68 PATTERNS AND PROBLEMS OF DEVELOPMENT 



ticular dye occurs differs in different organisms, and regional differences in 

 time and rate of reduction in the individual are associated with differences 

 in rate of oxidation. With gradual decrease of oxygen after staining, such 

 differences are most clearly seen. In embryonic stages and even in adult 

 individuals of many animal species these differences appear not as sharply 

 defined and bounded areas but as reduction gradients, that is, gradients of 

 change or loss of color in definite and characteristic relation to physiologi- 

 cal axes of the whole, of organ systems, and of loci of developmental activ- 

 ity, such as buds. If injury by the dye has not occurred, return of color 

 after reduction may be brought about by readmission of oxygen. With 

 small organisms in a small amount of water reoxidation may occur almost 

 instantaneously or within a few seconds, but a gradient of return of color 

 is usually temporarily visible. If not injured by lack of oxygen or dye, the 

 same material may serve for repeated reduction and reoxidation. 



A number of the basic dyes which are available as oxidation-reduction 

 indicators can be prepared as reduced, colorless leucobases with either a 

 reducing agent, sodium hyposulphite, or its formaldehyde compound, ron- 

 galite, or by catalytic reduction with molecular hydrogen by a platinum or 

 palladium catalyzer." The leucobases penetrate more readily than the 

 oxidized dyes, and the color appears in the material when it is exposed to 

 oxygen. Leucobases prepared with hyposulphite, however, are much 

 more toxic than the oxidized dyes, even those which stain readily in oxi- 

 dized form; consequently, great care is necessary in their use. In regions 

 of the living material injured by them reduction is retarded or does not 

 occur at all. Leucobases catalytically prepared are less toxic, but oxidize 

 rapidly on exposure to oxygen and enter living material without oxidation 

 only under anaerobic conditions. 



With many organisms it is possible to distinguish definite and constant 

 regional differences in rate of appearance of color after penetration of the 

 leucobase and exposure to oxygen or with hyposulphite-leucobase, as it 

 enters the cells in an oxygen-containing medium. In other words, the leu- 

 cobase is more rapidly oxidized in some regions than in others; an oxida- 

 tion gradient may appear temporarily. Regional differences in rate of in- 

 tracellular oxidation of leucobase have been regarded as indicating differ- 

 ence in oxidase activity; but since the leucobases oxidize rapidly on expo- 

 sure to oxygen without a catalyst, it may be questioned whether this reac- 

 tion is always an adequate indicator of oxidase activity alone. But wheth- 

 er the dye enters the cell or organism as leucobase or in its usual form as 



" See Fischer und Hartwig, 1937, and their citations. 



