244 Biological Stains 



with a definite proportion of K2Cr207 in acid solution, a process 

 which he finds to yield a more uniform product. 



Giemsa's and MacNeal's modifications are somewhat different. 

 In order to start with a more definite compound than polychrome 

 methylene blue, Giemsa (1902) used more carefully controlled 

 methods of oxidation and obtained a product which he considered 

 to be the same as Bernthsen's Azure I. This he combined with 

 eosin Y, to obtain a more definite compound than in the case of 

 stains of the Leishman type. Subsequently he added methylene 

 blue to the azure before combining with the eosin, and in that way 

 secured better differentiation. He did not state his method of 

 preparing azure I, but apparently gave it secretly to Dr. Grubler's 

 Laboratory, which subsequently became K. Hollborn and Sons; 

 and the latter company made much of this "trade secret" in 

 claiming their Giemsa stain to be the only authentic product of 

 that name. Besides azure I, this company put on the market an 

 azure II which was a mixture of Azure I and methylene blue in 

 equal parts. The eosinate of this mixture, known as azure-II- 

 eosin, was the chief ingredient of Giemsa stain. There probably is 

 no real secret involved in the composition of these azures. Giemsa 

 stated that he used Bernthsen's methylene azure, and Bernthsen 

 described this as having an absorption maximum at 650 m^t; as this 

 is approximately that of present day azure B, we can assume that 

 Giemsa either had the latter or a mixture of higher and lower 

 homologs having optical properties similar to azure B. There has, 

 however, been considerable confusion on this subject of recent 

 years; and in part this has grown out of certain erroneous in- 

 ferences derived from MacNeal's excellent work (discussed below) 

 on these stains. 



The formulae recommended by Giemsa for preparing solutions of 



this stain are: 



For blood For tissue 



Azure-II-eosin 3- g- 3. g. 



Azure II 0.8 g. 0.8 g. 



Glycerol, c.p 250 g. or 200 ml. 125 g. or 100 ml. 



Methyl alcohol, neutral, acetone free 250 g. or 312 ml. 375 g. or 457 ml. 



There is apparently a reason for the quantity of glycerin called 

 for in the first of these formulae (50% by weight or about 40% 

 by volume). Recent work in one of the Stain Commission labora- 

 tories indicates at least 40% by volume to be necessary to give a 

 reasonably stable solution. 



Substituting American dyes for those in the old Giemsa formula, 



and following the recommendations of Lillie, the following seems 



advisable : 



Azure A eosinate 0.5 g. 



Azure B eosinate 2.5 g. 



Methylene blue eosinate . . . 2.0 g. 

 Methylene blue chloride ... 1.0 g. 



Glycerol 375. ml. 



Methyl alcohol, reagent. . . .375. ml. 



