2380 Journal of Applied Microscopy 



A Review of the Methods of Staining Blood. 



IX. 



D. Neutral Stains. — Continued. 



Goldhoni (1901), who has obtained neutral staining of the blood by succes- 

 sive staining with eosin and his polychrome methylen blue, has since combined 

 the two into a neutral mixture and isolated the neutral dye. This he does as 

 follows : To his polychrome methylen blue (III D, 5, i), made strongly acid with 

 glacial acetic acid, is added 5 per cent, eosin solution till the mixture is transformed 

 into a pulpy mass. This is filtered through two layers of filter paper, and the 

 mass left on the paper is dried in a hot-air oven, after which it is dissolved in 

 wood alcohol. If the solution thus prepared is too acid, staining the erythrocytes 

 too deeply and the leucocytes very little or not at all, the desired reaction may 

 be obtained by adding gradually wood alcohol, made alkaline with potassium 

 carbonate, until the dye gives the required results. If the stain is made strictly 

 neutral or alkaline, the red corpuscles will stain a greenish hue and the granules 

 and " anaemic degenerations " will be shown while they will not be seen when 

 the dye is acid. 



To use the stain the preparation is flooded with the dye for a few seconds 

 and then washed with water. Acidophile granules are differentiated by allowing 

 the stain to act for about 15 seconds. The strength of the stain may be altered 

 by varying the amount of alcohol. The red corpuscles stain pink, eosinophile 

 granules more or less red, nuclei of leucocytes from blue to purple, and granules 

 of the mast cells most prominently metachromatic, malarial parasites as by 

 Plehn's solution, the nucleus of the young form being well shown. Blood 

 platelets are also stained. 



Giemsa (1902) considers the neutral precipitate from the eosin and methy- 

 len blue mixture isolated by Renter to consist of eosin salts of methylen blue, 

 methyl violet and azure, the two latter being impurities of the methylen blue. 

 He has prepared a stain of eosin and pure azure which he says gives a clear and 

 sharp staining with all the differentiations of the Romanowsky-Nocht stain. His 

 formula is as follows : 



0.05 per cent, solution of eosin (Hdcht), - - 19 c. c. 

 0.8 per cent, solution of azure, - . - . 1 c. c. 



Stain the preparations a few minutes and then wash them with water. 



Wright (1902) has modified Leishman's method. His staining solution is 

 prepared as follows : To a )4 per cent, solution of sodium bicarbonate in water 

 add 1 per cent, of methylen blue (Griibler). This mixture is steamed in an 

 Arnold steam sterilizer for one hour, and when cool one to one thousand solution 

 of eosin (Griibler, yellowish, soluble in water) is added until the mixture, losing 

 its blue color, becomes purple in color and a scum with yellowish metallic luster 

 forms on the surface, while, on close inspection, a fine-granular black percipitate 

 appears in suspension. (This requires about 500 c. c. of the eosin solution for 



