MICROSCOPIC STUDY AND STAINING 109 



its originator, the following being the formula given by him in 1904: 

 The substance referred to as az'ur II and purchasable 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:' 1 



Azur II-eosin 3 gms. 



Azur II 8 gms. 



This mixture is thoroughly dried over sulphuric acid in a desiccator, 

 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 G. This mix- 

 ture 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 ex- 

 udates containing bacteria, ten ,to fifteen minutes are sufficient. For 

 the staining of Negri bodies or Spirochsete pallida, one or more hours 

 of staining should be employed. After staining, wash in running tap 

 water and blot. 



WOOD'S METHOD. 2 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. 



When an intense stain is desired, the solution of eosin and azur II 

 may be flooded over the preparation together, using an excess of azur 

 II. They are then left on from five to ten minutes. At the end of this 

 time washing and drying as before completes the process. 



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

 purchasable under the name of "Giemsa Losung fur Romanowsky Farbung." 

 * Wood, Med. News, 83, 1903. 



