12 ON THE DIFFERENTIAL REACTION TO VITAL DYES EXHIBITED BY 



to receive them, rests essentially on four lines of evidence, which may be enumer- 

 ated as follows: 



1. The number and size of the dj r e granules, or stained structures, within the 

 cell, other things being equal, increases with increased dye dosage. 



2. Instances of metachromasia displayed by some few of these dyes prove con- 

 clusively that the dye slowly concentrates in the segregation-apparatus until it is 

 thrown out of solution, the solid form showing a different color from the fluid. 



3. By appropriate dosage, a true crystallization of some of these dyes can be 

 made to occur within the cell. 



4. Double staining, i. e., the superposition of one acid dye of marked color 

 difference from a preceding one (the effects of which have been previously deter- 

 mined), demonstrates that both substances are handled together in the same place 

 of deposit (i. e., in the segregation-apparatus), unless the diffusion rate of the two 

 dyes, ease of segregation or crystallization, or other physical attributes dispose 

 them to produce a different vital-dye picture in the cell. 



We may now consider in appropriate detail the nature of this evidence. 



1 . As regards the macrophages, very different effects are obtained in accordance 

 with the method of administration of the same dye, and again, equally pronounced 

 differences, in accordance with the dye chosen for experiment. In the case of 

 many of those benzidine dyes which diffuse easily in vivo, a daily intraperito- 

 neal administration of 1 c. c. of a 0.1 per cent solution leads Only to the most 

 gradual attainment of macroscopic color; nor are the macrophages in the areolar 

 tissues of such an animal at all similar to those of an animal which has tolerated 

 an acute high dosage of the same dye. It will be remembered that the acute high- 

 dosage effect is to give us macrophages with large and small vacuoles which show 

 great disparity in size (fig. 20) . The macrophages of animals treated with very low 

 dosages of the same dye are remarkable for the fact that their vacuoles are small and 

 of strikingly uniform size (fig. 22). 



Protocol: Rat 30, injected intraperitoneally with a 1 per cent solution of dye T 148, a 

 brilliant red, diffusible, true vital stain, synthesized for our comparative studies on 

 the azo dyes. The dye results from the combination in alkaline solution of 1 molecule 

 of o-tolidine with 2 molecules of alpha naphthylamine 4.8 disulphonic acid, and 

 hence has the formula 



NH 



J = N- ^ )\ \ n = N- 

 CH. 



NqOjS 



March 1 to 7, inclusive, 2 c. c. each day with the exception of March 5. 



March 8: Animal is stained a deep red. General condition of animal somewhat affected by 

 dye dosage. Subcutaneous tissues arc stained light red. Under the low-power, more color is present 

 in cells than is usual with this dye. The bright-red deposits seen in great numbers are apparently 

 in macrophages, but fibroblasts are easily identified and contain a large amount of dye. They are 

 not, however, as bright a red as the macrophages. 



I'nder the oil, differentiation of cell types is confirmed. The reaction is essentially a macro- 

 phage reaction, i. c, the macrophages contain the greatest amount of dye, as is shown in every way 

 by the size and number of their vacuoles and by the deeper red color they possess. These cells vary 



