Injury, Recovery and Death in Relation to Conductivity and Permeability

conductivity of Laminaria, experiments were made by adding small amounts of nicotine, caffeine and cevadine sulfate to sea water (and then making the solution the same conductivity as sea water). The experiment was not successful in the case of nicotine, owing to the for- mation of a visible precipitate, which was apparently due to the presence of calcium and 'magnesium in the sea water. In the case of caffeine (0.01 to 0.04 M) and of cevadine sulfate16 (0.0006 to 0.0025 M) a distinct decrease in permeability was found I (as shown by the rise in resistance) ; this was followed by an increase.

18 This is regarded as two molecules of cevadine united to one molecule of H2S04. It was purchased from Merck under the name of veratrine sulfate ( C^H^NO, ) a.H2SO4. It is well known that death is accompanied by an increase of permeability. Thus a slice of red beet kept in water will live for a long time without giving off pig- ment, but as soon as it is killed the color begins to escape from the cells. In this case the coloring matter is dis- solved in the large central vacuole which fills the interior of the cell. In order to escape, it must pass out through the layer of protoplasm which surrounds the vacuole. As long as the protoplasm remains in its normal condi- tion it is impermeable to the dissolved pigment, but as soon as death occurs it become freely permeable and the color escapes.

We meet the same condition if we study the penetra- tion of substances from without. It is a matter of common observation that cells may resist the penetration of certain dyes as long as they 'are alive, but absorb them readily as soon as they are killed. The increase in permeability which accompanies death is paralleled in a striking manner by a simultaneous increase in electrical conductivity.1 This suggests that 1 An apparent exception to this statement is found in two articles by Galeotti (1901, 1903) who states that death produces an increase in the electrical resistance of muscle, kidney, etc., followed by a decrease. He suggests that the increase is due to the fixation of electrolytes by the tissues. In Galeotti's experiments the tissues were not immersed in solu- tions and in consequence the electrodes had to be applied directly to the surfaces of the tissue. It is possible that his results were due in part to faulty technique (bubbles of gas readily form in dying tissue, increasing the resistance). The matter requires further investigation.

Kodis ( 1901 ) whose technique seems to be decidedly preferable to that of Galeotti, (see page 21) found that dead frog muscle had less electrical resistance than living. The writer has confirmed this, using the method employed by Kodis. the two phenomena may be closely connected. If this is the case it may be possible to use electrical conductivity as a measure of permeability. Let us consider this from the standpoint of the per- meability of protoplasm to salts.

When an electrical current passes from a salt solu- tion into a living cell, ions must enter the protoplasm.2 An increase in the permeability of the protoplasm to ions must decrease its electrical resistance, and vice versa. The electrical resistance of the protoplasm may there- fore be regarded as a measure of its permeability to ions. 'In this connection it should be noted that experiments have been made with direct currents. Cf. Stiles and Jorgensen (1914).

If we attempt to measure the electrical resistance of the protoplasm we must first consider the structure of the tissue. Very useful for experiments on tissues are plants which form membranes consisting of a single (Fig. 84) or a double layer (Fig. 85) of cells. In measuring the conductivity of these plants we obtain much the same results as with the more complex tissues of Rliodymenia (Fig. 86) and Laminaria (Fig. 87). We may therefore conclude that the complexity of structure is not a factor of importance in the interpretation of the results. As a matter of fact in the case of Laminaria the resistance appears to be due chiefly to the rounded cells lying at and below the surface, while the elongated cells which occupy the center of frond have large spaces between them through which the current can easily pass.

If we consider the structure of the individual cells, we find that in Laminaria (as in the other plants employed in the experiments of the writer) the protoplasm of each cell forms a thin layer, which surrounds a large central vacuole filled with cell sap. When tissue (either with or without previous treatment with liquid air) is ground with powdered quartz (so as. to open the cells) and a little sea water is added, and the juice is subsequently expressed, it is found to have a little higher conductivity than sea water. Since this expressed juice consists to a

considerable extent of cell sap we may conclude that the conductivity of the sap does not differ very much from that of sea water. The fact that the conductivity is higher, may be due in part to evaporation during the manipulation. When the juice is obtained by merely heating the plants to 100° C in a tightly stoppered bottle (without addition of sea water) the conductivity equals that of sea water. This is confirmed by some observations on Valonia. This marine alga forms a large, multinucleate cell consist- ing of a cell wall, within which is a delicate layer of protoplasm, forming a sac which encloses a very large central vauole. By drying the exterior and pricking the cell, the sap can be made to spurt out and may then be collected for examination. It was found by Wodehouse (1917) that the sap of uninjured cells gives little or no test for S04 : the contents of each cell were accordingly tested by this method by Dr. Crozier, who kindly collected the sap for these experiments, and rejected those which contained more than a minimum amount of S04. Deter- minations of the electrical conductivity of the sap, by the writer, showed that it was not much higher (in no case more than 20%) than that of the surrounding sea water.

Since the cell sap of Laminaria has about the same electrical resistance as the solution which bathes the cell, it is evident that if the electrical resistance of the cell increases when it is transferred from sea water to another solution of the same conductivity, the change must be due to an increase in the resistance of the thin layer of proto- plasm which bounds the cell. This has led the writer to assume that the resistance is proportional to a substance, 7lf, at the surface of the cell ; if M forms a layer at the sur- face, it is obvious that an increase in the thickness of this layer will increase the resistance, and vice versa. It is therefore assumed that the resistance depends upon the amount of M which is present in the surface.3

Since the protoplasmic masses (cells) are separated from each other by thin layers of substance (cell wall) a part of the current goes through the protoplasm and another part passes between the protoplasmic masses, in the substance of the cell wall.4 Consequently when we employ the electrical method we must ascertain whether we are investigating the permeability of the protoplasm or merely that of the cell wall. Obviously the best method of attacking this problem is to kill the tissue by such means (e. g., partial drying, heating to 35 °C., weak alcohol, etc.) as cannot alter the cell wall, and then investigate its behavior under the influence of various reagents. We find that all of these methods produce the same result. After death the tissue no longer shows the changes in resistance which are observed when living tissue is subjected to the influence of reagents. It is therefore evident that the changes are due to the living protoplasm.

The cell wall appears in all cases to have practically the same conductivity as the surrounding solution. If we subject living tissue to solutions of the same conduc- tivity, but of different chemical composition, the resist- ance of the cell wall remains unaltered, while that of the protoplasm undergoes great variations. If, for example, living tissue is placed in a solution of NaCl or CaCL, (of the same conductivity as sea water) its behavior differs. In NaCl the resistance falls ; in CaCl2, it rapidly rises and later falls to a minimum. We infer that the permeability

4 As previously explained (Cf. Osterhout, 1918, (7), a part of the current must pass through the protoplasm; this is shown by the fact that CaCl2 (which has little effect on the resistance of the cell wall) raises the resistance of the tissue and that the temperature coefficient of electrical conductivity is not the same for dead as for living tissue. of the protoplasm increases in NaCl; and that in CaCl2 there is a decrease followed by an increase.

This is in complete agreement with results obtained when permeability is measured by such methods as plasmolysis, specific gravity, tissue tension, exosmosis, diffusion through living tissue and direct determination of penetrating substances. FIG. 88. — A vegetable cell showing plasmolysis. At left, normal; in the center, plasmolyzed; In order to show the bearing of these measurements on the problem, the methods employed will be briefly described.

The measurement of osmotic pressure by means of plasmolysis depends upon the fact that when the osmotic pressure within a cell is greater than that of the surround- ing solution, water is absorbed. In the case of plant cells, there is usually a central vacuole, the contents of which exert pressure against the protoplasmic sac which sur- rounds the vacuole : in consequence the protoplasmic sac is pressed against the cell wall. If, however, the osmotic pressure of the external solution exceeds that of the vacuole, water is withdrawn from the cell and the proto- plasmic sac contracts. This contraction is called plasmolysis (Fig. 88). At the moment when contraction begins, the osmotic pressure of the solution within the cell is regarded as slightly less than that of the external

solution. We therefore obtain in this manner an approxi- mate measure of the osmotic pressure of the cell.5 Strictly speaking, plasmolysis measures the osmotic pressure within the cell without telling us anything regarding the penetration of substances into the proto- plasm. We may, nevertheless, learn something about permeability by this method. When, for example, we find that a substance (such as alcohol) fails to produce as much plasmolysis as is expected, we infer that this substance penetrates the cell so rapidly, as to partially offset its own osmotic effect. Let us suppose that the osmotic pressure within the cell is 10 atmospheres. On placing the cell in a solution of alcohol, whose osmotic pressure is 11 atmospheres, plasmolysis might be ex- pected to occur, but if there is an immediate penetration of alcohol, which raises the osmotic pressure within the cell to 11 atmospheres, no plasmolysis will take place.

On placing the cell in a solution of alcohol strong enough to cause plasmolysis we should expect the alcohol to penetrate until the osmotic pressure of alcohol is the same inside and outside of the cell. When this has occurred, the osmotic pressure in the cell will be equal to 6 There are a number of disturbing factors which interfere with such measurements. Among these may be mentioned: 1. The plasmolyzing agent may have a toxic action and may alter the permeability of the cell.

2. Plasmolysis may produce mechanical injury due to the tearing of the outer layer of protoplasm. 3. Exosmosis of dissolved substances may occur during ex- posure to the reagent. 4. The time necessary to produce plasmolysis is an important factor which is frequently overlooked. 5. The shrinkage of the protoplasm away from the cell wall is preceded by a diminution of volume of the entire cell. In some cases, where the cellulose wall is considerably stretched by the internal osmotic pressure, this may be of considerable importance.

the original osmotic pressure, plus the increased pres- sure due to the withdrawal of water (thus increasing the concentration) plus the osmotic pressure due to the pene- tration of alcohol. The total osmotic pressure will there- fore be greater than that of the external solution and water will accordingly be absorbed by the cell. The result will be that the plasmolyzed cell will recover, and return to its original expanded condition.6 The time required for recovery is usually regarded as approximately propor- tional to the rate of penetration of the alcohol.

This method; was employed by Overton (1895) in the well-known studies on permeability which gave rise to the lipoid theory. He came to the conclusion that there is rapid penetration of alcohol, and of many other organic substances, but that inorganic salts do not penetrate. He attributed this to their insolubility in the lipoid layer, by which he supposed the cell to be surrounded. The writer repeated his experiments with salts, and came to the opposite conclusion.7 It was found in experiments with salts of NH4, Cs, Rb, Na, K, Li, Mg, Ca, Sr and Al, that the protoplast which is plasmolyzed and left in the solu- tion expands again to its normal size, showing that all these salts penetrate the protoplasm.

The following experiment will serve to illustrate the procedure. Filaments of Spirogyra were placed in 0.4 M NaCl solution. Within two minutes the protoplasts of the cells were so far plasmolyzed that they no longer touched the end walls of the cells. Several of these were 'Since the effect of the alcohol outside the cell is exactly balanced by that within the cell the final effect is the same as that of placing the cell in pure water. accurately sketched with the camera lucida and kept under continuous observation. In the course of ten min- utes, several of them had begun to expand and in thirty minutes all had expanded so as to completely fill their respective cells. To avoid the injurious action of the salt, the filaments were then transferred to 0.18 M CaCl2 solu- tion, and this was gradually diluted until its osmotic pressure was not greater than that of tap water. The cells were then transferred to tap water. They were examined the next day and found to be alive. On being placed in 0.4 M NaCl, they were plasmolyzed and they afterward expanded as before.

Certain facts may be worthy of mention which tend to obscure these results and which may have caused them to be overlooked. In the experiment just described, the cells were trans- ferred to a favorable solution as soon as expansion was complete. If this precaution be neglected and the cells be allowed to remain in the solution of NaCl, the injurious action of the salt soon causes the protoplast to shrink. In salts which are more toxic than NaCl, this contraction may be more rapid and more pronounced. This shrink- age, which may be called false plasmolysis,8 may also be produced by very weak (hypotonic) solutions and has nothing to do with plasmolysis, but may simulate it in very misleading fashion. If the cells are not continuously observed, but only examined at intervals, the expansion of the protoplast may easily be overlooked, and the sub- sequent shrinkage may be easily mistaken for plasmolysis. It is therefore desirable to keep the same individual cell under observation during the entire course of the experiment.

It was found that recovery from plasmolysis is much slower in CaCl2 than in NaCl, indicating that the latter penetrates more readily. This is in harmony with the results of measuring electrical conductivity. Other experiments indicate that the penetration of NaCl is more rapid in a solution of pure NaCl than it is in a mixture containing NaCl plus Cad2, in such propor- tions as to make a balanced solution. This is also in agree- ment with the results of measurements on electrical conductivity.

Another method of measuring permeability was used by Loeb,9 who has shown that eggs of Fundulus will float for a time in NaCl 3M , but not, as a rule longer than 3 hours. " Before sinking they lose water, as is indicated by the collapse of the membrane and the shrinkage of the yolk sac. Probably some NaCl enters the egg. ' ' In CaCl2 1.25 M, they sink in about half an hour. If, however, they are placed in a mixture of 50 cc. NaCl 3 M + 2 cc. CaCl2 1.25 M they float for three days or longer. Loeb interprets this as showing that the membrane of the Fundulus egg is practically impermeable to water and to salts in a physiologically balanced solution. But when transferred to a hypertonic non-balanced solution the natural impermeability of the membrane is gradually lost, so that water diffuses out of the egg and its specific gravity is increased to such an extent that it sinks. This action, and likewise the entrance of NaCl into the egg, is prevented by the addition of small amounts of CaCl2.

A very interesting series of experiments was made by S. C. Brooks, using the following methods: (1) diffusion through living tissue, (2) exosmosis, (3) change of curva- ture of strips of tissue. In the first of these methods10 different solutions were placed on opposite sides of a piece of tissue. The appa- ratus used is shown in Fig. 89. The diffusion of salts through the tissue was then measured. In the first , -I experiments, a solution of NaCl 0.52 M

0.14 M, as well as sea water and sea water diluted with one volume of dis- tilled water were employed in the same way. As the stronger solution diffused into the weaker the increase in the elec- trical conductance of the latter was measured. In Fig. 90, the rate of change of the electrical conductance is plotted against time. It will be observed that NaCl diffuses through the tissue FIG. so —Apparatus for more rapidly than sea water, while CaCU at first diffuses more slowly than sea ?»' Voided water and then more rapidly :

which is sealed to the living, we find that the rate of diffusion of vaseline and beeswax, is very much more rapid in all cases, F. The lower cell is pro- rubber uibhig* cTnd °a ml^ ^at a^ the solutions pass through pinch cock, 'D. a^ about the same rate of speed. These results are precisely what would be expected in view of the results of the electrical experiments. In the second of these methods,11 tissues of the dande- lion (Taraxacum officinale) were placed for a short time in a salt solution and the rate at which salts subsequently diffused out of the cell was measured by placing the

tissue in distilled water and observing the increase in conductance of the latter. The results are shown in Fig. 91, in which the ordinates denote the amount of exosmosis. It will be seen that there is more exosmosis from tissue FIG. 90. — The ordinates denote the rate of diffusion of NaCl, CaCla and sea water through living tissue of Laminaria. NaCl diffuses more rapidly than sea water, while CaCh at first which has been previously treated with NaCl 0.22 M than from tissue treated with a balanced solution.12

Tissue treated with CaCU 0.17 M showed less exos- mosis than that treated with the balanced solution.13 These results are also in harmony with the experi- ments on electrical conductivity. 12 For exosmosis of pigments of Rhodymenia in relation to electrical resistance see Osterhout (1919, C). 13 The concentrations of NaCl, CaCl, and of the balanced solution were chosen so as to be approximately isotonic with the tissue. In the third method employed by Brooks,14 strips of the peduncle of the dandelion were placed in hypertonic solutions and the rate of penetration of the salt into the protoplasm was calculated from the rate at which the

FIG. 91. — The ordinates denote the amount of exosmosis into distilled water from living tissue of Taraxacum officinale which had been previously treated with various solutions and then placed in distilled water. Treatment with NaCl produces more exosmosis than treat- ment with a balanced solution, while treatment with CaCh produces less exosmosis. The experiments indicate that the measured exosmosis is largely due to salts present in the cells before the application of reagents. If it were caused by the reagents (by diffusion out of the cell walls and intercellular spaces) it would be greater in CaCh than in NaCl.

strips recovered their normal shape after being curved by the action of hypertonic solution (the strips remaining in the solution during recovery). This gives the same kind of information as plasmolysis, but avoids the most serious errors of that method. In Fig. 92, the rate of recovery is plotted against time. The more rapid rate of recovery in NaCl, KN03, and NH4C1 shows that in these salts penetration is more rapid than in the balanced solution. On the other hand,