ABSTRACT
The pH indicator, methyl red, as a vital stain (from a medium at pH 5.3) and having a relatively low pK value, was used for testing the penetration of acids and of salts of organic acids into cells. The cells were those of gill strips of Modiolus and Mytilus and the fertilized eggs of the sanddollar, Echinarachnius parma.
The penetration of various acids into cells was studied by immersing the cells in artificial, unbuffered sea water acidified with the given acid at 3 ×10−3M concentration in experiments with gill strips and at 10 × 10−3M concentration in experiments with sanddollar eggs. The colour change indicating intracellular acidification was almost instantaneous with benzoic acid (5 sec. or less), slightly less rapid (5–20 sec.) with the fatty acids, valeric to acetic, and slow
with HCl. The rate of the return of the methyl red to the original alkaline colour upon the transfer of the cells to artificial acid sea water (pH 5.3) was in the same order for the various acids.Intracellular acidification with a fatty acid was diminished when salts of the acid were present in the medium. Since the counteracting effect of the salts does not appear until the concentration of these salts exceeds that of the fatty acid by at least three times we may conclude that when both acid and salt are penetrating, the acid does so more rapidly than its salts.
The penetration of salts of acetic, butyric and benzoic acids also occurred in the absence of added acid and at an alkaline pH approximating that of normal sea water. However, the rate of penetration was much slower than in the presence of added acid. Evidence for penetration of the salts in absence of added acid was obtained by exposing the cells to the salts prior to treatment with the corresponding acid.
Effects obtained by prior treatment with the salts alone were enhanced by increasing the concentration of potassium in the medium.
The gills of Modiolus and Mytilus during the summer months are orange in colour because of the accumulation of diatoms. In the late autumn and winter the Mytilus obtained in New York City had colourless gills which were better suited for colorimetric experiments.
In order to test the continued viability of the eggs by observing their subsequent cleavage it is necessary to fertilize the eggs prior to the acid treatment since it has been found (cf. Clowes & Smith, 1924) that a high percentage of sanddollar eggs cannot be fertilized after even moderate acid treatment.
The concentration of free acid formed by dissociation of the salts is of a very low order, by calculation ca. 1·6 × 10−6M for 0-05 M benzoate and 1·1 × 10−6M for 0·05 M acetate and butyrate solutions.
On the basis of a pK value of 4·53 for butyric acid (cf. Drucker, 1905 and Michaelis & Drucker, 1931) this acid, by calculation, is about 84 per cent undissociated, while the amount of undissociated acid in the presence of 9 × 10−3M concentration of its salts approaches 100 per cent. While there is an appreciable difference in concentration of undissociated acid in the two solutions, the experimental findings indicated greater acidification of the eggs in the absence than in the presence of the salts.
