THE MITOCHONDRIAL CONSTITUENTS OF PROTOPLASM. 87 



that dimethylsafraninazodimethylauilin did not stain the filaments specifically. 

 He found that, on reduction of the dye in the tissue itself or in the test-tube, the 

 dimethylanilin group splits off, leaving the red diethylsafranin. According to him 

 the diethylsafranin formed in this way does not stain the filaments specifically. 



(C2 Hs) N 



-N= N-/\ 



I J N(CH3l 



Fig. a. 



Laguesse immediately recognized the importance of Michaelis's discovery, 

 obtained some janus green from Griibler (not Hoechst), and used it in his inves- 

 tigations on gland-cells. It stained some structures which he called "vermicules" 

 and "ergastidions." His results, however, were not uniformly satisfactory, prob- 

 ably on account of the fact that he obtained the dye from the wrong source. 



Accordingly, janus green was soon forgotten as a vital stain for mitochondria, 

 and it remained for Benslej' (using the janus green of the Farbwerke Hoechst Com- 

 pany) (1911, p. 304) to revive interest in the discovery of Michaelis 



The reaction of mitochondria to janus green may be conveniently described 

 in three stages: the staining of mitochondria with janus green, the reduction of the 

 janus green with the formation of diethylsafranin, and the production of the leuco- 

 base. 



If one injects the pancreas of a guinea-pig through the blood-vessels with a 

 solution of 1:25,000 janus green in 0.85 per cent sodium-chloride solution, the 

 mitochondria become intensely stained in the course of 15 minutes or more. The 

 other structures in the cell, like the nucleus and zymogen granules, remain lui- 

 colored unless the stain is applied in too great concentration for too long a time. 

 The staining is facilitated by exposing the pancreas to the air. 



If, now, portions of the pancreas are mounted in salt solution on a slide and 

 are covered with a cover-glass, the dye is slowly reduced by the tissues, with the 

 formation of diethylsafranin (see, however, Michaelis, 1902, p. 101), which has 

 a bright pink color. The change first takes place in the more central parts of the 

 tissue remote from the oxygen of the air and surrounding salt solution and proceeds 

 slowly towards the periphery. It is hastened by ringing the preparation around 

 the edge of the cover-glass with vaseline, which excludes the air and prevents 

 evaporation. The faintly green-stained ground substance first changes to pink 

 before the intensely stained mitochondria are affected. The change passes like 

 a wave across the tissue from the center to the periphery. The mitochondria in 

 the part of the cell nearest the middle of the j^reparation change their color first. 

 Those in the remainder of the cell then follow suit. There is no evidence that 

 mitochondria of different sizes change at a different rate, or that different parts of 



