THE BLOOD DYES 267 



that the best result was obtained when a mould was growing on 

 the surface of the stock solution of methylene blue. He remem- 

 bered that Unna ^^^ had investigated the changes undergone by 

 methylene blue solutions on standing or treatment with dilute 

 alkali. A new substance appeared, which Unna called methylene 

 red; the 'ripened' solutions coloured Mastzellen red. Unna tried 

 various ripening agents, and found certain carbonates, especially 

 potassium carbonate, the best. From these researches resulted his 

 polychrome methylene blue. 



Nocht ^^^ tried a methylene blue/eosin solution in which the 

 polychrome variant had been substituted for pure methylene blue. 

 He found it necessarv to neutralize the alkaline solution. When this 

 was done, the nucleus of the malarial parasite was regularly dyed 

 red. The basiphil cytoplasm, however, was violet, not blue, and 

 this gave insufficient contrast with the red of the nucleus. Xocht 

 overcame this difficulty by simply adding pure methylene blue to 

 his polychrome neutral dye : the basiphil cytoplasm was now blue. 



Nocht worked with a freshly-made solution containing a small 

 excess of the basic dyes. The Romanowsky effect could be pro- 

 duced with certainty, but one practical defect remained: the 

 proper balancing of the eosin with the basic dyes had to be care- 

 fully done every time one wanted to dye blood- smears. The 

 obvious necessity was a stable stock solution of the dyes, requiring 

 nothing but dilution. 



The problem was solved independently in Germany by 

 Renter "^^^ and in England by Leishman.-^^ Wright's ^^^ well- 

 known mixture is a mere variant of Leishman's. All used a partly 

 polychromed methylene blue and dissolved the precipitated neutral 

 dye in alcohol. Renter used absolute ethanol, while the others made 

 use of Jenner's discovery that absolute methanol is a particularly 

 good solvent for methylene blue/eosin. Leishman, an Assistant 

 Professor in the British Army Medical School, made the important 

 discovery that a single solution could act first as fixative for the 

 blood-smear, because the solvent was methanol, and subsequently, 

 on dilution on the slide with distilled water, as a quickly-acting 

 dye. Leishman's and Wright's techniques are often used to the 

 present day and generally give excellent results, though they are 

 not quite so uniform as the dyes made up entirely from known 

 ingredients, in accordance with the principles that will now be 

 explained. 



It is necessary to know what substances besides methylene blue 



