APPENDIX II 



Methylene blue and Janus green have been found most useful in work on differ- 

 ential dye reduction. The former has been used both as an oxidized dye and as a 

 leucobase, the latter in oxidized form. A considerable number of samples from differ- 

 ent sources have given essentially similar results. With most forms tested, methylene 

 blue is less toxic than other dyes used, except, perhaps, brilliant cresyl blue. Janus 

 green has proved particularly useful because of the change in color from the blue green 

 of the oxidized form to brilliant red with partial reduction. This makes it possible 

 to distinguish slight differences and slight degrees of reduction more clearly than does 

 the mere loss of color on reduction of methylene blue and various other dyes. With 

 some forms the further reduction of Janus green to the colorless leucobase has been 

 observed. In all samples used, Janus green has been found much more toxic than 

 methylene blue, and great care has been necessary in its use in order to avoid altera- 

 tion of the normal reduction pattern by injury of the more susceptible regions and 

 consequent retardation of reduction or complete failure to reduce in them. Thionine 

 and cresyl echtviolett have been used for comparative purposes with results similar 

 to those obtained with Janus green and methylene blue. In fact, in every case thus 

 far, reduction gradients have been the same with different dyes. 



The procedure followed with small forms — protozoa, and embryonic and larval 

 stages of echinoderms and other animals — consists in sealing a considerable number of 

 individuals in a cell of small volume, usually made by drawing a ring of melted vaseline 

 of the desired size on a slide and covering the material in water without air bubbles. 

 The oxygen uptake of the animals brings about gradual oxygen decrease; consequent- 

 ly, reduction occurs gradually, and slight differences are distinguishable. With oxi- 

 dized dyes the material is either stained before sealing or a concentration of dye 

 determined by preHminary experiment to be nontoxic within the period preceding and 

 during reduction is used as medium in the cell. With this latter procedure staining 

 and oxygen decrease are occurring at the same time; and with certain concentrations 

 of dye and rate of oxygen decrease, reduction may occur in one region while another 

 is becoming more deeply stained. For elongated hydroids of considerable size, such 

 as Corymorpha, elongated cells may be made; or for these and for earthworms and 

 other large annelids glass tubes large enough to admit the animal may be used. Mineral 

 oil has been used in some cases to exclude air. With many forms rapid staining with a 

 relatively high concentration of oxidized dye and rapid reduction following have been 

 found less likely to produce toxic effects than slow staining with a lower concentra- 

 tion. Toxic effect seems to depend on accumulation of the dye on certain cell constitu- 

 ents which appear as deeply stained granules, rather than on its mere presence in the 

 cell. When the deeply stained granules become visible, reduction is retarded. With 

 slow staining the granular accumulation may occur before reduction, and the reduc- 

 tion picture may be altered; with rapid staining and quick reduction this effect may 

 be avoided. That this holds for all material is not asserted; it appears to hold for 



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