and Laboratory Methods -177 



Neutrophiles, nucleus takes the stain quite darkly, granules either a dull pink- 

 ish color or a bright red, "denoting that they are slightly acidophile.'" Myelo- 

 cytes take a faint blue stain. Nuclei of normoblasts take the basic stain. Mala- 

 rial plasmodim stains bluish green with pigment granules in contrast. 



3. Picric Acid^Rosaniline. — Ehrlich .• This dye is insoluble in cold water, 

 soluble in alcohol or glycerin. A glycerin solution gives in 12 to "24 hours 

 fairly satisfactory results. A quicker staining can be obtained by allowing a hot 

 water solution to drop from a filter on the heated dry preparation for several 

 seconds, wash in water, dry and mount. The neutrophile granules stain quite 

 well, the acidophile granules are easily recognized but are not so well stained as 

 by the glycerin solution. 



4. The Triple Stain of Ehrlich and its Modifications. — Ehrlich in 1883 pub- 

 lished his first formula for his acid fuchsin-orange G. -methyl green neutral com- 

 pound. This formula was as follows : To 300 c. c. of a mixture of equal parts 

 of a saturated solution of methyl green, acid fuchsin and orange G, add 60 to 

 90 c. c. of alcohol. This solution is to be employed only after long standing. 

 Nuclei stain greenish, erythocytes orange, acidophile granules very dark (red) 

 and neutrophile granules violet. 



There have been many modifications of Ehrlich's triple stain. 



Biondi (1888) prepared at room temperature saturated watery solutions of 

 orange G, acid fuchsin and methyl green (100 c. c. of water will dissolve about 15 

 grams of orange, 60 grams fuchsin and 10 grams of methyl green). The fluid 

 was shaken often and allowed to stand several days, then filtered. They were 

 then mixed, 10 parts of the orange G solution, 12 parts of the acid fuchsin and 

 3 parts of the methyl green solution. They must be mixed in order given, and 

 the methyl green added drop by drop with constant stirring. This mixture 

 stains dry preparations in a few minutes. 



Heidenhain (1888) modified Biondi's solution. 100 c. c. of orange G is 

 mixed with 20 c. c. of acid fuchsin, and 50 c. c. of methyl green added with 

 constant stirring. 



The Biondi Heidenhain triple stain can be obtained prepared in the pow- 

 der at dealers in stains. 



Bergonzini (1890) used a mixture of a basic dye (methyl green) and two acid 

 dyes (Wieget's acid fuchsin and gold orange after Griesbach). 20 grams of each 

 of these dyes were dissolved in 100 grams of distilled water. One part of the 

 red solution, two parts of the green solution and two parts of the yellow solution 

 were mixed, then filtered through cotton. The resulting dark brown fluid 

 remained good many months. 



Aronson and Philipp (1892) used the following modification of Ehrlich's 

 triple stain for the study of leucocytes in sputum : 



Saturated aqueous solution of orange G (extra) - 55 



" " " " acid rubrin - 50 



Distilled water - - - - - - 100 



Alcohol - - 50 



