METHODS OF PHYSIOLOGICAL ANALYSIS 65 



represents one aspect of an axial differential which expresses itself in 

 various ways is indicated by the fact that it parallels gradients shown by 

 other methods in the same material. 



Benzidin has been used as a test for peroxidase activity in sea-urchin 

 eggs and embryos;'' but since the method involves killing the material, the 

 significance for the living organism of observed differences or absence of 

 difference remains a question. 



DIFFERENTIAL STAINING BY VITAL DYES 



The so-called "vital dyes" which stain intracellular constituents in the 

 living organism are more or less toxic and, with sufficient staining, kill. 

 Certain of these dyes undergo reduction in living cells under anaerobic 

 conditions or in low oxygen, becoming colorless (methylene blue, etc.) or 

 changing color in the course of reduction (Janus green). Use of dye-re- 

 duction methods is considered in the following section. For the present, 

 staining with oxidized basic dyes is the chief concern. In water of suffi- 

 ciently high oxygen content to prevent intracellular reduction of the dye, 

 organisms or parts with little regional differentiation along the polar axis, 

 coelenterate blastulae and planulae, echinoderm blastulae and gastrulae, 

 the body of hydra, the naked stem of Corymorpha, hydroid tentacles, etc., 

 stain uniformly or almost uniformly at first; that is, there is little or no 

 evidence of differential penetration of dye in the ectoderm of such forms. 

 With continued staining, however, an axial differential often appears. The 

 depth of staining becomes greatest apically or anteriorly, decreasing basip- 

 etally or posteriorly, and cytolysis and death produced by the dye follow 

 the same course. A study of staining gradients in relation to susceptibility 

 gradients and to the gradient problem in general has been made by J. W. 

 MacArthur (1921) with protozoa, hydra, planarians, other fiatworms, and 

 annelids as material. Like others before him, he finds the basic dyes much 

 more effective, both as dyes and as toxic agents, than the acid dyes. Acid 

 dyes in neutral solution do not stain and are rarely sufficiently toxic to 

 kill. Increased acidity increases effectiveness of acid dyes. In a planarian 

 which shows certain minor differences in relative susceptibility to basic 

 and acid agents (pp. 11 2-13), MacArthur finds that basic dyes act like 

 acid agents, and acid dyes, when effective at all, like basic agents. He also 

 finds that the staining gradient shows the same axial relations as the later 



' Ranzi e Falkenheim, 1937; Pitotti, 1939. 



