potassium chloride to 100 c.c. sustains normal contraction for a still

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1 CONCERNING EXPERIMENTS TO TEST THE INFLU- ENCE OF LIME, SODIUM AND POTASSIUM SALTS ON THE DEVELOPMENT OF OVA AND GROWTH OF TADPOLES. BY SYDNEY RINGER, M.D., F.R.S. A FEW years back I drew attention to the effect of calcium and potassium salts on the contraction of the frog's heart', and showed that the heart contractility is sustained by an adequate mixture of calcium and potassium salts in saline solution. I first employed calcium chloride and potassium chloride with bicarbonate of soda, but I subsequently found that a saturated solution of tribasic phosphate of lime2 in 60/o saline solution with the addition of 1 c.c. to 1-5 c.c. of 1 /o solution of potassium chloride to 100 c.c. sustains normal contraction for a still longer time. I also tested the action of distilled water and distilled water containing sodium, or potassium or calcium salts' on fishes. Minnows placed in distilled water die on an average in about four hours. Sodium chloride, sodium bicarbonate and potassium chloride added singly to distilled water sustain life much longer and for about the same time, about two days; sodium bicarbonate prolonging life rather longer than either of the other two salts. Calcium chloride added to distilled water sustains life much longer than corresponding quantities of either sodium or potassium salts. The addition of sodium bicarbonate greatly enhances the life-prolonging effect of calcium chloride. The destructive action of distilled water appeared to be chiefly due to its disintegrating action on the exposed tissues of the fish and especially of the gills. With Dr Buxton4 I then tested the effect of minute quantities of sodium and calcium salts on one of the simplest forms of contractile tissue, namely cilia. We found that distilled water quickly disintegrated the tissues, 1 This Journal, Vol. ]v. p This Journal, Vol. vii. p This Journal, Vol. v. p This Journal, Vol. VI. p. 158.

2 80 S. RINGER. separating and breaking up the ciliated cells, and so quickly arresting ciliary movemen-ts, whilst sodium and calcium salts prevent their disintegration and greatly prolong ciliary activity, sodiuim salts being more efficacious than calcium salts. We have here obviously an altogether different action to that by which simple saline suspends contractility of cardiac muscle. When the frog's heart is fed with simple saline contractility is lost in a few minutes, and can be restored by the addition of a calcium salt. In these experiments however, ciliary action persisted for about twenty-four hours, and the distilled water produced complete disintegration, so that contractility could not be restored by adding salts to the distilled water. Further, sodium bicarbonate cannot sustain cardiac contractility, only a lime salt can do this, whereas in the above experiments sodium bicarbonate was certainly as efficient as calcium chloride in preventing disintegration and sustaining ciliary action. With cilia it appears that distilled water affects the cement substance binding the cells together, and in a less degree disintegrates the cells themselves. I next tested the action of distilled water and distilled water cointaining calcium, sodium and potassiuim salts on Laminaria'. Laminaria swells prodigiously in distilled water, and this swelling is not lessened by the addition of sodium or potassium salts, though it is greatly conltrolled by the addition of a minute quantity of a calcium salt. In the Fucoid Algae the cell consists of two parts, the outer being composed of a degradation compound, namely bassorin or tragacanth, and it is this material which binds the cells together and which swells up so enormously when the dried plant is placed in water. It appeared to me probable that the cement substance which unites the animal cells together might be of a similar nature, and behave in a similar manner; and to some extent this is true, for the cement substance in both vegetable and animal tissues appears to be similarly affected by lime, thus preventing their imbibition of water, and so preventing swelling in the case of Algae and disintegration of tissue in the case of the freshwater mussel's gills. Recently I have continued the investigation of the action of distilled water alone or containing sodium or potassium or calcium salts on animal tissues, and shall speak first of the action on the ova of frog's spawn. In all these experiments the fluid was renewed at the end of twenty- I This Journal, Vol. vii. p. 118.

3 INFL UEANE OF SALTS ON DEVELOPMENT. 81 four hours to remove any salts that had diffused from the ova or its mucilaginous envelope. Frog's spawn placed in distilled water does not develope at all or very little. The mucilaginous envelope swells up to four to six times the size of ova in tap water or in distilled water containing calcium or sodium salts. In two or three days the vitelline membrane separates from the rest of the ovum and enlarges so that the ovum is seen lying in a large cell, about twice or three times its own size, and a little later between the swollen membrane and the rest of the ovum a white opaque substance forms which obscures the ovum, but is not developed equally in all parts of the sac formed of the distended vitelline membrane, so that some parts of the ovum are more obscured than others. The opaque substance under the microscope looks like a low form of organized membrane containing a few fibres. I next tested the effect of various lime salts added to distilled water, using the chloride, the nitrate, the sulphate and the tribasic phosphate salts. With the chloride and nitrate development proceeds further than with distilled water, but not so far as with calcium sulphate, whilst this in its turn is far inferior to tribasic phosphate of lime; these results correspond to the influence of lime salts on the frog's heart, whose contractility is far better sustained by a saturated solution of tribasic phosphate of lime in.60/! saline solution than by calcium chloride or calcium sulphate. I used solutions of various strength of calcium chloride, adding in different experiments, respectively, 5 c.c., 10 c.c., 20 c.c. and 40 c.c. of 20/0 solution to 1000 c.c. distilled water. The ova developed to a slight extent but did not usually escape from the mucilaginous envelope. They developed slowly, and did not appear to increase in size for the most part beyond the third or fourth day, but occasionally growth continued till the seventh or eighth day. The enlarged ova, as with distilled water, becomes surrouinded by a sac composed apparently of the separated and enlarged vitelline membrane, and some of the opaque white material was formed between the wall of the sac and the contained ovum, though much less than in the experiments with distilled water. In the experiments with calcium nitrate I employed 20 c.c. of 2 0/0 solution to 1000 c.c. distilled water. The ova slowly developed and ceased to grow on the eighth to the eleventh day. With sulphate of lime I employed a saturated solution and one part PH. XI. 6

4 82 ks. RINGER. of a saturated solution to two parts of distilled water. The difference in quantity of the lime salt did not modify in any way the rate or amount of development. For the first three or four days the ova developed in these solutions as rapidly as ova placed in tap water, afterwards they grew much more slowly so that on the seventh to the tenth day the ova were only one half the size of ova in tap water. Most escaped from the mucilaginous envelope and lay almost motionless on their side. Most died on the tenth or eleventh day, one or two living till the thirteenth day. With a saturated solution of tribasic phosphate of lime in distilled water development progressed much better, indeed almost as rapidly as with tap water. After twenty days all were alive in the phosphate solution, but only half the size of ova placed at the same time in tap water, and they lived as long as tadpoles in tap water. In these experiinents the tadpoles were of course unfed, or this would have vitiated the experiments by supplying, them with other salts than the phosphates. These experiments prove that lime salts greatly assist development of ova but not in an equal degree, phosphate of lime being far nmore helpftul than other preparations of lime. The amount of tribasic phosphate dissolved by distilled water is very small, only just discoverable by the oxalate of ammoniumi test, and it may be urged that the difference in the efficacy of the different forms of lime salts was due to the strength of the solution and not to the difference in the form of the salt. This view is certainly not correct, for with calcium chloride the weaker solutions did not contain more lime than the phosphate of lime solution, judging roughly by the visible deposit on precipitating the lime with oxalate of ammonium. I also tested the action of potassitum chloride on the development of ova, adding 20 and 40 c.c. of 1 0/0 solution to 1000 c.c. distilled water. Very little development occurred, scarcely more than with distilled water. The mucilaginous envelope swelled a little more than the envelope of ova placed in tap water, but much less than of ova in distilled water. It may be suggested that distilled water destroys the ova by diffusing out potassium, sodium and calcium salts which are necessary for development and growth. This explanation however fails, for in the first place salts will not diffuse from living tissues probably because they are combined with the tissues, otherwise fish would be destroyed and the tissues of the gastro-intestinal tract would constantly be

5 INFLUENCE OF. SALTS ON DEVELOP3MENT. destroyed after taking water. Moreover sulphate or nitrate or chloride of calcium would prevent diffusion of limne salts as cornpletely as phosphate of limne, and indeed should do so more effectually, as in most of these experiments, chiloride, sulphate and nitrate of lime solutions containied far more lime than the phosphate of lime solution. As the ova were in most of the experiments comripletely covered with the solution, it imay be surmised that the superiority of tap water is due to its containing more air than the various saline solutions made with distilled water. This view however fails to explain the great superiority of phosphate of lime to other lime solutions. The greater quantity of air in tap water may perhaps explain the more rapid growth of ova and tadpoles in tap water than in phosphate of lime solution, but I suggest that the more rapid developmernt is in part due to the more abundant growth of low forms of vegetable life from the germs deposited by the- dust of the room, for after many days tap water became of a slight greenish colour, and this was much more pronounced in tap water thani in pbosphate of lime solution. We have seeni that with distilled water and some lime solutions the vitelline membrane separates from the rest of the ovum and becoines greatly distended. Is this due to diffusion or imbibition? Not to diffusion, as phosphate of lime solution containing a very minute quantity of lime prevents this, whilst it occurs in strong solutions of chloride and of sulphate of lime. Finally, the destructive action of distilled water cannot be due to its causing great swellinrg of the mucilaginous coating of the ovum, for phosphate of lime solution causes much more swellino than stronger solutions of chloride or sulphate of lime, and yet as we have seen ova in phosphate of lime solutions develope well. I next performed a series of experiments of a similar kind with tadpoles. The tadpoles were for the mnost part hatched and kept unfed in tap water in the physiological laboratory. Some came from the country. Those hatched in the laboratory were about three weeks old. Whilst kept in the testing solution they were unfed'. Placed in distilled water tadpoles die in twelve to eighteen hours, and one sees the epithelium covering then separating in flakes showing, that as in previous experiments distilled water disintegrates tissues. Placed in distilled water containing 5 c.c., 10 c.c., 20 c.c. and 40 c.c. of 1 /o solution of sodium bicarbonate in 500 of water they die in less 1 They had therefore to eat only the albuminous covering of the ova and any low organisms that grew in the exposed fluids from spores deposited from the air in the room

6 84 than twenty-four hours. S. RINGER. Hence bicarbonate of soda is unable to sustain life. Placed in a solution consisting of 5 c.c. of lime water to 1000 c.c. of distilled water tadpoles die in a few hours. Calcium chloride in solutions containingf 5 c.c., 10 c.c., 20 cc. and 40 c.c. of 20oi solution in 1000 c.c. of distilled water does not sustain tadpole life, for they died, some in six hours and the rest were found dead in twenty-four hours. The epithelium soon desquamated and was easily rubbed off. Tadpoles placed in a saturated solution of sulphate of lime or in a saturated solution diluted with two parts of distilled water speedily die, not living more than twenty-four hours and often dying much sooner. Carbonate of lime and tribasic phosphate of lime on the other hand sustain life for a considerable time. The solution with carbonate was formed by mixing precipitated carbonate of lime with distilled water. Of course carbonate of limne is insoluble and if tadpoles are at once placed in this fluid they speedily die, but if the mixture is exposed several days then the fluid can sustain life, due no doubt to the formation of some bicarbonate of lime. When placed in the solution exposed some days, all the tadpoles were alive after ten days but were very inactive, much more so than tadpoles kept much longer, unfed in tap water. Tribasic phosphate of lime too is well able to sustain life. This has been already shown, for ova mature and hatch in such a solution and then continued to live from three weeks to a month. Further experiments were made. Tadpoles reared in tap water were placed in tribasic phosphate solution and lived from ten to twenty days. In conclusion, I draw attention to the interesting fact that those lime salts most efficacious in sustaining function of cardiac tissue are those best adapted to sustain life and growth in ova and tadpoles; phosphate of lime being far more efficacious than sulphate and the sulphate somewhat more efficacious than chloride. I have not made sufficient experiments with carbonate of lime to test its influence in cardiac muscle. It would appear that those salts of lime where the lime atom is least saturated by the acid are the most capable of sustaining function. 1 This is shown by the precipitate that occurs when oxalate of ammonsum is added to the fluid.