THE MITOCHONDRIAL CONSTITUENTS OF PROTOPLASM. 89 



cells and in protozoa, and in organs which may be stained by the injection of janus 

 green through the blood-vessels. 



Janus green occasionally stains other structures in addition to mitochondria, 

 of which a few may be enumerated: cement substance between the cells of the 

 testis, lipoid inclusions, blepharoplasts, chromosomes, granules of islet-cells, etc. 

 Rut it is important to note that it is specific for mitochondria when used in great 

 dilution, 1 : 500,000, for instance. 



The toxicity of janus green varies in different cells of the same animal and in 

 (lifTerent animals. The same is true in plants. Fresh and old solutions of the dye 

 have the same toxicity. Lewis found that in cultures of heart-muscle cells the 

 mitochondria stained with janus green while the muscle continued to beat. Ship- 

 ley (1916, p. 441) found that trypanosomes retained their motility for some time 

 after the mitochondria in them were specifically stained with janus green. 



I have been able to stain the mitochondria with janus green in the polymor- 

 phonuclear leucocytes of man and several other vertebrates. Such leucocytes, 

 with their mitochondria stained, move around in an amoeboid fashion. The tip of 

 the pseudopod is generally free of both mitochondria and the specific granula- 

 tions. It is followed by a mass of mitochondria and granules. The nucleus gen- 

 erally comes last. Cells stained in this way engulf foreign particles and show no 

 deviations from their normal behavior. They will continue to do this for from 

 half an hour to an hour, depending upon the temperature of the warm stage, the 

 rate of evaporation, etc. Their rate of disintegration is not greatly accelerated by 

 the action of the stain. 



When applied subcutaneously it remains localized at the point of injection. 

 Intraperitoneal injection causes death in a short time and intravenous injection 

 in a very few minutes. Feeding experiments gave no results and attempts to 

 increase the tolerance of protozoa to it proved futile. 



To what is the toxicity due? There are a number of possibilities. It may be 

 due to the presence of some toxic material used in the manufacture, but rather 

 against this is the fact that different samples of janus green B possess the same 

 toxicity when applied to the same type of cell. The toxicity varies, however, mark- 

 edly in different kinds of cells, and it is not a question of penetration. The idea of 

 obtaining, in some way, a completely non-toxic janus green is an inviting one, but 

 janus green can never approximate to the azo dyes like trypan blue in this respect, 

 for the reason that it quickly undergoes chemical change in the body, with the 

 liberation of a number of potentially toxic substances. 



Before taking up the specificity of the janus-green reaction, it will be of 

 interest to bring out a few more facts about janus green itself. 



The name may or may not be associated with the Roman deity Janus, gen- 

 erally represented with two faces looking in opposite directions. The dye certainly 

 shows two colors, green and red, of quite opposite character. 



According to IMichaelis (1902, p. 51), janus green is made by (he action of 

 nitric acid on safranin, one of the two amido groups being changed into the diazo 

 group. To this dimethylanilin is added according t(i the following eciuation: 



