and Laboratory Methods. 2229 



A Review of the Methods of Staining Blood. 



VI. 



D. Neutral Stains — Continued. 



5. The Development of the Neutral Eosin=Methylen Blue Method of Staining Blood. 



— For years the neutral triple stain and its modifications have been the stand- 

 ard stains for the blood. They produce a considerable differentiation of the 

 complex elements of the blood, but they have weak basic properties and do not 

 stain the basophile granules of the red cells, the granules of the mast cells, or 

 the malarial parasites and bacteria when present in the blood. Recently, how- 

 ever, students of the blood have been devoting their attention to staining with 

 eosin and methylen blue. We have in these two dyes the most powerful and at 

 the same time the most precise acid and basic stains that are known for staining 

 the blood, and hematologists have succeeded in combining the two into a neutral 

 stain with which we can obtain a complete differential staining of the blood. 



As we have already seen in the section on double staining with acid and 

 basic stains, several hematologists, including Chenzinsky, Plehn, Aldehofif, Lav- 

 eran, Gabritschewsky, Canon and Pielicke, and Mannaberg, have employed eosin 

 and methylen blue, either successively or in combination, for staining the acido- 

 phile and basophile elements of the blood. But Romanowsky (1891) appears to 

 be the first to obtain a neutral stain from the combination of these two dyes. 



Romanoivskfs stock solutions consisted of : 



(1) Saturated watery solution of methylen blue. 



(2) 1 per cent, watery solution of eosin. 



These two solutions can be kept for a long time ; the older the methylen blue 

 solution the better its action. Dry preparations heated for 30 minutes at 105° 

 to 110°C. are floated on the surface of a mixture of 1 part of the filtered methy- 

 len blue solution to two parts of the filtered eosin solution, contained in a watch 

 glass, for from 2 to 3 hours, the preparations being covered by another inverted 

 watch glass, and the whole by a bell jar moistened on the inside. The prepa- 

 rations are then washed in water, or, if overstained, first in alcohol. 



When the two stains are mixed a heavy precipitate falls which should be 

 used together with the solution. Romanowsky assumes that a third neutral color 

 is formed, for which the nuclear network of the malarial parasites shows the 

 greatest affinity. The malarial parasites are stained Prussian blue, the nuclear 

 chromatin carmin violet. Presumably the basophile, acidophile and neutrophile 

 elements of the blood are also stained. 



Romanowsky's method of staining has, however, given variable and unsatis- 

 factory results and has been superseded by improved methods. These improve- 

 ments have consisted first in the use of a solvent for the neutral dye. Ehrlich 

 found that the neutral precipitates from the mixtures of acid and basic dyes were 

 at most but slightly soluble in the fluid of the mixture. He obtained a solution 

 of the neutral dye by the addition of an excess of the acid dye, but consequently 

 obtained a weak basic staining. Those who have worked with the Romanowsky 



