24 ON THE DIFFERENTIAL REACTION TO VITAL DYES EXHIBITED BY 



The case is chiefly notable for lack of much blue in fibroblasts (indeed often not detectable 

 at all) and for the crystals of trypan blue which have been able to form in T 148 vacuoles, the blue 

 color of which is never very marked. 



It is notable, then, that the trypan blue crystallizes out with the same readiness 

 in the vacuolar system created by dye T 148 as would characterize it were the pre- 

 ceding red dye not present. 



The cell's tendency to handle low closes of trypan blue and its biological homo- 

 logues at relatively few places in the protoplasm is evidenced by the insignificant 

 enlargement of the segregation-apparatus which, as we have already mentioned, such 

 dosage produces. We have repeatedly employed, for the first treatment in our 

 "double-staining" experiments, dye-stuffs which, either from their essential nature 

 or from the dosage with which they are employed, produce an abundant and large 

 vacuolar segregation-apparatus. Though this apparatus is consequently, as it 

 were, waiting in all parts of the protoplasm to receive the blue secondary dye 

 (dilute dosage with try pan-blue homologues), yet the latter does not avail itself of 

 many more of these sites for anchorage that it would when employed in this dos- 

 age as the sole stain. It is apparent from this that the character and number of 

 vital-dye deposits with any one method of dosage of a particular vital stain is not 

 greatly influenced by the type of segregation-apparatus which already happens to 

 have been produced in the cells treated. T\ T o data would appear to be more con- 

 clusively in favor of the view that "dye bodies" are dependent chiefly on the 

 nature of the vital dye and on the exact method of its administration. Such 

 experiments also furnish, it would appear, irrefutable evidence that even the con- 

 ditions for crystallization are, for each dye substance, laws unto themselves, and 

 that the tendency for the second dye to crystallize is not seriously interfered with 

 through the fact that it must occupy vacuoles already filled with the first dye sub- 

 stance and apparently in more soluble form. These ideas, however, gained their 

 most convincing demonstration by the production of analogous pictures when 

 simultaneous dosage with an actual mixture of the same dyes was employed. 



Protocol: Rat 22G9, injected intraperitoneally with a mixture of equal parts of a 0.1 per 

 cent solution of trypan blue and a 0.1 per cent solution of dye T 148 April 18 to 27, 

 inclusive, 1 c. c. each day; April 28,0.8 c. c; April 29, 30, May 1,3,5,7,9, 11, and 13, 

 1 c. c each day. 



May 14: Films from right thigh examined. The animal is stained in general a light plum-color. 

 Under the low-power the macrophages only are seen. They carry brilliant blue deposits, which, 

 even at this power, can be identified as crystalline. The fibroblasts can be seen only with difficulty, 

 but appear to display a pale-blue color. 



Under the oil, the macrophages are seen to exhibit their trypan-blue deposits exclusively in 

 the form of pale-blue, seed-shaped structures, sharply pointed at both ends, having a dense-blue 

 single or double edge. Instances of a central deep-blue crystal, with or without lateral ones, are 

 also seen. Cases of bodies which are almost spherical and possessing on one side a sickle-shaped 

 condensation are also frequent. Instances of free, delicate, needle-shaped crystals, while seen after 

 much search, are rare. Neutral red shows only a small number of other vacuoles. Janus green 

 (1:10,000, four minutes) stains normal mitochondria. 



The fibroblasts are full of pale-yellow, irregular-shaped T 148 vacuoles which fill the proto- 

 plasm, including its chief processes. In a portion of these (always a half dozen instances) the 

 trypan blue has accumulated so as to give a marked blue hue and in some cases to produce a minute 

 blue crystal. Instances of these minute crystals unrelated to the T 148 deposits are not seen, and the 

 blue, when most emphatic, is so difficult to see that the case must be continued for at least another 



