Methods for Testing Stains 327 



aqueous solution of the ripened hematoxylin, rinsed in tap water, diflFerentiated 

 in the above iron alum solution, controlling the diflFerentiation by microscopic 

 examination, washed in running water about 5-10 minutes, counterstained with 

 0.1% eosin Y in 25% alcohol 2-5 minutes, dehydrated, cleared and mounted in 

 balsam. The criterion of a satisfactory sample is that the nuclei should appear 

 black. 



Ehrlich's method is as follows: Dissolve 2 g. hematoxylin in 100 ml. 95% ethyl 

 alcohol, and add 100 ml. distilled water, 3 g. AINH4 (804)2 -121120, 100 ml. glycerin, 

 and 10 ml. glacial acetic acid. Ripen by exposure to air and sunlight in a paper- 

 capped vessel for 4-6 weeks or immediately by addition of 0.4 g. NalOs. Sections 

 are stained 2-5 min. in this solution, washed in tap water until section appears 

 blue, stained 1 min. in 0.5% aqueous eosin Y, rinsed in water, dehydrated in 2 

 changes each of 95% and 100% alcohol, cleared and mounted in balsam. A satis- 

 factory sample should show dark blue nuclei. 



Orcein 



Identification: The exact formula of orcein is unknown. The following method of 

 identification is employed: Dissolve 50 mg. in 250 ml. of 0.01 N NaOH. Dilute 

 10 ml. of this solution to 200 ml. with 0.01 N NaOH. Read in Beckman spectro- 

 photometer. Absorption maximum 576-582 mpt; ratio P-15/P-|-15 is from 1.01 to 

 1.18. 



Method of analysis: Since the exact formula of orcein is unknown, determination 

 of dye content is impossible. 



Biological Tests: Orcein is tested as an elastic tissue stain on Zenker's, acetone, 

 formalin, or Bouin's fixed tissue. The paraffin is removed in the usual manner. The 

 sections are stained 30 min. in orcein (orcein, 0.4 g. in 70% alcohol + 1 ml. cone. 

 HCl), rinsed briefly in 70% alcohol, then in distilled water, stained 5 min. in diluted 

 methylene blue solution (methylene blue, 1 g.; borax, 1 g.; distilled water, 100 ml.; 

 for use, diluted with 9 vol. of distilled water), differentiated and dehydrated in a 

 dish of 95% alcohol to which has been added a few drops of 10% alcoholic rosin, 

 keeping the section in constant motion about 2 min.; dehydration is quickly com- 

 pleted with absolute alcohol, followed by clearing in xylene and mount in balsam. 

 A satisfactory sample should show even the finest elastic fibers either a dark purple 

 or reddish purple. 



Wright Stain 



Identification: Wright stain is a compound of eosin with an oxidized and partly 

 de methylated methylene blue. Because of its complexity no satisfactory chemical 

 methods have been yet worked out for its identification or analysis. 



Biological Tests: The dye is dissolved in methyl alcohol (1 to 600) and is applied 

 to a dried blood film for 1-3 minutes. Then 1 ml. of distilled water is added. After 

 standing twice as long as the undiluted stain, this solution is poured off and the 

 slide is washed with distilled water, until the thin portions of the stained film are 

 pink. The smear is dried by blotting carefully. A satisfactory sample should show 

 the following picture: erythrocytes, yellowish red; polymorphonuclear neutro- 

 philic leucocytes, with dark blue nucleus, reddish lilac granules, and pale pink 



