THE ELECTRICAL CONDUCTIVITY OF PROTOPLASM By SAMUEL GELFAN From the Zoological Laboratory, University of California Received for publication, February 27, 1928 The determination of the electrical conductivity of protoplasm not only establishes a physical property of living matter but the solution of other problems in cell physiology may be simplified by an accurate knowledge of this property. The questions, for example, of the electrolytes in the interior of the cell and their state of combination can be answered by a knowledge of the specific conductance of the cell interior apart from the plasma membrane. As we shall see farther on, conclusions concerning the structure of protoplasm must necessarily follow from the conductivity data. The conductivity method has been used in biology in the study of problems of absorption and excretion of salts, effects of anesthetics and other agents, general problems of permeability, effect of injury and death, and even in the study of enzyme actions. The attempts to measure the electrical conductivity of protoplasm proper, or the cell contents, have not, however, in most cases been very exact. This is due to the difficulty presented by the plasma membrane whose high resistance accounts for the low conductivity results, and because of the minuteness of the living cells, a special and refined technique is required. That the high resistance of living cells is due to the plasma membrane has already been demonstrated by the work of HOBER (1910, 1912, 1913), PHILIPPSO~ (19~0, 1921) and McCLE~DON (19~7) by the use of high frequency currents. The theory here is that the higher the oscillatory frequency the less difference should the presence of the membrane make. In a previous paper (1927), the writer described a method which enables one to directly determine the electrical conductivity of protoplasm by means of non-polarizable micro-electrodes. The points of these

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electrodes are minute enough to penetrate into a single living cell without greatly injuring the cell. It was pointed out that at the point of entrance, especially in the plant cell Nitella, a small amount of protoplasm congeals around the electrode so that a seal is made. The results obtained by measuring the internal specific conductance of four ciliated Protozoa and the vacuolar sap of the plant cell Nitella were also given. These studies have been extended to other Protozoa, Amoeba p~'oteus, Euplotes , Spirostomum teres, and Frontonia. The conductivities of starfish (Pisaster) o(icytes and the protoplasm of Nitella have also been determined.

Nitella It was surprising to find that the protoplasm of Nitella has a somewhat lower conductivity than the vacuolar sap. Whereas the sap was previously found to have a conductivity that was equivalent to a 907 N KC1 solution, the conduc~vity of the protoplasm was this time found t o be equivalent to a "04 N KCI solution. The protoplasmic layer in the cell of Nitella, which is very thin, lies close to the cell wall. The rest of the interior of the cell is taken up by the large vacuole containing the cell sap. Because of the extreme thinness of the layer of protoplasm, it is very difficult in the case of small cells to insert the electrode into the protoplasmic layer and to make certain that they are not in the sap vacuole. But by using cells from 3 to 5 mm in length it has been found possible to determine whether the minute points of the electrodes are in the protoplasmic layer or in the vacuole. It was possible therefor to measure with certainty the conductivity of the protoplasm itself as well as that of the cell sap. Whether the protoplasm is streaming or not appears to have no appreciable effect upon the conductivity, whether it is the conductance of the protoplasm or sap that is being measured. The insertion and withdrawal of the electrodes from the cell causes the streaming, as a rule, to stop. Unless the cell is killed, the streaming will soon commence again, and after about two minutes will have attained its normal rate. With the electrodes inside the cell, the already streaming protoplasm can be caused to stop streaming by a slight movement of the electrode. Because of the minuteness of the tips of the electrodes, the current applied by a dry cell becomes almost infinitely small, since the resistance of tile electrodes is so great. This current in going through the Protozoa as well as through Nitella seems to have no observable effect on the Protoplasma. IV

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protoplasm or the cell as a whole. With larger platinum electrodes, however, and a stronger current, the protoplasmic streaming will stop when the circuit is closed, but after about two minutes it will have commenced again as usual, irrespective of whether the circuit remains closed or not. The breaking of the circuit has no effect, but making it always stops the streaming, provided that the current is of a great enough intensity. With a still stronger current, the streaming that has stopped will not commence again unless the circuit is broken. The cessation of protoplasmic streaming in Nitella appears to be a gelation phenomenon caused by the mechanical or electrical stimulus. By a quick thrust of the needle, thus causing a wound in the cell in which the protoplasm was normally streaming the protoplasm that flows out is obviously of a fluid nature since it quickly diffuses inta the water. But if a puncture is made immediately after the streaming has been caused to stop, the small globule of protoplasm that may flow out does so slowly and adheres to the cell wall, appearing to be quite viscous. That a gelation phenomenon takes place when the streaming' stops was also indicated by the observation that the Brownian movement stops for a brief time upon the cessation of protoplasmic streaming. The significance of this observation will be brought out in the discussion. The following table lists the forms, the concentration of KC1 to which their internal conductivity corresponds, and the specific conductance in reciprocal ohms at 18 o C. The four ciliates and the vacuolar sap of N i t e l l a (with asterisk) reported in the last paper are included here for sake of comparison and completeness. :Normality KC1 A m o e b a proteus "01 *Stentor . . . . . . "035 Euplotes . . . . . . "0375 S p i r o s t o m u m fetes "0385 *Blepharisma . . . . "04 *Oxytricha . . . . . "05 Frontonia . . . . . "053 '+Paramecium . . . . "06 Nitella protoplasm "04 *Nitella sap . . . . . "07 Starfish (Pisaster) o6cytes "25

Specific Conductance ohms-1 CIIl.-1 1"295 X 10-3 4"1 X 10 -3 4"4 X 10-3 4"5 }( 10-3 4"7 }( 10-3 5"8 X 10-3 6"1 X 10-3 6"9 X 10 -3 4.7 X 10 -3 7.9 X 10-3 26"2 X 10-3.

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PROTOZOA Of those Protozoa studied Amoeba proteus has the lowest internal conductivity and Paramecium the highest. In all, however, as the conductivity data indicates, the electrolytic content is markedly greater than that of their fresh water medium. No difficulty is encountered in inserting the electrodes into the protozoan cells. The injury caused by the micro-electrodes can not be great, since these animals survive and continue to multiply after mechanical treatments that are much more severe than the one required for the conductivity measurement.

STARFISH O(}CYTES Ovarian eggs taken from the starfish Pisaster, that occur on the piling of San Francisco Bay, showed a surprisingly high conductivity. Several hundred eggs from different individuals coming from the same locality were measured, but they all had a specific conductance equivalent to that of a "25 N KC1 solution. The conductivity of the sea water from that part of the Bay in which the starfish were found was determined by the same method that was used for the eggs, and was found to be equivalent to a "~5 N KCI solution. The conductivity of the eggs is therefor the same as the seawater of this particular part of the Bay. To the writer's knowledge there are no published data on the electrical conductivity of the sea water of this coast. But RIVERS-MOORE (1919) published some data on the electrical conductivity of samples of sea water from the open sea on the "South Coast". The specific resistance of his sample at 150 C. is 24"3 ohms. According to our measurements, the specific resistance of the water from that part of the San Francisco Bay in which the starfish were found, at 18 o C. is 38"1 ohms. Professor C. B. LIPMASTin his yet unpublished data has found a considerable difference in the salt content between the Bay and the open sea water of this coast. The water in the San Francisco Bay, according to chemical analysis is markedly more dilute than the open sea water. According to the report of SUM~En, LOUDERBACK, S C ~ I T T and JOHNSTON (1914), the salinity of that part of the Bay from which the starfish were taken is considerably lower than that of the open sea. McCLENDON (1910) and G~Au (1916) using the conductivity method for studying fertilization of Echinoderm eggs, found that the normal unfertilized eggs had a high resistance. Although the conductivity increases upon fertilization, it is even then considerably lower than that 13.

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of the sea water. The high resistance of the normal unfertilized eggs is to be expected since in this case the measurements are made of massed suspensions of eggs in sea water. The eggs normaly not being permeable to ions, they serve in this case mostly to obstruct the passage of the current through the sea water. Upon fertilization, the resistance is lowered because of increased permeability. CONDUCTIVITY AND STRUCTURE OF PROTOPLASM I t is obvious then, that the internal conductivity of living cells, or of the protoplasm apart from the plasma membrane is fairly high. The average specific conductance of the protoplasm of the forms here reported, except for the unusually high one in the starfish eggs is practically equivalent to a "05 N KC1 solution, and the latter is one of our best inorganic electrolytes. The element of viscosity, which is a universal characteristic of protoplasm, has as yet not been taken into account in the work on the conductivity of protoplasm. There are no complete data on the effect of viscosity on the conductance of solutions. B u t there are sufficient indications from the work of K~AUS (]952) and others that in the case of aqueous solutions which exhibit a negative viscosity effect, the conductance is in direct proportion to the fluidity and in salt solutions which exhibit a positive viscosity effect, the correction for the viscosity change is slightly smaller. W h e t h e r the dispersion medium in protoplasm is water, however, is not definitely established. According to ETTISCtt and PI~TERFI (1955), it is water. But the effect of viscosity, especially large increases in the viscosity of the medium in case of non-aqueous solutions, is j u s t as effective in lowering the conductance. Though the work on the viscosity of protoplasm is not very exact, yet simple microscopic examination will reveal that protoplasm has a viscosity at least higher than that of water. Viscosity was one of the first characteristics to be observed about protoplasm, SEIFRIZ (1924) measured the viscosity of the sand dollar eggs by introducing minute nickel particles into the eggs, and comparing the rate of migration in a magnetic field in this case with the rate of migl"ation of the same particle in a substance of known viscosity. He finds these echinoderm eggs to possess a viscosity that is slightly lower than that of concentrated glycerine. The latter has a viscosity of 8007 based on a value of 1 for water. HEmBaONN (1922) had already utilized the magnet method without micromanipulation on myxomyccte plasmodia. For the several species used, he gets viscosity values ranging from 9 to 18 tflncs that of water.

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I-[EILBRUNN(1926 a, b), using the centrifuge method, and applying" STOKES'law for the movement of a spherical particle through a fluid, finds the viscosity of the granule-free protoplasm of sea-urchin (Arbacia) eggs to be approximately two times that of water. The gTanule-free protoplasm of the eggs of the clam Cumingia he found to have a viscosity about four times that of water. In both forms, the viscosity of the entire protoplasm of the eggs is about two to three times that of the granule-free protoplasm. Because HEILBRUNN'svalues for viscosity are so much lower than the value obtained by SEIFRIZ, the former doubts the accuracy of the magnet method used by SEIFRIZ. ~{EILBRUNNhimself, however, is aware of the fact that in his own method, the application of STOKES~ formnla cannot be very accurately made in viscosity measuremeuts of protoplasm. It is also quite possible that the Echinaraehnius eggs used by SEIFRIZ and the Arbacia eggs used by HEILBRUNN,although belonging to the same class of echinoderms possess markedly different viscosities. From microscopical examination and from mierodisseetion studies, one would expect the Protozoa to have a greater viscosity than the eggs. That this is probably true is indicated by the results of MIss FETTER (1926). She used HEILBRUNN's centrifuge method and calculated the viscosity of the protoplasm of Paramecium to be from 8000 to 8700 times that of water. F r o m the considerations of the effect of the increase of viscosity on conductance and the viscosity m e a s u r e m e n t s of protoplasm the conductivity data would not indicate the true electrolytic content of the living cell. If the electric current, during conductivity m e a s u r e m e n t s in protoplasm is carried by simple ions, to find the exact electrolytic content in the cell, a viscosity correction would have to be made. B u t if we do apply viscosity correction for protoplasm, the resulting figures become impossible. F o r Paramecium, for example, after the viscosity correction, the electrolytic content would be equivalent to about 500 N KC1. Not only from chemical considerations would the results of viscosity corrections be impossible, but from chemical analysis of living tissues, the salt content is of the same general order of magnitude as is indicated by the conductivity measurements. I t appears then, that in protoplasm the conductance is not affected by the viscosity as would be expected. I f we assume t h a t the viscosity is due to a coarse structure or a loose network, the mobility of the ions or even larger charged carriers, if such exist in the cell, would not be hindered and the conductivity would consequently be independent of the viscosity. Such a structure has been postulated by MCBAIN (1994) and his coworkers for soap sols and gels. ZSlG~ONDY and BACHMANN (1919) had already pointed out the existence of a fibrillar structure in addition to the grainy structure in dilute gels of gelatin, agar, and silica. SEIFRIZ (1996) has adopted this " b r u s h - h e a p " structure for protoplasm in order to explain the measurable elasticity in protoplasm.

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MIss LAING and MCBAIN (1920) found that the conductivity, as well as several other properties, are identical in soap sols and gels. The viscosity may change a thousand fold during gelation yet the conductivity is unaffected. We have also observed in Nitella that the conductivity of the protoplasm does not materially change when the streaming is caused to stop, and as has been pointed out, the cessation of streaming appears to be a gelation phenomenon. In soap solutions, according to McBAIN (1923), half of the conductivity is due to a colloidal electrolyte, the ionic micelle. "A colloidal electrolyte is a salt in which one of the ions is replaced by ionic micelle; that is, highly charged and solvated colloidal particles." Whether the electric current in case of protoplasm is carried by ions or ionic micelles is difficult to prove. But the structural part of the protoplasm, the open network that imparts the apparent viscosity but does not hinder the movement of the charged particles, whether ionic or colloidal, must be made up of similar protein units as is postulated for the soaps and other elastic jellies, These units are the neutral micelles or aggregates of micelles. We are proposing then, the miccllar theory for the structure of protoplasm, the theory that was originally proposed by N~GELI. With the introduction of the ultra-microscope this theory was resuscitated by ZSIGMONDY and BACIIMANN (1919) for the explanation of the structure of gels. According to LAI~G and McBAIN (1920), the quantitative identity of conductivity in sol and gel is irreconcilable with all theories of gel structure hitherto advanced, with the exception of the micellar theory of Ns As WEISER (1924) states, this two phased solidliquid conception of the structure of gels is now held by the majority of colloidal chemists. The contents of the living cell cannot be considered as a rigid gel but the properties of protoplasm are characteristic of hydrophile colloids like gelatin, soap solutions, gums, &c. Hydrophobic solsl), like metal suspensions, and hydroxides of metals, are little hydrated, and unstable. They are coagulated by even small traces of salts like KC1 and NaC1. From the conductivity data and chemical analysis, the salt content in protoplasm would easily precipitate a hydrophobic colloid. A high measurable conductivity is a distinct property of hydrophilic colloids (see FREUNDHCH, 1926). Hydrophilic colloids possess the property of surfacd tension that is markedly different from hydrophobic colloids. Whereas in the latter 1 FREUNDLICtt (1926) uses the terms tyophobie and lyophilic.

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the surface tension is not different from that of water, the surface of hydrophilic colloids is very active. They can from solid films, while on hydrophobic sols no solid films are formed. Films, like plasma membranes and nuclear membranes are universal among living organisms. It is known that new films will be formed in living protoplasm at surfaces freshly exposed by cutting or other injury. Sol-gel transformation, a common phenomenon in protoplasm, is characteristic of hydrophilic colloids only. The property that characterizes hydrophilic colloids more than any other is that of viscosity. Even very dilute solutions are appreciably more viscous than water. That protoplasm is more viscous than water cannot be questioned. As a result of a mathematical treatment HATSCttEK (1917) concludes that the structure of a gel cannot be that of an emulsion, since a liquidliquid system cannot account for the elasticity. FREU~DLICIt and SEIFRIZ (1923), using a micromagnetic technique found that sols possess elasticity as well as gels, although the latter to a greater extent. They found measurable elastic properties in a dilute soap solution, the viscosity of which was less than twice that of water. Using this method SE~FRIZ (19~4) has demonstrated elasticity in sand dollar eggs. He has also demonstrated it in other animal cells by stretching with micro-needles. The ultramicroscopic structure of protoplasm can therefore not be that of a fine emulsion. Because of its elasticity, SEIFRIZ (1926) regards protoplasm as a gel or at least as possessing the physical properties of jellies. To account for the elasticity in gels, the structural units are conceived as being linear in configuration, long tenuous fibres which interlace. In the case of soaps, MCBAIN (1924) postulates such a filamentous structure. Although microscopically protoplasm may appear to be a suspension or emulsion or both it follows from the above considerations of its properties that its fundamental structure must be similar to the type of colloid that is exemplified by gelatin and soaps. It has already been pointed out that the significant properties in sol and gel are identical. The difference is only in degree of elasticity and rigiditity, since even dilute sols possess elasticity. The colloidal units are therefore the same, except in the gels they become larger. A high conductivity in protoplasm in spitn of the viscosity, and its hydrophililic properties forces us to a conception of the structure of protoplasm that is based on the micellar theory.

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Gelfan, The electrical conductivity of protoplasm SUMMARY

T h e specific c o n d u c t a n c e of the protoplasm, a p a r t from the cell m e m b r a n e of Amoeba proteus, Euplotes, Spirostomum teres~ Frontonia, of the p l a n t cell Nitella and the starfish o~igonia has been determined. T h e conductivities of all these forms except the starfish eggs, v a r y from one t h a t is e q u i v a l e n t to a '01 N KCI solution to one t h a t is e q u i v a l e n t to a "06 N KC1 solution. T h e a v e r a g e is e q u i v a l e n t to a b o u t a "05 N KC1 solution. T h e c o n d u c t i v i t y of the starfish eggs is v e r y high, b e i n g e q u i v a l e n t to a "25 KC1 solution. I t is pointed out t h a t the c o n d u c t a n c e of the p r o t o p l a s m is n o t affected by the viscosity. This and other evidence is a d v a n c e d p o i n t i n g to a micellar s t r u c t u r e of protoplasm. LITERATURE

CITED

ETTISCH, G., and P]~TERFI, T., Pfliig. Arch. Phys., 208, 3./4. Heft, 467, 1925. FETTER, Dorothy, Jour. Exp. Zool. 44, 279, 1926. FREUNDLICH, ]:I., Colloid and Capillary Chemistry, 1926. - - and SEIFRIZ, W., Zeitschr. f. phys. Chem. 10~, 233, 1923. ~ELFAN, S., Univ. Cal. Publ. Zool., 29, no 17, 435, 1927. GR.XY, J., Phil. Trans. Roy. Soc. Lon., 207, B, 481, 1916. HATSCHEK, E., Trans. Faraday Soe., 12, 17, 1917. HEILBRONN, A., Jahrb. f. wissensch. Botan., 61, 284, 1922. HEILBRUNN, L.V., Jour. Exp. Zool., 44:, 255, 1926. -- Am. Naturalist 60, 143, 1926. HOBER, R., Pfliig. Arch. Phys., 183, 237, 1910. - - Ibid., 1~8, 189, 1912. - - Ibid., 150, 15, 1913. KRAUS, C., Properties of Electrical Conducting Systems, 1922. LAING, M. E., and MCBAIN, J., Trans. Chem. Soe., 117, 1506, 1920. MCBAIN, J., Union Internationale de la Chimie Pure et Appliqu6e, 1923. - - Bogue's Colloidal Behavior, 1, 1924. lV[CCLENDON, J. F., Am. J. Physiol. 27, 250, 1910. -- Protoplasma, 8, 7, and 71, 1927. PHILIPPSON, ]~., C. l=~. Paris Soc. Biol., 83, 1399, 1920. - - Bull. Brussels Acad. Roy. Belgique, 7, ser. 5, 387, 1921. RIVERS-MOORE, H. R., Electrician, 82, 174, 1919. SEIFRIZ, W., Brit. Jour. Exp. Biol. 2, 1, 1924. - - Am. Naturalist, 60, 124, 1926. SUMNER: F. B., LOUDERBACK,D. D., SCHMITT,W. L., and JOHNSTON, E. C., Univ. Calif. Publ. Zool., 14, no. 1, 1, 1914. WEISER, ~I., Bogue's Colloidal Behavior, 1, 1914. ZSIGMONDY, :R., and BACHMANN,~u Koll.-Zeitschr, 11, 150, 1912.