METHODS OF PHYSIOLOGICAL ANALYSIS 63 



more recently studies on respiration and glycolysis have been made on 

 small fragments of amphibian embryos with the Cartesian diver "ultra- 

 microrespirometer" (Boell et at., 1938, 1939). 



In so far as they involve separation of the material into pieces, the 

 methods for determining regional differences in glycolysis present the 

 same difffculties and raise the same questions as those for oxygen consump- 

 tion and CO2 production. 



DIFFERENTIAL REDUCTION OF POTASSIUM PERMANGANATE 



Potassium permanganate is a powerful oxidizing agent and is readily 

 reduced by living protoplasms to oxides of manganese, which are precipi- 

 tated and stain the protoplasm brown or, in high concentration, opaque 

 black. As might be expected, this substance is highly toxic and may be 

 used as a lethal agent to show death gradients. In very low concentrations, 

 however, intracellular reduction and coloration of the protoplasm take 

 place slowly; and axial differentials in rate and, in some cases, in depth or 

 density of coloration can be observed. Concentrations used in this way 

 range from m/2,000 to m/ioo,ooo, according to material and rate of colora- 

 tion desired. The method is chiefly of value for small more or less trans- 

 parent forms, such as protozoa, some eggs, blastulae, planulae, and gas- 

 trulae, but may also be used to show differential reduction in the ecto- 

 derm of hydroids, medusae, and various larval forms and on cut surfaces 

 at different levels of an axis. Even small embryonic or larval stages of 

 some species become opaque black if the reaction is allowed to continue to 

 completion and final death of all parts. In some of these, however, the 

 organism becomes more or less translucent after dehydration and clearing, 

 and a gradient in depth of color is visible ; but in the clearing oil or in bal- 

 sam mounts the color gradually disappears. It is perhaps scarcely neces- 

 sary to point out that observation of the color through different thick- 

 nesses of tissue in the same organism may lead to incorrect conclusions. 

 In organisms killed by boiling water or by various fixing agents the amount 

 of reduction, as indicated by depth of color, and the rate of reduction are 

 greatly decreased; and either no gradient appears or a slight gradient is 

 present immediately after killing but soon disappears. 



The gradient in rate of reduction might conceivably result from a gra- 

 dient in rate of penetration of KMn04, but evidence indicating appreciable 

 differential in penetration has not been obtained. The reduction color is 

 at first uniform, as reduction occurs superficially in the cell or cells, and 



