PHYSIOLOGICAL GRADIENTS. 165 



ently begins on the external surface of the protoplasm, often 

 within a few seconds after the organisms are brought into the 

 solution and the differences in rate of precipitation and staining 

 at different levels of an axis are usually very marked. Penetra- 

 tion into the protoplasm occurs rather slowly, its rate depending 

 somewhat on concentration and it is certainly not to any large 

 extent dependent on permeability of living membranes but rather 

 on the killing of the protoplasm from the surface inward. 



If the reaction is allowed to continue to completion in excess of 

 permanganate the whole organism may become opaque black and 

 no gradient is visible, but many small organisms thus stained, 

 e.g., blastulas, hydroid planulse, small monosiphonous algae, can 

 be made more or less transparent after such staining by harden- 

 ing, clearing and mounting, and in such cases the gradient in 

 staining appears. Under these conditions the gradient repre- 

 sents a gradient in the total amount of reduction of permanganate 

 of which the protoplasm is capable and it is highly significant to 

 find that the protoplasm of the apical region of a blastula or an 

 alga axis, for example, is capable of reducing more permanga- 

 nate than more basal levels. If the organism is first killed by 

 some other agent, e.g., various histological fixing agents, heat, 

 etc., reduction and staining are uniform, or in some cases slight 

 traces of the gradients are still present, but after a few days in 

 alcohol they are completely absent in all cases examined. 



Since the chemical reaction concerned here is an oxidation- 

 reduction reaction, the rate and amount of reduction of perman- 

 ganate must be associated in some way with the oxidative activity 

 of protoplasm, the regions of higher rate of oxidation showing 

 a higher rate and greater total amount of reduction. We find 

 that the permanganate gradients correspond with those demon- 

 strated by other methods, i.e., the more active and more suscep- 

 tible regions reduce permanganate more rapidly and in larger 

 amount than the less active and less susceptible. This method 

 is a very delicate one and, I believe, of considerable value as a 

 means of determining regional and axial differences in physio- 

 logical state, particularly in small organisms. 



In certain cases the indophenol reaction has been used to 

 demonstrate the existence of axial gradients in blastulae and 



