2260 Journal of Applied Microscopy 



A Review of the Methods of Staining Blood. 



VII. 



D. Neutral Stains. — Continued. 



Willebrand (1901) and Becker (1901) obtained a neutral stain by adding 

 acetic acid to a mixture of eosin and methylen blue. In a mixture of eosin and 

 methylen blue diffuse staining is obtained, but if dilute acetic acid be added 

 drop by drop, a dififerentiation takes place. Becker believes that the acetic 

 acid acts as a solvent of the neutral precipitate. 



Equal volumes of a 0.5 per cent, solution of eosin in 70 per cent, alcohol and 

 of a concentrated watery solution of methylen blue are mixed. To 50 c. c. of 

 this mixture ten to fifteen drops of a 1 per cent, acetic acid solution is added. 

 The solution should be filtered before using. Preparations which have been 

 fixed by dry heat, absolute alcohol or 1 per cent, formol alcohol, should be 

 stained five to ten minutes, washed in water without decolorization. Red cor- 

 puscles stain red, nuclei dark blue, acidophile granules red, neutrophile granules 

 violet, and granules of the mast cells intense blue. 



Ewmg (1901) also uses acetic acid as a solvent of the neutral dye. 



1, To 30 c. c. polychrome methylen blue (Griibler) add five drops of a 3 per 

 cent, solution of acetic acid (V. S. P. 33 per cent. ). 



2, Make a saturated 1 per cent, aqueous solution of methylen blue (Griib- 

 ler), dissolve dye by gentle heat. This solution should be at least a week old. 



3, Make a 1 per cent, aqueous solution of Griibler's aqueous eosin. 



To 10 c. c. of water add four drops of the eosin solution, six drops of the 

 polychrome methylen blue solution, and two drops of the 1 per cent, methylen 

 blue solution. Mix well. Preparations should be stained two hours, blood side 

 down. 



Several hematologists have obtained a neutral staining by successive stain- 

 ing with eosin and methylen blue, the neutral stain being precipitated in the 

 protoplasm of the cells. 



i5'//^^/ (1901) stained fixed dry preparations five minutes in an eosin solution 

 (eosin 1 g., water 90 g., alcohol 10 g.), washed in water and stained thirty 

 seconds in a concentrated watery solution of methylen blue or Loffler's alkaline 

 methylen blue. It is important that the methylen blue should not act too long 

 on the preparation. All of the basophile, acidophile and neutrophile elements 

 of the blood are stained. Engel thinks that failure to get a differentiation of 

 the basophile, acidophile and neutrophile elements by successive staining with 

 eosin and methylen blue is due to too long action of the methylen blue. He be- 

 lieves that a previous preparation of a neutral dye is unnecessary. 



Japha (1901) also stained successively with eosin and methylen blue. He 

 fixed dry preparations for one minute in 1 to 2 per cent, formalin in alcohol, 

 stained well with a strong watery solution of eosin, then several seconds with 

 dilute — but not too dilute — watery solution of methylen blue. Judgment must 

 be used in staining with the methylen blue. Japha examined the washed prep- 

 aration from time to time to determine the degree of staining. 



