128 BIOLCKJY AND TECHNIQUE 



spirilla, protozoa, and Negri bodies. The stain has been modified 

 several times by its originator, the following being the formula, given 

 by him in 1904: The substance referred to as azur II and purchas- 

 able under that name, consists of pure methylenazur chloralhydrate 

 combined with an equal quantity of methylene-blue chloralhydrate. 

 The substance referred to as azur II-eosin is a combination of this 

 substance with eosin. 



The staining fluid is made up as follows : 35 



Azur II-eosin 3 gins. 



Azur II 3 gms. 



This mixture is thoroughly dried over sulphuric acid in a desic- 

 cator, finely powdered, and rubbed through a fine sieve. It is then 

 dissolved in 250 gms. of C. P. glycerin (Merck), at 60 C. To this 

 is added methyl alcohol (Kahlbaum) 250 c.c., previously warmed 

 to 60 C. This mixture is well shaken and allowed to stand at room 

 temperature for twenty-four hours. The mixture is now ready for 

 use. 



For use 10 c.c. of distilled water are poured into a test tube and 

 one to two drops of a one per cent potassium carbonate solution 

 are added. Ten drops of the staining solution described above (one 

 drop to the c.c.) are mixed with this slightly alkaline water. The 

 preparation which is to be stained is fixed in methyl alcohol, dried, 

 and covered with the diluted staining solution. For the staining 

 of protozoa and exudates containing bacteria, ten to fifteen minutes 

 are sufficient. For the staining of Negri bodies or Spirochgete pallida, 

 one or more hours of staining should -be employed. After staining, 

 wash in running tap water and blot. 



WOOD'S METHOD. 36 "Wood has devised a simple staining method 

 based on the principles of the Giemsa stain, in which azur II and 

 eosin may be used in separate solutions. Preparations are fixed in 

 strong methyl alcohol for five minutes and are then stained in a 

 0.1 per cent aqueous solution of eosin until the preparation is pink. 

 The eosin is then poured off and the preparation is covered with a 

 0.25 per cent aqueous solution of azur II for one-half to two minutes. 

 Following this, it is washed in tap water and dried by blotting. 



35 It is best not to attempt to make up the undiluted staining fluid, since this 

 is purchasable under the nume of "Giomsa Losnng fiir Romanowsky Farbung. " 

 38 Wood, Mod. News, 83, 1903. 



