Injury, Recovery and Death in Relation to Conductivity and Permeability

the slower rate of recovery in MgCl2, CaCl2, A12C1G and Ce (N03)3 shows that in these salts penetration is slower than in the balanced solution. This is in complete agreement with the results of the electrical experiments (even the order of penetration of the salts of Mg, Ca, Al and Ce, is the same as that found by the electrical method). F IG. 92. — The ordinates denote rate of recovery from the effect of hypertonic solutions of strips of the peduncle of the Taraxacum officinale. The rapid rate of recovery in NaCl,

KNOs and NH4C1 shows rapid penetration of these salts. On the other hand in MgCh, CaCh, AhCle and Ce (NOs)s penetration is slower than in the balanced solution. Recently the writer has had an opportunity to test some of these conclusions by direct determinations of the penetration of substances into the cell sap. The advantages of this are obvious, when we consider the uncertainty of most indirect methods. Some investigators have sought to avoid the diffi- culties of indirect methods by making analyses of tissues, but these must obviously include too much intercellular material to be satisfactory. Analysis of the solutions

in which the tissue lies is open to the objection that sub- stances are absorbed on the surfaces of the cells and in the spaces between them, and it is impossible to say what actually penetrates the protoplasm. Others have attempted to analyze the cell sap. The most favorable cells for this purpose are those of plants, since they contain, as a rule, vacuoles filled with sap. In general, the method has been to crush the tissues and express the sap, but it is obvious that this procedure involves too many possibilities of error.15

The entrance of dyes has been extensively investi- gated, but this method is beset by many pitfalls,16 and the results hitherto obtained are confusing. "Among these may be mentioned contamination of the cell sap by substances present in the cell walls or intercellular spaces and chemical reaction between the cell sap and the crushed protoplasm and cell walls. The degree of pressure used in expressing has a marked influence on the concentration of the sap. Cf. Mameli (1908), Dixon and Atkins (1913) Gortner and Harris (1914), Gortner, Lawrence and Harris (1916). The investigation of blood and other body fluids is open to the objection that we do not know to what extent substances penetrate between the cells in reaching these fluids. In many cases penetration into these fluids seems to present very special features.

" To a great extent the coloration of the cell by a dye shows the extent to which the dye can combine with substances within the cell rather than the rate at which the dye penetrates. Thus many cells contain substances which combine with methylene blue so that it becomes far more concentrated within the cell than in the external solution (Pfeffer 1900, 1:96). Unless the cell has this power it often fails to appear colored even though it may contain the dye in the same concentration in which It exists outside. In such cases it may sometimes be detected by plas- molyzing the cell and thus concentrating the dye. A further complication is that a cell may appear to have taken the dye into its interior when in reality only the surface or the cell wall is stained. There are many other difficulties, which need not be discussed here, such as toxic action of the dye and changes in the dye ( decolorization etc.) as it enters the cell. A very serious objection to this method is that it does not give quantitative results. A review of the literature will be found in Hober (1914).

The penetration of acids and alkalies has been studied by employing organisms containing natural indicators, or by introducing indicators into the cell.17 In some cases the penetrating substance may cause a visible precipitate with the cell: this is especially the case with alkaloids.18 The absorption of Ca19 has been detected by observing the formation of crystals of Ca-oxalate within the cell. It is evident, however, that these methods have but limited application and that in many cases they are open to the objection that the penetrating substance injures the cell.

The penetration of a substance may often be demon- strated by observing its effect upon metabolism, but this method is unsatisfactory from a quantitative standpoint. Some investigators state that substances may produce effects on metabolism by their action at the surface, with- out actually penetrating the cell. It is evident that the most satisfactory method would be to place the cell in a solution containing the substance whose penetration was to be investigated, and, after a definite time of exposure, to obtain the cell sap without contamination and test it for the presence of the sub- stance. Experiments of this sort have apparently not been carried out, though interesting results have been obtained by Meyer,20 Hansen,21 Wodehouse,22 and Crozier,23 by comparing the cell sap of Valonia (which can be obtained without contamination) with the sea

water. Janse24 found that filaments of Spirogyra which had been kept for a time in a solution of KN03, gave a test for N03 after being rinsed and caused to burst in a solution of diphenylamine. It is evident that in his method, there is serious risk of contamination by the substance in and upon the cell wall (or between the cell wall and the protoplasm). In order to avoid this difficulty, the writer has employed the cells of a species of Nitella, some of which reach a length of four or five inches and a diam- eter of a thirty- second of an inch.

Within the cell wall the protoplasm forms a thin layer in which are imbedded the chlorophyll bodies. Inside this layer of protoplasm is the large central vacuole filled with cell sap. It is possible to obtain the cell sap without contamination in various ways. The writer has made use of the following methods: The cells are placed for the desired length of time in a solu- tion containing the substance whose penetration is to be tested. They are removed, washed in running tap water (followed in many cases by distilled water) and dried by means of filter paper. The cells are so large and turgid, that this manipulation presents no difficulty. A cell is then placed on a piece of glass or filter paper and pierced with the point of a capillary tube (which has been drawn out to a fine tip). The cell sap is drawn up into the tube (by capillary action) quite free from protoplasm or chlo- roplasts.25 Another method, which is preferable in many cases, is to suspend the cell by a pair of forceps attached to the upper end, cut off the lower end and bring it in contact with a glass slide, and then grasp the upper end

K During the manipulation care should be taken to prevent the sap from running out of the cell and coming in contact with its outer surface. gently with another pair of forceps, which is slowly moved downwards. The cell sap then flows out on to the glass slide in contact with the drop. By uniting the drops from a number of cells it is possible to get a sufficient amount for qualitative chemical tests, and in many cases approx- imate quantitative results may be obtained.

Since in previous investigations the writer had employed indirect methods of testing permeability, it was of considerable interest to compare these results with those obtained by direct tests of the cell sap. An investi- gation was therefore made in which the permeability of Nitella was tested by the direct method, as well as by determinations of plasmolysis and of electrical conduc- tivity. This may be illustrated by a series of experi- ments26 with NaN03 and Ca(N03)2.

Experiments on plasmolysis were carried out by placing the cells in a hypertonic solution and observing the time required to recover from plasmolysis (without removing the cells from the solution) on the assumption that the more rapid the recovery, the more rapid is the penetration of the salt. In these experiments the smaller .cells near the tip of the plant were largely employed. They were observed in Syracuse watch glasses or placed on glass slides and covered with large cover glasses, the edges of which were sealed with vaseline.

Plasmolysis may be harmful to many cells, even in a balanced solution,27 while in an unbalanced solution there 36 All the experiments were performed at about 19° C. All the solutions were approximately neutral. Cf. Osterhout (1922). 2T For this reason penetration may be more rapid than would otherwise be the case. In order to reduce toxicity chemically pure salts should be used and the water should be distilled from quartz (or from glass which has been in use for some time), using cotton plugs in place of rubber or cork stoppers and rejecting the first and last parts of the distillate.

may be the additional injury due to the toxic action of the salt. For this reason many cells which would recover if very slightly plasmolyzed may not do so if plasmolyzed more strongly, since recovery may require so long a time that the process of injury gets the upper hand. It was found that recovery was more rapid in NaNO;; than in a balanced solution of NaN03 + Ca(N03)2 or in Ca(N03)2 alone. Similar experiments with RbCl, CsCl, and CaCl2 gave the same result. This indicates that in a solution of NaN03, NaCl, RbCl, or CsCl penetration is more rapid than in Ca(N03)2, CaCL, or in a bal- anced solution.

These results agree with those obtained in the study of Spirogyra.28 The experiments on conductivity were carried out by means of the apparatus described on page 34. As it was desirable to surround the cell by a solution of the same conductivity as that of the cell sap determinations of the latter were made by filling a small tube with sap and inserting an electrode at each end (taking great care to avoid the inclusion of air bubbles). It was found that the sap had approximately the conductivity of sea water plus three parts of distilled water (this will be called for convenience 0.25 sea water). The cells were accord- ingly placed in this for some time before beginning the determination of the conductivity of the living cell. Under these circumstances it was assumed that altera- tions in conductivity during the course of the experiment must be due (in great part at least) to changes in the protoplasm, rather than in the cell sap.

In general, it was found that in 0.25 sea water, the resistance remained unaltered for a long time, while in NaN03 of the same conductivity it soon began to fall. Direct tests of the sap were made by determin- ing N03, since it was found that the cells normally give tests for Na and Ca. The method employed was not sensitive enough to detect N03 in the sap of the control cells under any circumstances, so that if a test was obtained after exposure to a solution containing N03, it must have been due to penetration from without. The sap was tested by placing it on a glass slide, adding a drop of a solution of nitron in 10% acetic acid, and observ- ing it under the microscope. If NO3 is present, it may be recognized by the formation of characteristic crystals.

Cells kept for 24 hours in 100 c.c. NaN03 0.05 M + 10 c.c. Ca (N03)2 0.05 M gave no test, which shows conclu- sively that the method is safe as far as contamination by NO3 on the surface is concerned. After 48 hours a test was obtained. As the cells continued to live in this solu- tion for 3 weeks (at which time the experiment was discontinued) and as they appeared normal in every way, it is evident that the penetration was not the result of injury.

It is probable that in 24 hours some N03 penetrated which was not revealed by the test. This, however, is of no significance in the present investigation which does not aim to determine the absolute amount of penetration, but merely to compare the relative penetration in bal- anced and unbalanced solutions. The results of such a comparison are very striking. After 3 hours in NaN03 0.05 M a good test was obtained. The cells had lost some of their turgidity ; if left in the the solution of NaN03 or if transferred to tap water they subsequently lost all their turgidity, indicating death. It is therefore evident that this rapid penetration was accompanied by injury. Similar results were obtained by Mrs. Brooks (1922) with Li, Ba, and Sr.

It may be remarked that the turgidity of the cells affords good indication of their condition. It is easily tested by lifting them partly out of the solution; if turgid they appear stiff, otherwise they collapse or appear flaccid. It is, however, necessary to distinguish between loss of turgidity in isotonic or hypotonic solu- tions, which indicates injury, and a similar appearance in hypertonic solutions, which may indicate nothing of the sort. In the latter case the cell promptly recovers its turgidity when placed in tap water; in the former it does not.

Another criterion of injury is afforded by the appear- ance of the chlorophyll bodies. In the normal cell they are arranged in regular rows and are of a clear trans- parent green color. Where injury occurs they lose their regular arrangement and the color becomes more opaque. In 0.05 M Ca (N03)2 the cells live for a week or more. During the first few days, at least, penetration is not more rapid (perhaps less so) than in a balanced solution. unqualified confirmation of the results obtained by the indirect methods. We find that penetration in injurious solutions is relatively rapid as compared with penetration in non-toxic solutions. This corresponds to the fact that recovery from plasmolysis is more rapid in injurious solutions as well as to the fact that conductivity increases in such solutions.

It would therefore seem that we may regard deter- minations of electrical conductivity, and, in some cases, of recovery from plasmolysis as reliable means of detect- ing alterations in permeability. It is, however, desirable to go further, if possible, and analyze the factors involved in electrical resistance. If we consider the behavior of the current from this point of view, it is evident that in the simplest cases, where the plant is a membrane only one cell thick (as in Porpliyra and Monostroma) and the current passes through this membrane at right angles to its surface, we need consider only a single cell and its adjacent cell wall, as shown in Fig. 93, A. The part of the current which goes through the protoplasm may be designated as Cp. while that which traverses the cell may be called Cw.

Experiments show that the resistance of the living tissue is much greater than that of tissue which has been carefully killed with all possible precautions to prevent any alteration of the cell wall.29 We therefore feel con- fident that the conductivity of the living protoplasm is less than that of the cell wall. In order to see how the current may distribute itself, let us suppose the protoplasm to be replaced by a wire,30 P, as in Fig. 93, B and the cell wall to be replaced by

30 We might consider the protoplasm to be replaced by two wires, one of which corresponds to the thin layers of protoplasm which are traversed by the current in a direction at right angles to their planes, the other corresponding to the similar layers of protoplasm in each cell (around the edges of the cell shown in Fig. 93, A) in which the current flows in the plane of the layer. It is evident, however, that these latter may be neglected in our calculations since they occupy such exceedingly small fractions of the cross-section.

If we neglect these we may say that in traversing a cell the current passes through a thin layer of cell wall and then one of protoplasm (in both cases at right angles to the plane of the layer), then through the cell sap, and finally through a layer of cell wall and one of protoplasm ( at right angles to their planes ) . It is evident that in this case we may neglect the effect of the cell wall and of the cell sap since their resistance is very small in comparison with that of the protoplasm and is in series with it. We may therefore consider the protoplasm to be replaced by a single wire having a resistance equal to that of the two layers of protoplasm which are traversed by the current in a direction at right angles to their planes.

a wire, W. The current flowing between the points X and Y in the wire P may be called Cp ; that in the other wire Cw. The total current, C, flowing between X and Y will be the sum of the partial currents, or, We may consider the current (conductance) as equal to the reciprocal of the resistance and write the resistance of the wire P, and Rw , that of W. Apply- ing this equation to Laminaria31 (and expressing the resistance in the usual way as the per cent, of the normal) we may calculate the values of Cw, CP, Rw, and Rp.

Under normal conditions in sea water, the resistance is taken as 100 and therefore C = l-r- 100, but in certain 81 So far we have considered only the simplest case, when the plant is only one cell thick. But it is evident that these considerations also apply when several membranes are placed together, forming a mass comparable to the tissue of Laminaria. The only difference is in that case the current would traverse a very thin layer of cell wall in passing from one protoplasmic mass to the next, so that what we have spoken of as the resistance of the protoplasm would be composed in part of the resistance of these cell walls. When the protoplasm is dead the total resistance is only 10.29 and the resistance of these cell walls must be only a small fraction of this. Consequently their resistance in the living tissue of Laminaria is undoubtedly less than 1 when that of the protoplasm is 140. The resistance of these cell walls may therefore be neglected.

solutions (having the same conductivity as sea water) the resistance may rise to 300 or more ; and in this case C would equal 1 -~ 300 = = .0033 (or less), and since some of it must flow in the protoplasm the amount which trav- erses the cell wall must be less than this. We are there- fore safe in putting it as low as 1 -f- 350 = .002857. All the experiments hitherto made indicate that the conductivity of the cell wall remains unaltered in spite of changes in the chemical character of the solution, pro- vided the conductivity of the solution remains the same. We may therefore take .002857 as the fixed value of Cw.

The changes in resistance thus far discussed have been treated as though they occurred in sea water; in this case the experiments indicate that the conductivity of the cell sap remains practically constant and hence need not be taken into account in our calculations. We may now ask whether this is also the case when the changes in resistance occur in other solutions. In order to investi- gate this, experiments were made with solutions of NaCl and CaCl2 (of the same conductivity as sea water). The tissue was placed in these solutions and removed after various intervals of exposure. It was cut into small bits and ground (so as to open the cells) and in some cases

32 The total conductance of the protoplasm is greater than that of the cell walls, but the protoplasm occupies a much greater fraction of the conducting cross-section than the cell walls, so that the actual conduc- tivity of the protoplasm is much less than that of the cell wall. the tissue was killed by heat: the conductivity of the expressed juice was compared with that of sea water. As no significant difference was found we may consider that the conductivity of the cell sap does not change sufficiently in these solutions to alter our calculations.

Let us now consider the changes in protoplasmic resis- tance which occur in toxic solutions. When tissue is placed in NaCl 0.52 M the net resistance falls rapidly. The death curve may be obtained by means of the formula83 in which T is the time of exposure, KA and KM are con- stants, and e is the basis of natural logarithms. We find by means of tin's formula that in a solution of NaCl 0.52 M (for which KA^ .018 and /f^=.540) the net resistance after 10 minutes is 87.76% of the normal ; after 30 minutes it is 64.26, and after 60 minutes it is 41.62. Knowing the net resistance we can calculate the protoplasmic resist- ance, as explained above. After 10 minutes the proto- plasmic resistance is 117.12% (corresponding to the net resistance of 87.76%). Since it is desirable to express all resistances as per cent, of the resistance in sea water we divide 117.12 by 140 (which is the protoplasmic resistance in sea water) and obtain 83.66%. Proceeding in this way we find that after 30 minutes the protoplasmic resistance is 56.22% and after 60 minutes 33.74%. In order to fit the formula to these values we must change the constants, put- ing JT,iP=0.0234 (in place of KA= 0.018) and KMP= 0.702 (in place of 7*^=0.54). It is therefore evident that in