STAIN TECHNIQUE 69 



to the solution, the color produced by the stain is more reddish 

 violet. 



Iron Haematoxylin is J per cent Haematoxylin in water or 

 dilute alcohol. The mordant, 1 to 4 per cent ferric ammonium sul- 

 fate, is applied separately. Heidenhain gives detailed directions for 

 the preparation and use of the stain (Martin Heidenhain, "Noch 

 einmal iiber die Hamatoxylinfarben," Zeit. Wiss. Mik. 13: 186- 

 199 (1896)). He reports the application of iron Haematoxylin 

 by Benda on nervous tissue, by Biitschli on cytoplasm, and by 

 Sobotta on tissues fixed in vapors of osmic acid. Heidenhain 

 recommends 2J to 4 per cent ferric ammonium sulfate, because 

 weaker solutions decompose quickly. Paraffin section on slides are 

 placed vertically in the mordant six to twelve hours. The stain is 

 given as Weigert's solution containing 1 gram of Haematoxylin in 

 10 per cent alcohol. Four weeks are needed for the ripening, and 

 then, the stain is diluted with an equal volume of distilled water. 

 Tissues are stained 24 to 36 hours, rinsed, differentiated in 2^ per 

 cent ferric alum, and washed in running water 10 to 15 minutes. 



Iron Haematoxylin is applied in two solutions : the mordant 

 ferric ammonium sulfate and an aqueous stain. 



A. The mordant. 



Distilled water 100 ml. 



Ferric ammonium sulfate, clear 



lavender crystals 1 gram, or as much 



as 4 grams 



B. The stain. 



Distilled water 160 ml. 



Alcohol, 95% 40 ml., the preservative 



Haematoxylin 1 gram 



The stain is ripened, in a liter Erlenmeyer flask plugged with 

 cotton, at 60° C. for two days. The 1 per cent ferric alum should 

 not be kept more than three days because a trace of sulfuric acid, 

 which appears in dilute solutions of ferric alum on standing, pro- 

 duces a greenish Hematein pigment. 



A definite correlation exists between the fixatives, tissues, and 

 stains. Using alum Haematoxylin, a tissue fixed in formalin 



