38 SHINICHI MATSUMOTU 



A. METHODS OF VITAL STAINING 



1. Methods of staining by immersing the whole animal in water 



containing dyes 



a. Neutral red. Neutral red, which was first used by Ehrlich 

 for vital staining, wa/S considered as probably being the most 

 suitable for the present purpose, and this proved to be the case. 



In the majority of the experiments "neutral red for vital 

 staining (Ehrlich)" was used, and the frogs were subjected to 

 various concentrations of the dye for various lengths of time. 



The experiments may be divided into two groups. In the 

 first the animals were subjected to a relatively concentrated 

 (1 : 20,000) neutral red solution for a short time (one to five 

 hours), and in the second they werr- kept in a more dilute solu- 

 tion (1: 100,000 to 1,000,000) for a much longer period (one-half 

 to four days). The frog was then carefully washed in running 

 water, the tissue removed and prepared in a manner similar to 

 that used in previous experiments (Matsumoto, '18). Both 

 modes of staining are adapted to the study of the movement 

 of epithelium. 



Though the frog could be stained an intense red without doing 

 it any apparent harm, still the dye proved to be more o*' less 

 harmful to the cells. In fact, an earlier degeneration of the 

 explanted tissue due to the injury of cells in such preparations 

 was frequently seen. As it was desirable to stain the tissue in 

 such a manner that the granules would be just evident enough 

 for observation, weak solutions (such as 1: 100,000 to 1: 200,000; 

 ten to twenty-four hours) were preferable. The use of the more 

 rapid staining, at times, brought on the disadvantage of having 

 the granules appear unevenly distributed. 



The arrangement of the neutral rod granules shows a more or 

 less marked difference in the several cell layers of the corneal 

 epithelium. Observing from the upper (outer) surface of the 

 corneal tissue in the culture, we see the following: 



1. The outennost layer (fig. 1, d) consists of flat polygonal 

 cells with large nucleus and unstained fine refractive grannies in 

 the cytoplasm. These cells, which may be present all over the 



