APPENDIX ON PROTOZOOLOGY 751 



continued for five to ten minutes, and the film is finally washed with 

 water. 



Gienisa's Stain. This should be procured ready made. Azure II 

 is a mixture of methylene azure and methylene blue. (Methylene 

 azure is sometimes known as Giemsa's Azure 1.) The formula given 

 by Giemsa himself in 1912 is : 



Azure II-eosin ... ... ... ... 3*0 grm. 



Azure II ... ... ... ... ... cr8 ,, 



Glycerine, pure ... ... ... ... I25'O ,, 



Methyl alcohol, pure ... ... ... 375'O ,, 



The film is first fixed in absolute alcohol. The proportion of 

 stain usually used is one drop of stain to i c.c. of water. Stain for 

 about ten minutes and then wash in water. 



The details of the application of the Giemsa stain to films fixed 

 wet and to sections must be sought in larger works on technique. 

 These works should also be consulted for information regarding 

 the use of Pappenheim's Panchrome mixture. 



There are numerous formulae of stains containing ripened Hccnia- 

 toxylin or its essential principle, Hcematein. A mordant is necessary, 

 one of the alums being usually employed. The mordant may be 

 included as an ingredient in the staining mixture, or it may be used 

 separately as in the case of the so-called iron-haematoxylins, \vherein 

 ferric ammonium alum is used separately and is followed by staining 

 with haematoxylin or hasmatein. A few of these stains of general 

 application may now be mentioned. 



Delafteld's (or Grenadier's) Hcematoxylin. 



Haematoxylin crystals ... ... ... 4 grm. 



Absolute alcohol ... ... ... .., 25 c.c. 



Saturated aqueous solution of ammonia-alum ... 400 ,, 



Mix these ingredients, and leave exposed to light and air for three to four days. Filter 

 and add 



Glycerine ... ... ... ... ... 100 c.c. 



Methyl alcohol ... ... ... ... ico ,, 



Allow the mixture to stand until the colour is sufficiently deep, 

 then filter and place in a stoppered bottle. The solution should be 

 allowed to ripen for at least two months before use. Dilute aqueous 

 solutions of the stain are of service for films and for sections. A 

 trace of acetic acid may be added at the moment of use, for sharp 

 differentiation. 



Ehrlich's acid haematoxylin, Mayer's haemalum, and Mayer's 

 glychaemalum are also useful. Their formulae will be found in larger 

 works. 



