8O C. M. CHILD. 



I have made no attempt to determine at all exactly the length of 

 time necessary for completion of the reaction in given concentra- 

 tions but have merely made certain that length of time was 

 sufficient for the purpose. Usually preparations of this sort 

 remained 24-48 hours in a solution of m/iooo or higher. My 

 observations indicate, however, that in small organisms, such as 

 hydroids, and in embryonic stages the reaction is in most cases 

 complete in much shorter time, e.g., 2-4 hours in w/iooo. 



All concentrations of KMnO 4 high enough to give any appre- 

 ciable deposit of the oxide on or in the protoplasm are highly 

 toxic. Even in m/2o,ooo ciliary activity ceases within a short 

 time, ranging from a few seconds to about one minute in forms 

 examined and traces of the color begin to appear on the external 

 surfaces within two or three minutes. Except in those concentra- 

 tions in which cytolysis and disintegration take place it is im- 

 possible to determine exactly when death occurs. Undoubtedly 

 it occurs before reduction and staining proceed very far, for 

 alteration in aggregate condition, apparently a coagulation, can 

 often be observed in the cells in early stages of coloration. 



Certain precautions necessary in the use of the permanganate 

 may be noted. Certain low concentrations produce cytolysis 

 and disintegration in many, perhaps in all, organisms. In such 

 cases susceptibility gradients corresponding to those observed 

 with other agents and to the reduction gradients appear. In 

 concentrations producing disintegration the disintegrated mass 

 may show more or less distinct gradients in rate or amount of 

 reduction, provided it holds together, but commonly cytolysis is 

 followed by loss of continuity and by distribution of the cell 

 substance through the water. In higher concentrations the 

 reduction gradients appear without disintegration. 



In order to avoid irregularities in staining it is necessary to 

 agitate the solution frequently or to move the organisms about 

 in it. Reduction of the permanganate at the surface of the 

 organism often occurs much more rapidly than its diffusion, and 

 a zone of low concentration of permanganate may appear about 

 the organism, particularly about its more rapidly reducing 

 regions. Parts in contact with the glass or lying near other 



