The British Journal of

Experimental Biology

AN ELASTIC VALUE OF PROTOPLASM, WITHFURTHER OBSERVATIONS ON THEVISCOSITY OF PROTOPLASM.*

BY WILLIAM SEIFRIZ.

I. Introductory.

T H E physical properties of protoplasm have been the subjectof keen investigation ever since the discovery of the livingsubstance nearly a century ago. The earliest descriptions ofprotoplasm refer to its physical nature. Thus, Dujardin *characterised the "living jelly" as a glutinous transparentsubstance insoluble in water, and von Mohl7 described it asa "viscous liquid mass."

Viscosity is that physical property of protoplasm which hasbeen most intensively studied of late. Relative and actualvalues of the consistency of protoplasm have been determinedby a number of investigators and by a variety of methods.The literature on protoplasmic consistency is now fairlyextensive, but there is little reference to the elastic properties ofthe living substance. Apparently no attempt has been madeto actually measure with precision the elasticity of protoplasm.

Some interesting experiments were done by Pfeffer9 on the" elastic stretching capacity " of protoplasm. Pfeffer was able,by attaching weights to protoplasmic threads, to determine theload which a strand of myxomycete plasmodium can support.What Pfeffer was measuring was not the elasticity of protoplasmbut its tensile strength, or as he expressed it, the cohesive

• Received January 21st, 1924.VOL. 11.—NO. 1. I A

William Seifrizforce. Further, as Pfeffer points out, it is the cohesive forceof the protoplasmic membrane and not that of the inner proto-plasm of the plasmodium which he was measuring in hisingenious experiments. That the cohesive force of the surfacelayer of a plasmodium exceeds that of the inner protoplasm isreadily demonstrated by microdissection,11

2. Technique.

Freundlich and Seifriz4 recently devised a method formeasuring the elasticity of dilute jellies and sols which theyhoped would prove applicable to protoplasm. The method wasapplied by them to non-living colloidal systems only. Theapplication of this method to the living colloid is here reported.The technique is as follows.

A minute particle of a magnetic metal is suspended, withthe aid of microneedles, in the colloidal substance (sol, jelly orprotoplasm). The particle is then attracted by an electro-magnet until the colloid is stretched a maximum amount. Ifthis amount is not exceeded, the particle will, on release of themagnetic force, return to its original position. The distanceover which the particle has travelled in one direction is ameasure of the stretching capacity of the substance. The forcenecessary to produce this stretching is a measure of theelasticity.

In order to handle exceedingly minute particles with ease amicrodissection instrument must be used. In the experimentsof Freundlich and Seifriz a Pe"terfi micromanipulator wasemployed. Such an apparatus is convenient in even relativelycoarse work on non-living systems, and is an absolute necessityin experiments on living protoplasm. With the aid of amicrodissection instrument, one can bring a minute metalparticle (as small as 7 M in diameter) into the living or non-living jelly with a minimum amount of disturbance of thecolloid. In the experiments on protoplasm an instrument ofthe Barber type was used. The complete set-up of microscope,dissection instrument, and magnet as employed in the experi-ments reported in this paper, is pictured in the accompanyingtext figure.

The metal particles are selected from nickel powder (from

An Elastic Value of ProtoplasmKalbaum) which has been screened through a very fine coppergauze (10,000 meshes to a sq. cm.). Particles 16 M in diameter

A Barber microdissection instrument attached to a microscope, with an electro-magnet inposition for making elasticity measurements of living protoplasm. To the microdissectioninstrument «re clamped two glass needles the right angle tips of which, together with the metaltip of the magnet core, project into a glass moist chamber under the microscope objective.

are chosen, of as near spherical shape as possible. Such aparticle is picked up from among the screened particles on a

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William Seifrizslide by means of a fine rigid glass microneedle which has beenpreviously dipped in warm gelatin (or a mixture of gelatin andglycerine). The gelatin acts as a suitable adhesive. With theparticle thus attached to the tip of a microneedle, it is broughtinto the material and dislodged by a second needle. Theparticle is thus left freely suspended in the colloidal mass.The needle must be so formed as to perform this operation4

to advantage. The technique of making microneedles is fullydescribed in publications by Chambers1 and P£terfi.8 Thenickel particle must be held by the gelatin coated needlewith sufficient security so that it can be forced through theresistant membrane of the jelly or protoplasm, and yet not sosecurely held that it cannot be readily dislodged by the secondneedle when within the non-living or living colloid. This isparticularly necessary when working with protoplasm, so as toavoid excessive disturbance and consequent degeneration. Toaccomplish this, when working with echinoderm eggs, forexample, necessitates considerable practice. At best, the taskof bringing a 16 M nickel particle into an echinoderm egg sothat the protoplasm remains living and normal, and so that theparticle is freely suspended within the egg out of contact withthe egg membrane, is an exceedingly trying undertakingwhich is successful only after many attempts. The task isinfinitely easier where the material is a non-living colloidsuch as gelatin.

Non-living colloidal sols and jellies are conveniently putinto a small glass receptacle placed under the microscopeobjective. Living material must be suspended in a hangingdrop on the under side of the cover slip, which forms the top ofa small glass moist chamber into which the microneedles andthe magnet point project, as indicated in the text figure.

When a i6yti particle has been successfully brought intoliving protoplasm or into gelatin, the especially constructedpoint of the electromagnet is adjusted to a position so thatthe particle and the magnet pole are within about i mm.of each other. The distance between pole and particle isdetermined by the strength of the magnetic field, which shouldbe great enough to attract the particle the maximum possibledistance (the maximum stretching capacity of the living or non-

4

An Elastic Value of Protoplasmliving jelly), without immediately tearing the particle throughthe material. This distance varies from i to 2 mm.

In order to bring the end of the magnet pole into closeproximity to the material, the core is lengthened so that thetip is 5 cm. from the coil, and consists of but a single wire ofthe core bundle. (See fig.)

The electro-magnet used in the experiments on protoplasmwas a specially constructed one built for use with either highor low voltage.* The magnet was of two separate coils, eachhaving 2900 turns of No. 25 B. and S. gauge enamelledmagnet wire. The resistance of each coil at Jj" F. was32.6 ohms, or a total of 72.4 ohms for the whole magnet whenthe coils are operated in series, and 18.1 ohms when operatedin parallel. In the experiments reported upon here the coilsof the magnet were used in parallel with a current of 35 volts.When so used such a magnet is sufficiently strong to hold55 gms. at the tip of the extended magnet core, and strongenough to hold 4.2 kgms. when a metal plate covers the fulldiameter of the core.

3. Material.

In selecting suitable material for the determination of anelastic value of protoplasm by the method here described, onemust fully appreciate the difficulties and the limitations of thetechnique. The mass of protoplasm must be sufficiently largeto permit free movement of the metal particle, and be sufficientlyresistant to tolerate the unavoidable mechanical disturbancecaused by the depositing of a particle in the protoplasm.Myxomycete plasmodium is excellent material for this purpose,and was used with success by Heilbronn' who, with a techniquesimilar to that of Freundlich and Seifriz but without the aidof micromanipulation, made determinations of the viscosity ofprotoplasm. The material selected for the present work wasthe mature unfertilised eggs of the sand-dollar, Echinarachniusparma. These eggs average about 140^ in diameter, and arevery resistant to ill-treatment. The chief difficulty which they

* The magnet used was wound for the writer by the Trenton Office of TheGeneral Electric Co. through the courtesy of the resident agent Mr L. S. Harrison,to whom the writer is indebted for the assistance rendered.

VOL. II.—NO. I. 5 A 2

William Seifrizpresent as material is the presence of a firm and gelatinousmembrane through which the metal particle must be carried,and from which it must be freed in order to leave it freelysuspended in the protoplasm in the interior of the egg.*

4. Viscosity Data.

After the writer had succeeded in getting a 16 M nickelparticle freely suspended in the protoplasm of an Echina-rachnius egg, and had established a magnetic field, insteadof there being a slight forward movement of the particledue to stretching of the protoplasm, the particle immedi-ately rushed across the central region of the egg towardthe magnet and came to a standstill a short distance fromthe surface of the egg membrane. It was quite evidentthat the protoplasm making up the interior of the egg isof considerable lower consistency than that constituting thecortical layer. The fact that the protoplasm of the core ofan echinoderm egg is of lower viscosity than the peripherallysituated protoplasm was first pointed out by Chambers * as aresult of observations made with the aid of microdissection.

After the metal particle has rapidly traversed the centralregion of the egg and become partially imbedded in the highlyviscous protoplasmic cortex, the egg can be reversed by theneedles until the particle is on that side of the egg nowfurthest away from the magnet. If the magnetic force isagain applied, the particle is rapidly drawn a second timethrough the inner protoplasm, until its hurried forward move-ment is stopped by the cortical jelly. This procedure wasrepeated three times in one egg without any observable changein the consistency of the protoplasm.

The depth of the highly viscous cortical layer of the eggprotoplasm is somewhat less than one-tenth of the diameter ofthe egg.

Since the rapid forward movement of the particle throughthe inner protoplasm of the egg is suddenly checked when theparticle reaches the egg cortex, it is probable that the high

• This research was carried on at the Mt. Desert Biological Laboratory, Maine,where the writer enjoyed the privileges of a research room through the courtesy ofthe Director, Professor Ulrich Dahlgren, to whom thanks are due.

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An Elastic Value of Protoplasmconsistency of the peripheral protoplasm is rather sharplydelimited from the dilute inner protoplasm.

An attempt to measure the actual viscosity value of theinner protoplasm of an echinoderm egg was made by comparingthe rates of travel of a particle through the protoplasm andthrough a substance of known viscosity, namely, glycerine. Itwas found that the consistency of the inner protoplasm of theEchinarachnius egg is slightly less than that of concentratedglycerine. The viscosity of concentrated glycerine (sp. gr. 1.25)is 800, based on a value of 1 for water. It is this con-sistency, namely "barely" that of glycerine, which the writer10

in his earlier investigations in microdissection attributed tothe protoplasm of the ova of the echinoderms Tripneustes andEchinarachnius.

The consistency of the peripheral protoplasmic layer ofthe egg can be expressed only crudely on the basis of itselastic value. The viscosity of the cortical protoplasm is thatof a soft jelly.

5- Elasticity Data.

With a 16 M nickel particle imbedded in the corticalprotoplasm of a mature unfertilised Echinarachnius egg, andwith the tip of the electro-magnet 0.8 mm. from the metalparticle, the current is applied to the magnet for a secondor two. The distance which the particle is drawn towardthe magnet is then quickly noted on the ocular micrometerand the current released. The particle should return to itsoriginal position. If it fails to do so the distance betweenthe original and the final resting-place must be subtractedfrom the total distance travelled toward the magnet. Thisremainder is equal to the distance travelled in the returndirection.

In the experiments of Freundlich and Seifriz the colloidalmass was in a small receptacle which was held stationaryunder the microscope objective. There was here no questionof a movement of the mass as a whole. But in the caseof a freely floating egg, the magnetic force acting on themetal particle in the protoplasm is sufficient to draw theentire egg toward the magnet pole. In order to prevent

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William Seifrizthis movement of the egg in toto, the two microdissectionneedles are so placed as to block the path of the egg. Evenwith this precaution there is a slight movement of theegg which must be noted and subtracted from the observedmovement of the particle.

The distance which the cortical layer of the protoplasm ofthe Echinarachnius egg is stretched under the conditions ofexperimentation here described, is 9 M. This value representsthe stretching capacity of the protoplasm. The elastic valuemust be expressed in terms of the force applied to causethe stretching. Such a value cannot be given mathematicallywith the data at hand ; we can only convey an idea of theapproximate elastic value of the protoplasm by comparisonwith some standard, and even here we are handicapped by thelack of any recognised standard. The study of the elasticityof elastic jellies is a neglected field.

The extent to which an elastic material is stretched isdependent upon the force applied, other factors, such astemperature, remaining constant. In the method hereemployed for making elasticity measurements, the magnetforce, P, is dependent upon the mass (strength), m, of themagnet, the mass, m', of the metal particle, and the distance,r, between the particle and the magnet, in the following

ratio: P = — - j — . # An attempt was made to substantiate

this ratio as applied to protoplasm.At a distance of 1.5 mm. from the magnet, the particle is

attracted, i.e., the protoplasm is stretched 5/u, while at a distanceof 0.8 mm. the stretching value A, is 9p. The expected resultis thus partially verified. The distance stretched, A, variesinversely as the (square of the) distance between the particleand magnet, r. Our data here is altogether too limited andinexact to warrant forming any very conclusive deductions;but in the experiments of Freundlich and Seifriz * on gelatin,it was found that the ratio between the distance stretched, A,and the square of the distance between the magnet and the

* That the magnetic force is inversely proportional to the square instead of thecube of the distance is true here, because the distance between particle and magnetis greatly in excess of the distance between the poles of the magnet

8

An Elastic Value of Protoplasmparticle, r*, held very accurately for 35 values of r,remaining constant.

Owing to the close proximity of the particle in the corticalprotoplasmic layer to the egg membrane, it is doubtful if theelasticity measured represents the maximum stretching capacityof the protoplasm. Our data is, at best, only approximate.The presence of elasticity is conclusively demonstrated; itsprecise value is only suggested by the data obtained.

A difficulty with which the worker on living protoplasm isconstantly confronted is that of determining with certaintythe condition of the living substance at the time of observation ;that is, whether the protoplasm is living and normal or not.Approximately a dozen readings were taken of the elasticityof the cortical protoplasm in one echinoderm egg without anynoticeable change in the values obtained. Suddenly, therewas a complete cessation of all movement of the particle,clearly indicating that the protoplasm had instantly coagulated,i.e. that death had resulted. The coagulated state wassubsequently verified by microdissection.

The stretching value of O,M obtained is, in itself, of littlesignificance as a value. Only by comparison with somestandard will such a value mean anything. Unfortunatelythere is no accepted standard among colloidal jellies. Theelastic properties of jellies have received little attention.Comparisons of elastic values of protoplasm with those ofknown concentrations of, for example, gelatin, would give abasis for standardisation. It would, however, be unwise tomake any such comparisons until an extensive series ofcomparative measurements are made of protoplasm and ofgelatin. Different grades of gelatin, and also differentregions in the same preparation, vary greatly in theirelastic values,* even when such influencing factors as H-ionconcentration and temperature are kept constant. Untilsuch experiments are done no more precise comparison canbe made than that based on some few preliminary experiments,which show the elastic value of the protoplasm of the corticallayer of the Echinarachnius egg to be approximately that ofa soft gelatin jelly, a jelly liquid enough to flow slowly.

William Seifriz

6. The Elasticity of the Egg Membrane.Owing to the conditions under which the egg membrane

exists, surrounding, as it does, a semi-liquid sphere, it is quiteout of the question to measure its elasticity in the living statewith any precision. But a rough estimation of its stretchingcapacity can be made.

The stretching capacity of the egg membrane is very muchhigher than that of the protoplasm which it surrounds. Thehigh extensibility of the membrane is readily demonstrable withmicroneedles—the membrane can be stretched, when free fromthe egg, until it is so fine that it is bearly distinguishable.11

7. Discussion.The demonstration and measurement of elasticity in

protoplasm is not only of interest in itself as one of thephysical properties of protoplasm, but also because of itsbearing on the general problem of the structure of protoplasm.It has been customary for some time to regard protoplasmas a fine emulsion, not merely in its microscopically visiblestructure, but in its ultramicroscopic colloidal structure as well.There are many reasons to doubt this hypothesis, but we shallconsider here only the bearing of the elastic property ofprotoplasm on the problem.

Lyophilic colloids are, for the most part, prominently elastic.That this is true is evident from a superficial knowledge ofthe properties of a "trembling" jelly such as a 10 per cent,concentration of gelatin. Emulsions, on the other hand,are not elastic. This is readily demonstrated experimentally.Hatschek6 has further shown that a mathematical analysis of aliquid-liquid structure will not account for the elastic propertiesof trembling jellies. He concludes that " the theory that gelsconsist of two liquid phases must be pronounced untenable."

Since emulsions are not elastic and jellies are, and since wehave data clearly demonstrating the presence of elasticity inprotoplasm, the unavoidable deduction is, that the ultra-microscopic structure of protoplasm is not that of a fine emulsion.Protoplasm is not comparable, therefore, either in structure orbehaviour, to an emulsion.

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An Elastic Value of Protoplasm

8. Summary.

1. A technique for measuring the elastic value of livingprotoplasm is described.

2. An elastic value of the cortical layer of the protoplasmof the Echinarachnius egg is given.

3. The presence of an elastic property in protoplasm isincompatible with the emulsion hypothesis of protoplasmicstructure.

9. References.1 Chambers, R. (1922), "New Apparatus and Methods for the Dissection of Living

Cells," Anat. Rec, 1-19.1 Chambers R. (1921), " Microdissection Studies—III. Some Problems in the

Maturation and Fertilisation of the Echinodenn Egg," Biol. Bull., 41, 318-35.* Dujardin, F. (1835), "Recherches sur lea Organismes inferieurs," Zoologie, 4,

2« serie, 343-76.* Freundlich, H., and Seifriz, W. (1923), "Ober die Elastizitat von Solen und Gelen,"

Zritschr. f. physik. Chem., 104, 233-61.* Hatschek, E. (1917), "An Analysis of the Theory of Gels as Systems of Two

Liquid Phases," Trans. Faraday Sac., 12, 17-23.8 Heilbronn, A. (1922), " Eine.neue Methode zur Bestimmung der Viskositat lebender

Protoplasten,"/<**/•£./. wissensch. Botan., 01, 284-38.7 Mohl, H. von (1846), "Ober die Saftbewegung im Innern der ZeUen,"Bot. Zeit, 4,

73-78, 89-94.8 Peterfi, T. (1923), "Das mikrurgische Verfahren," Die Naturwissen., 11, 81-87.0 Pfeffer, W. (1890), " Der Plasmahaut und der Vacuolen," Leipzig.

10 Seifriz, W. (1920), "Viscosity Values of Protoplasm as Determined by Micro-dissection," Bot. Gas., 70, 360-86.

n Seifriz, W. (1921), "Observations on Some Physical Properties of Protoplasm byAid of Microdissection," Ann. Bot., 187, 269-96.

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