Injury, Recovery and Death in Relation to Conductivity and Permeability

of tissue, L, are packed together like a roll of coins. At each end is a platinum electrode, A, fastened in an electrode carrier, D. By means of the screw, F, the electrode carriers can be drawn together, compressing the tissue and holding it firmly in place. tion. This screw engages an internal screw contained in the electrode holder at the right. This is not the case with the electrode holder at the left in which the screw passes through a sleeve, and in consequence this electrode holder is drawn toward the other only when the block, M9 is fastened in place by the set screw, N, and the screw, F, is turned in the proper direction.

An end view of an electrode holder, 7), is shown in Fig. 4. Its lower portion (which contains the platinum electrode) is shown inserted in a hard rubber support, G. The support is pierced by a series of seven holes The disks are cut from the fronds by means of a cork borer and have gether like a roll OI COinS (abOUt 100 tissue («een in1 section] asla in all). They are firmly held in place by the glass rods which surround them and by the electrode holders which press against them at either end. At the same time the spaces between the glass rods allow free circulation of liquid.

"It was at first thought that cutting might injure the tissues at the edge of the disk sufficiently to interfere with the results, but experiments proved that this is not the case. Not only do the cells adjoining the cut surface live as long as those in the centre of the disk, but it is found that experiments (made by another method) on intact fronds give the same results as experiments on the cut disks, from this the disks are transferred to the support 6r, which is submerged in sea water. They are arranged inside the glass rods by means of forceps, and care is taken to see that no bubbles of air are caught in the space around the electrode or in the opening at H.

When the effect of a number of different solutions is to be compared the following procedure is adopted. If there are seven solutions seven disks are cut from the same part of a frond: each disk is placed in a separate tumbler of sea water. A second lot of seven disks is cut, as close to each other as possible, and placed in the tumblers, so that each tumbler contains two disks. This is continued until each tumbler con- tains one hundred disks. By this means the material in the different tumblers is made as similar as possible. The disks in each tumbler are then packed together (like a roll of coins) to form a cylinder whose resistance is measured. Throughout the experiments the differ- ent lots are kept side by side and treated as nearly alike as possible, except that they are placed in dif- ferent solutions.

The electrode holders are now pressed against the ends of the roll of disks, the block, M, is firmly fastened by means of the set screw, N, and the screw, F, is turned until the electrode holders are tightly clamped against the roll of disks. The pressure used in this operation should be fairly uniform.6 8 It was at first thought necessary to use a dynamometer, but it was found that the operator soon becomes so proficient as to make it un- necessary. The resistance is very little affected 'by variations in pressure.

f In the earlier experiments the resistance was taken with the cylinder submerged in sea water, and this may be preferable in special cases. and allowed to drain8 for a definite time (not over one minute) after which the resistance becomes practi- cally constant. The current passes for a short distance through sea water before reaching the disks. There is a film of sea water between each pair of disks and likewise a film around the cut edges. Otherwise the current passes only through the tissue.

As soon as the resistance has been measured the apparatus is replaced in sea water; the set screw, N, is loosened so that the electrode holders can be moved apart and the disks separated from each other by means of forceps. After standing for a few minutes in sea water the resistance is again determined. The disks are then separated as before and allowed to stand in sea water. This procedure is continued until it becomes evident that the resistance is practically stationary.9

The apparatus is then transferred to another solu- tion (e. g., NaCl 0.52 M) having the same conductivity (and temperature10) as the sea water. There should be at least 1,500 cc. of solution, contained in a shallow dish of glass or enameled ware. The disks are at once separated by means of forceps and thoroughly rinsed 8 Each support rests on a block of paraffin. Care must be taken that there is no conduction between the blocks; e.g., along the wet surface of the table.

•Unless this is the case the material is rejected. With good material the resistance remains stationary for a long time; in one experiment it remained so for 10 days at about 20° C. In this case the tissue wa8 kept in running sea water and was only half-submerged, thus ensuring an abundant supply of oxygen. See Osterhout (1915, B). When placed on ice Laminaria can be kept in good condition for a much longer time. 10 All readings should be made at the same temperature or, if this is not practicable, should be corrected to the standard temperature. For the temperature coefficient, see p. 37.

in the new solution, the whole apparatus being moved about in the dish to secure thorough mixing. By means of a medicine dropper the sea water around the platinum electrodes is thoroughly washed out. In some cases it is desirable to transfer to a second dish to ensure against contamination by sea water. By this means a very rapid change is effected and, as the disks are thin, diffusion is soon completed (this is often the case in 5 minutes and should not in any event require more than 10 minutes). Since the outward diffu- sion of salts may take place at a different rate from the inward diffusion there may be an apparent rise or fall of resistance in consequence. This effect lasts but a short time and is found in dead as well as in living tissue. It is therefore easy to guard against error due to such causes.11

The resistance of the disks at the ends is much greater than that of those in the middle since the current spreads out after issuing from the small opening,12 H, in the rubber disk (Fig. 1). For this reason the best disks of tissue should be placed at the ends and their positions should not be changed. Care should be taken that they are not cut or injured by contact with the edges of the opening in the rubber disk.13 The inequal- ity between the disks at the end and in the center may be minimized by introducing at intervals rubber disks

13 It results from this that the resistance does not increase in direct proportion to the number of disks. If we plot the resistance as ordinates and the number of disks as abscissae, we obtain a curve which is concave toward the base line. The curve is approximately logarithmic. "These edges may be rounded by filing. A soft rubber disk may be placed between the hard rubber disk and the tissue. provided with openings in the center (Fig. 5). This is desirable in many cases and makes it possible to get a high resistance with less tissue.

Care must be taken to see that liquid does not leak out of the space around the electrodes while the appa- ratus is out of the liquid. If a leak should occur fresh liquid may be added by means of a medicine dropper. With a proper ad- V? justment of the rubber disks and suf- ficient tissue to give elasticity no leakage should occur. In regard to the accuracy of the readings it may be said at the outset Flo.5._Hard rubber disks- cessive readings on the same material do not vary more than 1% from the average. This is as great accuracy as can ordinarily be hoped for in biological work and there is no object in striving to get greater accuracy than this in the apparatus itself.

It is usually desirable to introduce a variable capaci- tance or an arrangement such as is suggested by Taylor and Curtis (1915), by Taylor and Acree (1916) or by McClendon (1920). In the writer's experiments the capacity of the apparatus, filled with living Laminaria and lifted out of the sea water, was about one thousandth of a microfarad. An advantageous arrangement suggested by Profes- sor G. W. Pierce is shown in Fig. 6. The frequency is of some importance. The writer has found a thousand cycles convenient; this may be obtained by means of an "audio oscillator" (such as is used in wireless telegraphy) as furnished by the General Radio Co., or by means of a toothed iron wheel re-

volving in a suitably arranged magnetic circuit such as is furnished by Leeds and Northrup. The results so obtained did not differ from those secured with a Vreeland oscillator. FIG. 6. — Diagram to show bridge and connections. S is an alternating source (1000 eyelet or more), A and B are the ratio arms of the bridge, C is the variable resistance of the bridge, X it the unknown resistance (tissue and holder), T, telephone, V, variable condenser, G, a ground wire from the centre of a high resistance (in case the ratio arms of the bridge are unequal the two parts of the high resistance should also be unequal).

The use of the ordinary lighting circuit (60 cycles) with a vibration galvanometer is recommended by Green (1917). The use of an alternating current galvanom- eter in connection with a recording device is suggested by Weibel and Thuras (1918). FIQ. 7. Electrode carrier, A, consisting of a glass tube provided with a series of side tubes to hold an electrode tube, D, and a thermometer, E, also an inlet tube and an outlet tube. To the right two glass cells, B, C, each with an inlet tube and an outlet tube, with disks of

We may now turn to another form of apparatus which may for convenience be called Type B. Fig. 7 shows one end of the apparatus, which consists of an electrode holder, A, and a series of glass cells, B, C, etc. The electrode holder consists of a glass tube pro- vided with side arms for the admission of the electrode tube, Z>, (which is similar to the tube used in Type A) as well as of a thermometer, E. In addition there is an " See Taylor, W. A. and Acree, S. P. (1916), and previoua papers in the same journal.

inlet tube and an outlet tube by means of which the solution may be changed. Each of the glass cells, B, C, etc., has a similar inlet and outlet tube. Each outlet tube has a rubber connection through which liquid can be discharged without wetting the outside of the cells. All of the inlet tubes are connected (by rubber tubing and FIG. 8. — Disk of tissue, M, the edges surrounded by vaseline, W, with an electrode carrier a system of Y- tubes) to the same funnel, so that all the cells can be filled simultaneously.

The edges of the glass cells are ground in a plane exactly at right angles to the long axis of the cell. When pieces of Laminaria are placed between them (as at F and G) and they are pressed together, a tight joint is formed. The series of glass cells (with pieces of mate- rial) and an electrode carrier at each end are placed in a V-shaped trough with rigid ends ; at one end is a screw by means of which they can be forced together and held with any desired degree of pressure. At the places where the pieces of material are located, the trough is cut away so that they do not come in contact with it. Care is taken to keep the current from leaking along the trough (its surface is covered with paraffin).

The current therefore flows through the glass cells and through the pieces of material placed between them. The advantages of this type of apparatus are: (1) the end pieces do not have more resistance than those in the middle; (2) the solutions may be changed without disturbing the material. Types A and B may be combined by substituting disks of Laminaria for the glass cells. Type C is shown in Fig. 8. It consists of two elec- trode carriers similar to those in Type A. The material is shown at M, its edges being completely surrounded by vaseline, F, F, so that the current cannot leak out. In many cases it is preferable to use chicle, grafting wax, or art gum in place of vaseline. The apparatus remains partly submerged (the water line being indicated at W, TF), thus keeping the temperature more nearly constant. The solutions are changed by siphoning through the openings which admit the electrode tubes. This makes it unnecessary to unscrew and separate the electrode carriers during the experiment.

Type D is shown in Fig. 9. It permits the use of in- tact plants. One end of the plant is inserted in each of the cells A and B and held in place by a split rubber stopper. The cells A and B are filled with solution. The free portion of the plant is bathed in any desired solution until a reading is to be taken, when the solution is allowed to drain off and the reading is made. Care should be taken to prevent the current from leaking through or around the stopper.

Fio. 9. — Two glass cells, A and B, each provided with an electrode with a strip of tissue stretched between. FIG. 10. — A disk of hard rub- ber, A, one of tissue, B, and one of celluloid, C, tied to- gether with rubber bands, D (all seen in section). Surface view at the left. FIG. 11. — Disk of hard rubber, D, with a mass of tissue, M, wedged in the central opening (seen in section). per and in the cell may be killed to lessen its resistance. Material which is too soft to be handled in the man- ner recommended for Laminaria may be treated as follows : If it forms sheets or membranes it may be fas-

tened to thin disks of hard rubber16 provided with a central opening as shown in Pig. 10, in which A repre- sents the rubber disk (seen in section), B the material, and C another disk of thin rubber or celluloid. These are fastened together by rubber bands, D. For this purpose three projecting knobs are provided as shown in the surface view at the left of Fig. 10. The disk is placed in the frame described under Type A, and the knobs fit in between the glass rods in the manner shown in Fig. 10 (where the rods appear in section). Every other disk is turned upside down so that the knobs of adjacent disks do not touch and interfere with the close packing of the disks. The disks are treated precisely like the disks of Laminaria as described under Type A.

Most of the experiments on frog skin and on Ulva were made with this type of apparatus. Material which cannot be handled in this way may be treated as shown in Fig. 11, where D represents a hard rubber disk with a central opening into which the mate- rial is tightly wedged. The disks are then handled like so many disks of Laminaria. A special type of appa- ratus has been used in experiments on Zostera17. Experiments were also made with large cells of Nitella, some of which reach a length of 5 or 6 inches and a diameter of a thirty-second of an inch or more. They were packed (Fig. 12) in a trough cut in a block of paraffin (this was then, covered with a plate of glass). The trough was previously filled with a solution: this could readily be changed after the cells were in place. The cur- rent could be sent lengthwise or across the cells : usually both methods were employed.

M The edges of each piece of tissue are protected by vaseline. 17 Cf. Osterhout (191'9, A). In order to ascertain the conductivity of the cell sap of Nitella small amounts were expressed (see page 212) and allowed to fill a capillary tube. Platinum electrodes were then inserted into the opposite ends of the tube, care being taken to exclude air bubbles. By means of these methods a variety of plant and animal material has been studied by the writer.18 Cer-

FIG. 12. — Apparatus for measuring the conductivity of Nitella. The cells, N, are placed in a trough in a block of paraffin, P, and covered with plate glass, G. The solution is poured in through the funnel, T, and runs out through the opening, O. At E and E are tain precautions have been observed in the choice of material. It is desirable that the intercellular space or substance shall be constant in amount. This is the case in tissues, such as those of Laminaria, where the cell walls are of a firm consistency and do not change during the experiment.19 On the other hand many flowering plants present difficulties, since the spaces between the cells are largely filled with gas, which is

18 Plasmolysis must be avoided since this increases the space between the protoplasmic masses. displaced to a varying extent when the tissue is placed in a solution, with the result that the conductivity is altered. In such cases we must select material in which the displacement is very slow or else we must get rid of the gas at the start by submerging the tissue and evacu- ating by means of an air pump. As the writer's investigations were largely concerned with alterations in permeability it was necessary to provide for quick changes of reagents and for rapid penetration. This was accomplished by the use of thin sheets of tissue. For example it was found that when Laminaria was transferred from sea water to sea water diluted with an equal volume of distilled water, diffusion was practically completed in 5 to 10 min- utes ; this was also the case with the other material used in his investigations.

It is desirable that the thin sheets of tissue should be stiff enough to be handled easily and that they should not adhere to each other, but should tend to separate spontaneously when the pressure is removed so as to allow a free circulation of liquid between them (this is assisted by choosing pieces with a slight curvature). The material should be able to stand laboratory condi- tions and the manipulation required by the experiments. It is desirable that it should be available throughout the year. All these requirements are so admirably fulfilled by the marine alga Laminaria agardhii (a common kelp of the Atlantic coast) that it has been largely used in the investigations of the writer. It forms fronds several feet in length, 3 to 6 inches wide (having somewhat the consistency and thickness of a thin leather belt). It remains in normal condition in the laboratory for several weeks if kept in sea water (near 0°C.) and is not injured

by the pressure and the weak electric currents to which it is subjected during the experiments. The solutions were made with all possible precautions. The salts used were the purest obtainable. The distilled water was, as a rule, twice distilled from quartz or glass,20 using cotton plugs in place of cork or rubber stoppers in the distilling apparatus. The first and last parts of the distillate were discarded. The reaction of the solutions is of great impor- tance. Unless otherwise stated it was close to neutrality.

It may be desirable to add a word of explanation regarding the treatment of results. Most of the curves here presented are time curves in which each point represents the average of several experiments. In such curves it is desirable (as indicated on page 68) to average times (abscissa}) rather than resistances (ordinates). The probable error of the mean has been calculated in all cases by Peter's formula and expressed as per cent, of the mean.21 Since, however, space is lacking to present all the data, a general idea of the accuracy of the results may be given by saying that there is no point on the curve whose probable error of the mean exceeds a certain per cent, of the mean.