranslat ional motion produced by the action of anonuniform electr ic field upon neu tr a l po la ri zab le bodies. I t is es-pecially useful in s tu die s of suspensions of cel l s Normal and abnor-mal ce l ls respond differently as do cells of different species andmay be separated on the basis of their differ ing polarizational response

Because the phenomenon depends upon the polarizat ion and notthe charge of the part ic les subtle new d iff erences a re ava ilable forexploitat ion by dielectrophoresis . Operations can be carried out ina high frequency e le ct ri c f ie ld . The technique is rapid and simple.The frequency spectrum of cellular response to dielectrophoresisvaries with the species and with the physiological s ta te of the in -dividual ce l l Cells may be studied i nd iv idua ll y o r in large numbersa t a t ime. The technique works best in media of low conductivitysuch as in f re sh water .I t is a familiar fact that in a uniform e le ctr ic f ie ld a forcei s exerted upon a charged object. I f the field i s made nonuniformthat type of force pers is ts and a new type of force ar ises due topolarizat ion effects . In the l a t t e r case of nonuniform f ie lds evenneutral objects become su bject to a force one p ropo rt iona l to theirpolarizabil i ty . The two phenomena are distinguished by th e termselectrophoresis for that on charged objects; and dielectrophoresisfor that of neutral objects being pulled along by a nonuniform f ie ld .We shal l be most concerned with the l a t t e r dielectrophoresisand polarizat ion e ffec ts in the present work. With i t one can obtaininformation as to the electr ical polarizabil i t ies and related biolo-gical mechani sms. Moreover i t is easily possible to use the non-uniform field forces to produce u se fu l p hy sic al s ep ara tio ns t o par-t icula te materials such a s mix tu re s of cells or other par t ic les


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Nonuniform l t r ~ fields are easily produced in a controlledfashion. Since the polarizabil i ty of material bodies, especial lyl iving cel ls varies strongly with th e app lie d frequency, i t is are la t ively s tra ightforward matter to obtain a spectrum for the d i-electrophoretic responses of cellular suspensions. I t is observedt ha t d if fe ren t species show dist inct spectra with up to t hr ee cha racter i s t ic peaks o r r el axati on times. s we shall see, Chlorellavulgaris appears to be unique in having four characteristic peaks orrelaxation times.I n g en er al , before dielectrophoresis is applied to a part icular t es tsystem, several decisions concerning the experimental approach must be made.The important considerations are the conf igura tion of t he e le ct rode s whichwil l produce the nonuniform f ie ld , the selection of a cr i t ica l parameter

which ref lects changes in the die1ectrophoresis and thus measure the effectof the f ield and the determina tion of the varible quanti t ies upon whichthe chosen cr i t ica l parameter depends.A nonuniform field will be produced by an electrode design other thanpa ra ll el p la te s. The number of possible configurations can be greatlyreduced by requiring that the electr ic field be easi ly calculable. Thisdesire for mathematical simplici ty leads to the standard geometries ofconcentric spheres and concentric cylinders. Other configurations 3 havebeen devised which can be approximated as cyl in de rs o r spheres but which areeasier t o c on st ru ct and offer experimental advantages. These include awire perpendicular to a f la t plate (pin-plate), a wire paral le l to a f la tplate (wire-plate), two coaxial wires end-to-end, (pin-pin) and two paral le lwires side-by-side w i r e ~ i r e . ne other calculable shape is that whichgives a constant dielectrophoretic force3 the isomotive f ie ld . The choicebetween these fiv e designs for a part icular experiment depends upon theexperimental characteristics desired. Probably the most popular type i sthe pin-pin arrangement due to i t s ease of cons tr uc ti on and observation.Once a field shape has been chosen, the next problem i s to select adependent variable which wil l give an i nd ic ati on o f the effect of the f ield.Several such cr i t ica l parameters exis t , including the dielectrophoreticforce, the velocity of a tes t part icle and the ra te a t which materials isbui l t up a t one of the electrodes. The force has been used as the cr i t ica lparameter on several occasions and i s convenient when dealing with asingle, large par t ic le The force can also be used for single microorganismsin a variation of the Millikan o il drop exper iment where the die1ectrophoresisforce is balanced by gravity. The vel oc ity o f a part icle has not been usedas the dependent variable, but there are no obvious reasons why i t wouldnot be u sefu l in certain systems. The most Widely used cr i t ica l parameteris the rate of collection of part ic les a t an electrode. I t i s most oftenexpressed as a yield , which is the amount coll ected wi th in a specifiedtime, and is b est su ite d to systems which are suspensions of large numbersof par t ic les Fo r use with very dilute suspensions of cel l s ; a countingprocedure of considerable precision i s available. I t c on sists o f collectingce l ls for a short period (one minute, say) at a driving voltage (20 volts ,say) then cutting the app li ed vo lt age to a holding value (2 volts , say)then count ing the number of cells touching t he e le ct ro de wire along a prechosen length. Using a mechanical stage, the count density o f coll ec ted(single) cells can be checked along several regions of the wire-wire electrodeand the results averaged. Cell suspensions containing only about 1 to1 cells/mm3 can be convenient ly s tudied in th is manner.

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A fter th e dependent variable has been selected, the various independent variables need to be ascertained so that the necessary equipment andprocedures can be provided with the yield as the cr i t ica l parameter, theseindependent variables are; the size and shape of the part ic les , the conduct iv i t ies and permit t ivi t ies of both the particles and the suspending medium,the concentration of part ic les , the frequency and s tr ength o f the appliedf ie ld, and the e lapsed t ime.In a typical experiment involving suspensions, the ranges of the parameters are: part ic le diameter, 1-50 microns ; f requency of applied f ield102 - 109 Hz; applied voltage 5-30 volts for closely spaced 3mm electrodes;suspension conductivity, less than 10-1 mho/m. Low frequencies and highconductivities lead to excessive heating and other disruptive effects. nimportant point is th e s trong dependence of the resul ts on frequency andconductivity._ The previous simple theoret ical treatment, which works wellfor insulating materials i n in su la tin g media, at t r ibutes motion to a di f fer -ence in permitt ivit ies only and has no provision for the inclusion of theseother two variables. A more general treatment has been given which includesthese parameters. The predicted results using a spherical four-medium modelagree with yea st cel l behavior. However, the approach has not been checkedaga in st o th er systems and so a t p resen t th e applicabil i ty of the specialmodel to them requires further study and, doubtless , modif icat ion.

To summarize, dielectrophoresis i s a new method for handling l ivingcel l s in fresh water. I t has been shown capable of dist inguishing not onlyl iving and dead cel ls but also cel ls which are different in kind or evenin thei r physiological s ta teI I I SPECIFIC OBJECTIVES

The original objectives of this study were to develop an assay methodspecif ical ly for a model organism, Chlorel la vu lgari s sui table for applicationto fresh water samples; and to develop a continuous broadly applicable NUFEsep r t ion procedureTo date progress has been made towards both objectives. Because of theusually low concentration of planktonic biota, i t was early re alized th atlarge volumes of fresh water at least 100 ml) would have to be sampledin order to obtain fair and representative analyses. Earl ier NUFE studies workedwith sample volumes of 0.01 ml to 0.5 mI. Accordingly, i t proved desirableto develop the NUFE method for the handling of rather larger volumes. Thismeant the development of the continuous type of dielectrophoresis to practicablelevels before the f i r s t object ive could be sat isfactori ly handled. The majoremphasis during this past year has been on the development of techniqueswhich could apply NUFE to large volumes. Concurrently, th e specific organism,Chlorel la vu lgari s was studied and shown to col lect readily using the NUFEThe collection ra te in nonuniform electric fields was found to depend stronglyupon the applied frequency of the ac e le ct ric f ie ld . A yield spectrum ofof this response has been measured to help guide the separat ion opera t ions .IV. EXPERIMENT L SECTION

A. Biologica l Materia lsThe microorganisms chosen for this study were a yeast Saccharomycescerevisiae) and an a lg a Chl or el la vulgaris) . The yeast was grown in

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Sabouraud broth. The culture was shaken gently during growth to minimizeclumping of the cel ls In a l l cases a 48 hour culture was used for experiments. Yeast was used in most experiments since i t has been studied inthis laboratory extensively and i t s dielectric characteris t ic and responsesare well documented. l ,2,3One of the areas of study of this project was to make a similar studyof algae. The a lg a Chl ore lla vulgaris was grown on a Wood s Hole formula7in 4 1. aspirator bott les under fluorescent l ight with st i r ing to minimizeclumping of the cel ls

B. The Dielectrophoretic Collect ion Spectrum of an AlgaThe algal response curve was studied using a batch method and a wirewire configuration for the electrodes. Figure 1 shows the dielectrophoreticcollection rate DCR vs. frequency curve for both normal Chlore ll a vu lgari s

and for Q. vulgaris stained with crystal violet . This data allows usto select appropriate frequencies and voltage in our l a ter separat ion exper im nts

C. The Development of Continuous SeparatorsThe main area of this research has been the development of an effectiveand dependable apparatus for continuous dielectrophoresis. A large numberof different flow geometries and electrode designs were investigated.Originally, a cylindrical electrode design originated by Poh14 and l a terused by Mason and Townsley5 in thei r studies was used. This design incorporatesa cylindrical electrode with a central wire Figu re 3 . This design concentrated a flowing suspension of cel ls about the central wire where theycould be collected. Another design consisted of a flow chamber through whicha pair of wire electrodes ran diagonal ly Figure 4A . Yet a third chamber

af ter Verschure and I j l s t6 was designed consisting of one f la t plate electrodeand a wire mesh e lect rode t o provide the nonuniform field Figure 4B .All of the above chambers were designed with one in le t port and twoout let ports . A flowing suspension of cel l s would be concentrated by theapplied non-uniform electr ic field toward one of the out let ports . Thusone out let port should have a greater concentration of cel ls than the other.The opi ca l densi ty of the two ce l l suspensions was measu red by a photometerto determine the effect of the f ie ld. Figure 2Another cel l design consisted of mul ti ple pairs of wire electrodes Figure4C . The amount of cel ls collected on th e wires i s a measure of i t s biomass.The designs discussed above were a l l su bje ct to some problems. A cel la t different positions in th e non-uniform field experienced differentdielectrophoretic forces. The cel ls also flowed at different speeds accordin g to position, those in mid-stream flowing faster and being subjected tothe f ield for a shorter period of time than those near the wall . Theseproblems were eliminated by a new method of great promise: Stream-centeredContinuous Dielectrophoresis SCD). Accordingly the previous designs weret hen d isca rded in favor of the SCD method.

TheDevelopment of Stream-centered Continuous DielectrophoresisIn this type of chamber Figure 5 , a central stream of very concentrated

ce l l suspension is introduced into a surrounding support s tr eam. The stream

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of cells experiences a lateral force due to the applied electr ic f ie ld.Different particles wil l experience a different force and therefore def lect a different distance. This method has the potential to sep aratemany different species of organism and allow thei r collection and enumeration from large volumes of liquid e.g. one l i t e r .Most of the past year was applied to th e development of the SCD method.The f i r s t chambers made were from 7 to 10 m long and used various arrange

ments of e le ct rodes to produce the non-uniform f ie ld. The most effectivedesign was an isomotive3 configuration. Unlike other field configurations,the isomotive force does not diminish with distance. The isomotive shapeis relat ively easy to approximate and construct Figure 3).The f i rs t small, successful chamber of this type produced a noticeabledeflect ion approximately 2mm of the central stream of yeast with only 3 to5 volts applied while running along a path of 5 cm A larger chamber withmultiple out le ts yielded very encouraging resul ts with a maximum deflect ionof 7 mm using an applied field of 8 volts Figure 6). These results obtained with small voltages 10 Vrms suggested that longer chambers with correspondingly longer times in the field for the cel ls should y ie ld excel lentresul ts with the same voltages.The larger chambers, however, exhibit greater problems with turbulencewhich destroys the flow pattern and masks effects of the e le c tr ic f ie ld .Turbulences ar ise through the geometry differences of the f low chambers,electrode charge injection, the flow rate of the l iquid, and temperaturedifferences due, in part , to dielectr ic heating) caus ing convect ion currents.Two larger chambers have been bui l t one 15 cm long and one 45 m long. Wea re cu rren tl y i nves ti ga ti ng the use of an agar gel f i l l ing in these chambersto achieve a simple cylindrical geometry throughout the system and to reduceelectrode charge injection. Preliminary resul ts with the use of de-ionized2 agar gel to par t ia l ly f i l l the electrode area Figure 7) show excellentresul ts to date. Despite some l imited turbulence, noticeable deflection 3to 5 mm has been observed in the larger chamber at 5 to 7 volts using S.cerevisiae. At present, a method of thermos tat t ing throughout the y t ~ isbeing stu died to help reduce th e remaining turbulence.V SUMM RY ND CONCLUSIONS

We have shown that the fresh water alga, Chlorella vulga ri s exhibi tsposit ive dielectrophoresis over a wide frequency range, 100 Hz to 4 MHz.This means that the cel ls are pulled toward the region of highest fieldstrength in an ac electr ic f ie ld. The collection is observed to be rapidand to vary strongly with the applied frequency. The later results providesa yield spectrum showing th e var ied polorizat ion responses as the appliedfrequency changes and is a diagnostic of the organism. Most cells haveyield spectra with three or fewer peaks. vulgaris is unusual in that i t syield spectrum has a t least four peaks. A comparison between the yieldspectra of normal and dye-stained algal cells showed strong differences.In order to develop an assay method using NUFE as applied to freshwater samples, we have needed to modify the conventional NUFE techniques.Previously these a l l dealt with only very small volumes a bout 0 .01 to 0.5 ml).For dealing with fresh wate r a ss ay s of b io ta , r ath er larger volumes circa100 ml o r larg er) are needed. To this end, our main thrust has been todevelop the NUFE technique so as to handle these larger volumes. This meantthe development of continuous methods whereby the larger volumes could be madeto pass through th e electrode treatment area.

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The development of a continuous NUFE process has come, after many t r i a lequipments were buil t and tes ted to one which we believe has much promise. t subjects th e desired water stream to a nonuniform electr ic field af te rf i r s t surrounding i t with a protective thick layer of suspending medium.The technique is referred to as stream-centered continuous dielectrophoresisSCD . Preliminary tests with suspended cells show i t to operate reasonablywell. The results are so encouraging as to deserve strong further efforts toperfect and standardize t he t echn ique .The stream-centered continuous dielectrophoresis method appears to haveenormous potentia l . Not only does i t seem well s uite d fo r b io -assa y problemsof fresh water but i t should also be app li cabl e to subtle i nvest iga ti ons i nhealth problems. t should be ideally suited to handling suspensions of many

components such as whole blood and could presumably even dist inguish s l ightdifferences among cells in a single species. Active development of th ispowerful technique appears to warrant intense study.VI. GOALS FOR FURTHER STUDY Perfect and s tandardize the stream-centered continuous dielectro-phoresis technique for the ana ly si s and separation of liv in g ce l l s2. Application of the seD technique to the problem of bio-assay.3. Exploration of the capabil i t ies of this SCDmethod. For examplepossible applicat ion to the determination of the population type anddensity in a body of water. Or a possible applicat ion to human health

problems such as leukemia or sickle cel l anemia.


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REFERENCES

1. Pohl, H. A. and 1. Hawk, Separation of Living and Dead Cells byDielectrophoresis , Science, 152, 647 (1966).2. Pohl, H. A. and J S. Crane, Dielectrophoresis of Cells , BiophysicalJ 11, 711 (1971).3. Pohl, H. A. and J . S. Crane, Biological Aspects of Dielectrophoresis ,Chapter 5 in A. D Moore s editing of EleCtrostatics and I tsApplications, Wiley and Sons.4. Pohl, H. A., J Appl. Phys., 29 , 1182 (1958).5. Mason, B. D and P. M Townsley, Dielectrophoretic Separation ofLiving Cells , Can. J Microbio1., 17 , 879 (1971).6. Verschure, R. H and L. I j l s t Apparatus for Continuous Dielectricmedium Separation of Mineral Grains , Nature, 211, 619 (1966).7. Nichols, H. W., Growth Media-Freshwater , pp 7-24 (1973). In :Handbook of Phycological Methods, Ed) J . Stein, CambridgeUniversity Press, Cambridge.

Pohl - Biological Assay of Fresh Waters

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