242 Biological Stains 



Thiazin Eosinates* 



The first worker to combine eosin and methylene blue was 

 Romano vsky (1891). He realized that a mixture of these two 

 dyes had great selective properties as a stain, and showed it to be 

 excellent for blood, particularly in bringing out the malarial para- 

 site. He also appreciated that it was more than a mere mixture of 

 the two dyes and that some new dye having the property of giving 

 the nuclei a red color was present. It was some time later before 

 the nature of this new dye was known, although it was subsequent- 

 ly named azure I or methylene azure; its true chemistry has 

 scarcely been understood until recently (see p. 107). Methylene 

 violet, and "methylene azure", which probably was also present, 

 had already been described by Bernthsen (1885). How these 

 new dyes were formed in the Romanovsky stain was not known 

 then; although Romanovsky stated that different lots of methylene 

 blue solution varied in their ability to give a good blood stain, and 

 that old solutions on which a scum had formed were best. 



One interesting point in connection with this early work is that 

 Bernthsen made the statement that the absorption maximum of 

 Azure I is at 650 m/z. This statement indicates, as can readily 

 be verified by consulting the absorption data of the azures given 

 in Chapter VI, that his azure I may have been primarily azure B. 

 In much recent work concerning the chemistry of azure I, this 

 early statement of Bernthsen's seems to have been overlooked. 



Present day blood stains are often spoken of as modified Roman- 

 ovsky stains; although the modifications are so great as to make 

 them of a very different nature. The first modification was made 

 by Nocht (1898) who concluded that the differential staining was 

 due to the formation of other dyes by the decomposition of meth- 

 ylene blue. Unna (1891) had already described what he called 

 polychrome methylene blue, made by heating a solution of meth- 

 ylene blue on a water bath with potassium carbonate. Nocht 

 decided to use this in the Romanovsky stain instead of untreated 

 methylene blue. He found that it gave very good results if prop- 

 erly neutralized before mixing with eosin; and then learned that 

 better results could be obtained by the use of a smaller amount of 

 alkali and a longer period of polychroming, without subsequent 

 neutralization. 



The next step in preparing blood stains was made by Jenner 

 (1899) who collected the precipitate formed when methylene blue 

 and eosin are mixed, and redissolved it in methyl alcohol. He did 

 not use polychrome methylene blue, and his stain lacked the 

 nuclear staining principle of Romanovsky 's and Nocht's stains 

 unless allowed to stand for some time before using; but it was an 

 important step in that he showed the possibility of collecting the 



*An account of the history of these blood stains is given by MacNeal (1906), 

 and by Conn et al. (1933). 



