92 THE MITOCHONDRIAL CONSTITUENTS OF PROTOPLASM. 



The properties of janus green B are: (a) in water, blue solution; (b) on addi- 

 tion of hydrochloric acid, soluble blue precipitate; (c) on addition of caustic soda, 

 black precipitate; (d) in concentrated sulphuric acid, olive-green solution, on dilu- 

 tion becoming green, then pure blue. 



The azodimethylaniUn has but Uttle to do with the specificity of the reaction, 

 because the diethylsafranin alone will stain the mitochondria more or less specifi- 

 cally. Moreover, I have prepared the safranin from the janus green of Griibler, 

 the dimethylsafranin from the janus green G of Hoechst, and the diethylsafranin 

 from the janus green B of the same firm, and I find that the diethylsafranin alone 

 will stain the mitochondria. 



The method of preparing the safranin is as follows :' 



1. Make a saturated solution of janus green in distilled water in a flask. 



2. Add a little zinc dust and a few drops of hydrochloric acid. The solution first assumes a 



bright crimson color and then bleaches, the hydrochloride of the leueobase of the safranin 

 being fonned. 



3. Filter. Shake the filtrate in air and thus reoxidize the leueobase. 



4. Precipitate the dye by saturating the solution with sodium sulphate. It is often necessary 



to use a little heat. A dark red precipitate is formed. 



5. Filter. Collect the precipitate on the filter. Wash with a saturated solution of soduun 



sulphate and drj- it. 



6. Dissolve out the dye from the dried precipitate with absolute alcohol. 



7. Filter and evaporate the filtrate to dryness. 



8. Dissolve the dye in the required concentration in distilled water or in salt solution. 



The diethylsafranin prepared in this way behaves in exactly the same fashion 

 as some pure diethyl safranin manufactured especially for me by the Farbwerke 

 Hoechst Company. 



There is, in addition to these janus greens, a large series of other janus dyes, 

 of which janus blue G and R, janus gray B and BB, janus black D, I, II, and O, 

 and janus yellow G and R are of particular interest because they are safranin 

 derivatives, the others being dyes of other series. 



Janus blue is diethylsafranin-B-naphthol and it stains mitochondria in hving 

 lymphocytes in a constant and specific fashion. It is inferior to janus green in 

 that it will stain mitochondria in these cells only in a dilution of 1 : 300,000, but 

 as an indicator of processes of reduction it is better than janus green, for the con- 

 trast between the blue of the dye itself and its red safranin base is more brilliant 

 than in the case of janus green. The marks G and R indicate, according to Schultz 

 (1914, p. 48), that the janus blue is made by two processes, from clematin (mark 

 G) and from safranin (mark R). It is worthy of note that this color contrast with 

 janus blue is particularly beautiful in the kidney, where the glomeruh may be 

 colored deep blue and the remainder of the tissue red; so sharp is the contrast that 

 the glomeruli in thick sections of the entire kidney may be easily counted with a 

 binocular. Janus green hkewise stains the glomeruh specifically. 



Janus black I also stains mitochondria in living blood-cells specifically, but, 

 on exammation, I find that it is not a pure dye, but a mixtm-e of two substances, 

 diethylsafraninazodimethylanilin and a brown substance, the nature of which I 



'Dr. R. R. Bensley, personal communication. 



