The quiescence, rounding, sinking of the granules, and paling of the nucleus are similar to the effects seen after the injection of potassium and sodium chloride (11). Since the sodium salts of the anions were used, it might be inferred that the sodium is the active agent in the injected solutions. This is not entirely the case, however, for the effective concentrations of NaCl required are many times greater than those required in the case of the sodium salts of the calcium-precipitating anions. The fact that practically the same effects can be obtained in both cases leads one to suspect that there is a relation between the results of an increase in sodium ions and a decrease in calcium ions. It has been shown that a M/416 CaCl2 solution will antagonize a M/1 NaCl solution and even a more concentrated solution of KCl inside the ameba (12). Therefore the reduction in amount of calcium may leave a comparatively high concentration of unantagonized sodium and potassium.
The fine, purplish red granules resulting from the injection of the alizarin are, no doubt, the insoluble calcium alizarinate. Recovery of an ameba from such an injection may be explained by the postulate that the free calcium ions in the living ameba are in equilibrium with a reserve supply of unionized calcium. The equilibrium is upset when the free calcium is removed by precipitation or by other means, and the system may possibly react in such a way as to counteract the effect of the change imposed. By mobilization of the calcium from a reserve supply the ameba can therefore gradually resume its normal activity. The time required for the recovery depends on the amount of alizarin injected. The diffuse red color which is seen immediately following the injection of alizarin probably represents that extra amount of dye which was not used in precipitating the immediately available calcium. Then, as the calcium is being liberated from the reserve, it is taken up by this surplus alizarin, resulting in a gradual loss of the diffuse coloration and an increase in the number of purplish
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red calcium alizarinate granules. Only when all of the injected dye has been precipitated can the mobilized calcium be used to carry on the normal physiological processes of the organism.
The need of calcium to effect ameboid movement has been shown by Pantin (13) in a series of immersion experiments. This fact is quite suggestive, because the first effect of the injection of any of the calcium precipitants is absolute quiescence. Furthermore, there is no return to normal movement until the calcium apparently becomes available to the protoplasm.
In support of the conception of a reserve supply of calcium is the presence of the large crystals which give a positive reaction with alizarin for calcium on the death of the ameba. Schewiakoff (14), from crystallographic studies, claims that they are calcium phosphate.
The effect of the injection of the calcium-precipitating anions on the calcium of the protoplasm may be shown in another way. In determining the relative toxicity of these salts an arbitrarily standardized injection, about one-fourth of the volume of an ameba, was used. This was introduced because of the necessity to avoid effects due to variable amounts of the solvent, viz., water.. Thus the water effect was kept constant, and the variations in actual amount of salt injected were obtained by using a graded series of concentrations.
Arranging the sodium salts of these anions in order of increasing toxicity in one column, and the in vitro solubility products of the corresponding calcium salts in another column, it is seen that as the toxicity increases, the solubility product decreases (Table 1). This fact strongly suggests that the toxicity depends on the ability of the salt to remove calcium ions from the protoplasm. The apparent deviation of the carbonate from the rule can be explained by the specific effect of 002 (10) which is always present from the hydrolysis of the carbonate.