The vital staining of the fern-prothallium of eleven species and the influence of hydrogen ion concentration on it was investigated, applying thirteen kinds of acid dyes and twenty of the basic ones. The results of the investigations will be summarised as follows: (1) Four kinds of the acid dyes and eighteen of the basic ones were able to stain the fern-prothallia in vivo. (2) Of the structural elements of the prothallia, the following four elements were generally stained : the cytoplasm (cell sap) and the membrane of the rhizoid, the cap of the glandular hair, and the spermatid ; and occasionally the prothallial cell membrane also. (3) while the case in which a single element of the prothallium was selectively stained by a single dye was found few, the case in which four elements were colored at the same time appeared ordinarily. (4) In the fern-prothallia, the regular and close relationships between the chemical natures (constitutions) of the dyes and the vital staining reaction were not clear. (5) The types of vital staining of the cytoplasm of the rhizoidal cell were classified into the diffuse (homogeneous) or the non homogeneous one, and rarely into the granular staining. And occasionally the polar staining (the polarity of staining) was observed. (6) The cytoplasm of some rhizoid indicated the vacuolar demixing, and the drop-like one in a few cases. (7) The influences of hydrogen ion concentration on the vital staining were not so remarkable in the fern-prothallia, whereas the staining was accelerated or disturbed within a certain value of hydrogen ion concentrations. The elements of the prothallia which were not stained in the dyes diluted in redistilled water, were not generally accelerated in the staining reaction by the alteration of pH concentrations in the solutions.
In Sargassum piluliferum C. AG., the liberated eggs were developed when they had been treated doubly at first with butylic acid sea water and then with CaCl2 hypertonic sea water, resulting in the rhizoid formation. While, it was not so when they had been treated with the other reagents or with none. that this development is the parthenogenesis and accordingly that the appearance of the polarity is not affected by sperm entrance. It is also possible that the calcium ion needs not only to accenturate the tonicity of the sea water, but also to make the developmental process easy.