An Elastic Value of Protoplasm, with Further Observations on the Viscosity of Protoplasm

The physical properties of protoplasm have been the subject of keen investigation ever since the discovery of the living substance nearly a century ago. The earliest descriptions of protoplasm refer to its physical nature. Thus, Dujardin⁸  characterised the “living jelly” as a glutinous transparent substance insoluble in water, and von Mohl ⁷  described it as a “viscous liquid mass.”

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

Some interesting experiments were done by Pfeffer ⁹  on the “elastic stretching capacity” of protoplasm. Pfeffer was able, by attaching weights to protoplasmic threads, to determine the load which a strand of myxomycete plasmodium can support. What Pfeffer was measuring was not the elasticity of protoplasm but its tensile strength, or as he expressed it, the cohesive force. Further, as Pfeffer points out, it is the cohesive force of the protoplasmic membrane and not that of the inner proto-plasm of the plasmodium which he was measuring in his ingenious experiments. That the cohesive force of the surface layer of a plasmodium exceeds that of the inner protoplasm is readily demonstrated by microdissection.¹¹ 

Freundlich and Seifriz⁴  recently devised a method for measuring the elasticity of dilute jellies and sols which they hoped would prove applicable to protoplasm. The method was applied by them to non-living colloidal systems only. The application of this method to the living colloid is here reported. The technique is as follows.

A minute particle of a magnetic metal is suspended, with the aid of microneedles, in the colloidal substance (sol, jelly or protoplasm). The particle is then attracted by an electromagnet until the colloid is stretched a maximum amount. If this amount is not exceeded, the particle will, on release of the magnetic force, return to its original position. The distance over which the particle has travelled in one direction is a measure of the stretching capacity of the substance. The force necessary to produce this stretching is a measure of the elasticity.

In order to handle exceedingly minute particles with ease a microdissection instrument must be used. In the experiments of Freundlich and Seifriz a Péterfi micromanipulator was employed. Such an apparatus is convenient in even relatively coarse work on non-living systems, and is an absolute necessity in experiments on living protoplasm. With the aid of a microdissection instrument, one can bring a minute metal particle (as small as 7 µ in diameter) into the living or non-living jelly with a minimum amount of disturbance of the colloid. In the experiments on protoplasm an instrument of the 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 accompanying text figure.

The metal particles are selected from nickel powder (from Kalbaum) which has been screened through a very fine copper gauze (10,000 meshes to a sq. cm.). Particles 16 µ in diameter are chosen, of as near spherical shape as possible. Such a particle is picked up from among the screened particles on a slide by means of a fine rigid glass microneedle which has been previously dipped in warm gelatin (or a mixture of gelatin and glycerine). The gelatin acts as a suitable adhesive. With the particle thus attached to the tip of a microneedle, it is brought into the material and dislodged by a second needle. The particle is thus left freely suspended in the colloidal mass. The needle must be so formed as to perform this operation to advantage. The technique of making microneedles is fully described in publications by Chambers ¹  and Péterfi.⁸  The nickel particle must be held by the gelatin coated needle with sufficient security so that it can be forced through the resistant membrane of the jelly or protoplasm, and yet not so securely held that it cannot be readily dislodged by the second needle when within the non-living or living colloid. This is particularly necessary when working with protoplasm, so as to avoid excessive disturbance and consequent degeneration. To accomplish this, when working with echinoderm eggs, for example, necessitates considerable practice. At best, the task of bringing a 16 µ. nickel particle into an echinoderm egg so that the protoplasm remains living and normal, and so that the particle is freely suspended within the egg out of contact with the egg membrane, is an exceedingly trying undertaking which is successful only after many attempts. The task is infinitely easier where the material is a non-living colloid such as gelatin.

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

When a 16 µ particle has been successfully brought into living protoplasm or into gelatin, the especially constructed point of the electromagnet is adjusted to a position so that the particle and the magnet pole are within about 1 mm. of each other. The distance between pole and particle is determined by the strength of the magnetic field, which should be great enough to attract the particle the maximum possible distance (the maximum stretching capacity of the living or non-living jelly), without immediately tearing the particle through the material. This distance varies from 1 to 2 mm.

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

The electro-magnet used in the experiments on protoplasm was a specially constructed one built for use with either high or low voltage.^* The magnet was of two separate coils, each having 2900 turns of No. 25 B. and S. gauge enamelled magnet wire. The resistance of each coil at 77° F. was 32.6 ohms, or a total of 72.4 ohms for the whole magnet when the coils are operated in series, and 18.1 ohms when operated in parallel. In the experiments reported upon here the coils of the magnet were used in parallel with a current of 35 volts. When so used such a magnet is sufficiently strong to hold 55 gms. at the tip of the extended magnet core, and strong enough to hold 4.2 kgms. when a metal plate covers the full diameter of the core.

In selecting suitable material for the determination of an elastic value of protoplasm by the method here described, one must fully appreciate the difficulties and the limitations of the technique. The mass of protoplasm must be sufficiently large to permit free movement of the metal particle, and be sufficiently resistant to tolerate the unavoidable mechanical disturbance caused by the depositing of a particle in the protoplasm. Myxomycete plasmodium is excellent material for this purpose, and was used with success by Heilbronn ⁶  who, with a technique similar to that of Freundlich and Seifriz but without the aid of micromanipulation, made determinations of the viscosity of protoplasm. The material selected for the present work was the mature unfertilised eggs of the sand-dollar, Echinarachnius parma. These eggs average about 140µ in diameter, and are very resistant to ill-treatment. The chief difficulty which they present as material is the presence of a firm and gelatinous membrane through which the metal particle must be carried, and from which it must be freed in order to leave it freely suspended in the protoplasm in the interior of the egg.^*

After the writer had succeeded in getting a 16 µ nickel particle freely suspended in the protoplasm of an Echina-rachnius egg, and had established a magnetic field, instead of there being a slight forward movement of the particle due to stretching of the protoplasm, the particle immediately rushed across the central region of the egg toward the magnet and came to a standstill a short distance from the surface of the egg membrane. It was quite evident that the protoplasm making up the interior of the egg is of considerable lower consistency than that constituting the cortical layer. The fact that the protoplasm of the core of an echinoderm egg is of lower viscosity than the peripherally situated protoplasm was first pointed out by Chambers ²  as a result of observations made with the aid of microdissection.

After the metal particle has rapidly traversed the central region of the egg and become partially imbedded in the highly viscous protoplasmic cortex, the egg can be reversed by the needles until the particle is on that side of the egg now furthest away from the magnet. If the magnetic force is again applied, the particle is rapidly drawn a second time through the inner protoplasm, until its hurried forward move-ment is stopped by the cortical jelly. This procedure was repeated three times in one egg without any observable change in the consistency of the protoplasm.

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

Since the rapid forward movement of the particle through the inner protoplasm of the egg is suddenly checked when the particle reaches the egg cortex, it is probable that the high consistency of the peripheral protoplasm is rather sharply delimited from the dilute inner protoplasm.

An attempt to measure the actual viscosity value of the inner protoplasm of an echinoderm egg was made by comparing the rates of travel of a particle through the protoplasm and through a substance of known viscosity, namely, glycerine. It was found that the consistency of the inner protoplasm of the Echinarachnius egg is slightly less than that of concentrated glycerine. The viscosity of concentrated glycerine (sp. gr. 1.25) is 800, based on a value of 1 for water. It is this consistency, namely “barely” that of glycerine, which the writer ¹⁰  in his earlier investigations in microdissection attributed to the protoplasm of the ova of the echinoderms Tripneustes and Echinarachnius.

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

With a 16 µ nickel particle imbedded in the cortical protoplasm of a mature unfertilised Echinarachnius egg, and with the tip of the electro-magnet 0.8 mm. from the metal particle, the current is applied to the magnet for a second or two. The distance which the particle is drawn toward the magnet is then quickly noted on the ocular micrometer and the current released. The particle should return to its original position. If it fails to do so the distance between the original and the final resting-place must be subtracted from the total distance travelled toward the magnet. This remainder is equal to the distance travelled in the return direction.

In the experiments of Freundlich and Seifriz the colloidal mass was in a small receptacle which was held stationary under the microscope objective. There was here no question of a movement of the mass as a whole. But in the case of a freely floating egg, the magnetic force acting on the metal particle in the protoplasm is sufficient to draw the entire egg toward the magnet pole. In order to prevent this movement of the egg in toto, the two microdissection needles are so placed as to block the path of the egg. Even with this precaution there is a slight movement of the egg which must be noted and subtracted from the observed movement of the particle.

The distance which the cortical layer of the protoplasm of the Echinarachnius egg is stretched under the conditions of experimentation here described, is 9 µ. This value represents the stretching capacity of the protoplasm. The elastic value must be expressed in terms of the force applied to cause the stretching. Such a value cannot be given mathematically with the data at hand; we can only convey an idea of the approximate elastic value of the protoplasm by comparison with some standard, and even here we are handicapped by the lack of any recognised standard. The study of the elasticity of elastic jellies is a neglected field.

The extent to which an elastic material is stretched is dependent upon the force applied, other factors, such as temperature, remaining constant. In the method here employed for making elasticity measurements, the magnet force, P, is dependent upon the mass (strength), m, of the magnet, the mass, m′, of the metal particle, and the distance, r, between the particle and the magnet, in the following ^* 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 µ, while at a distance of 0.8 mm. the stretching value Δ, is 9 µ. The expected result is thus partially verified. The distance stretched, Δ, varies inversely as the (square of the) distance between the particle and magnet, r. Our data here is altogether too limited and inexact to warrant forming any very conclusive deductions; but in the experiments of Freundlich and Seifriz⁴  on gelatin, it was found that the ratio between the distance stretched, Δ, and the square of the distance between the magnet and the particle, r³, held very accurately for 35 values of r, Δr³ remaining constant.

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

A difficulty with which the worker on living protoplasm is constantly confronted is that of determining with certainty the 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 elasticity of the cortical protoplasm in one echinoderm egg without any noticeable change in the values obtained. Suddenly, there was 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 was subsequently verified by microdissection.

The stretching value of 9 µ obtained is, in itself, of little significance as a value. Only by comparison with some standard will such a value mean anything. Unfortunately there is no accepted standard among colloidal jellies. The elastic properties of jellies have received little attention. Comparisons of elastic values of protoplasm with those of known concentrations of, for example, gelatin, would give a basis for standardisation. It would, however, be unwise to make any such comparisons until an extensive series of comparative measurements are made of protoplasm and of gelatin. Different grades of gelatin, and also different regions in the same preparation, vary greatly in their elastic values,⁴  even when such influencing factors as H-ion concentration and temperature are kept constant. Until such experiments are done no more precise comparison can be made than that based on some few preliminary experiments, which show the elastic value of the protoplasm of the cortical layer of the Echinarachnius egg to be approximately that of a soft gelatin jelly, a jelly liquid enough to flow slowly.

Owing to the conditions under which the egg membrane exists, surrounding, as it does, a semi-liquid sphere, it is quite out of the question to measure its elasticity in the living state with any precision. But a rough estimation of its stretching capacity can be made.

The stretching capacity of the egg membrane is very much higher than that of the protoplasm which it surrounds. The high extensibility of the membrane is readily demonstrable with microneedles—the membrane can be stretched, when free from the egg, until it is so fine that it is bearly distinguishable.¹¹ 

The demonstration and measurement of elasticity in protoplasm is not only of interest in itself as one of the physical properties of protoplasm, but also because of its bearing on the general problem of the structure of protoplasm. It has been customary for some time to regard protoplasm as a fine emulsion, not merely in its microscopically visible structure, but in its ultramicroscopic colloidal structure as well. There are many reasons to doubt this hypothesis, but we shall consider here only the bearing of the elastic property of protoplasm on the problem.

Lyophilic colloids are, for the most part, prominently elastic. That this is true is evident from a superficial knowledge of the properties of a “trembling” jelly such as a 10 percent. concentration of gelatin. Emulsions, on the other hand, are not elastic. This is readily demonstrated experimentally. Hatschek ⁶  has further shown that a mathematical analysis of a liquid-liquid structure will not account for the elastic properties of trembling jellies. He concludes that “the theory that gels consist of two liquid phases must be pronounced untenable.”

Since emulsions are not elastic and jellies are, and since we have data clearly demonstrating the presence of elasticity in protoplasm, 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 or behaviour, to an emulsion.