THE BLOOD DYES 269 



azure I. This was evidently azure B, or else azure B somewhat 

 contaminated with related dyes.^^^ He mixed this with eosin and 

 showed that the main features of the Romanowsky effect were 

 obtained, in the absence of methylene blue.^^^ Now azure B, unlike 

 methylene blue, is a metachromatic dye. Itself blue, it has a strong 

 tendency to impart a reddish colour to chromatin. Giemsa attri- 

 buted the reddish or red colour of the nuclei of leucocytes and of 

 the malarial parasite in Romanowsky preparations to the presence 

 of azure I in polychrome methylene blue. 



Giemsa subsequently came to the conclusion that his azure/ 

 eosin alone was imperfect, because the colour of basiphil cyto- 

 plasm was greyish rather than pure blue and therefore gave less 

 striking contrasts. It was for this reason that he put methylene 

 blue in his mixture, in addition to azure and eosin. This gave the 

 desired result. ^^^ He mixed his azure with an equal weight of methyl- 

 ene blue, and from this mixture ('azure IP) he made a neutral 

 dye by precipitation with eosin. He added a small extra quantity 

 of azure II so as to obtain a preponderance of the basic dyes. 



It is fortunate that azure B, a blue dye, gives a strongly meta- 

 chromatic colour to the chromatin of leucocytes, while methylene 

 blue has a special affinity for the basiphil cytoplasm of lympho- 

 cytes and monocytes. Otherwise it would have been difficult to 

 dye the chromatin of these cells in a different colour from the 

 cytoplasm. The acidic substance in the cytoplasm that is coloured 

 by these basic dyes is presumably RNA. In the form in which it 

 occurs in the tissues, RNA is luckily less apt to act as a chromo- 

 trope than DNA. 



Giemsa experimented also with methylene violet/eosin dissolved 

 in alcohol and water, but did not obtain the full Romanowsky 

 effect. On adding methylene violet to his azure mixture he found 

 that dyeing was inhibited and the tendency to precipitation in- 

 creased. For these reasons he never used methylene violet in his 

 later mixtures. 



Giemsa's subsequent papers i88,i89,i9i ^Lre mainly concerned 

 with solvents and practical details of technique. He followed 

 Leishman and Wright in using methanol as a solvent for his stock 

 solution, but added glycerol to increase the capacity to dissolve 

 the dyes and allow easier mixture with water. 



To rationalize Giemsa's technique still further, it was necessary 

 to use an azure of definitely known chemical composition. A com- 

 parison of azures C, IV, A, and B was undertaken in the U.S.A. 



