Staining 165 



the presence of most bacteria. The success of the method 

 seems to depend largely upon the quality of the reagents 

 used and a careful study of their effects. Hardening in 

 Zenker's fluid is highly recommended as a preliminary. The 

 details as given by Mallory are as follows: 



i; Stain paraffin sections in a 5 to 10 per cent, aqueous 

 solution of eosin for five to twenty minutes or 

 longer; 



2. Wash in water to get rid of the excess of eosin; 



3. Stain in Unna's alkaline methylene-blue solution 



(methylene-blue i , carbonate of potassium i , water 

 100), diluted i : 10 with water, for one-half to one 

 hour, or use a stronger solution and stain for a few 

 minutes only; 



4. Wash in water. 



5. Differentiate and dehydrate in 95 per cent, alcohol, 



followed by absolute alcohol until the pink color 

 returns in the section; 



6. Clear with xylol; 



7. Mount in xylol balsam. 



The nuclei and micro-organisms will be colored blue, the 

 cytoplasm, etc., red. 



Zieler* recommends for the staining of the typhoid, 

 glanders and other difficultly stainable bacteria, the follow- 

 ing method of demonstration in the tissues :- 



1. Fix and harden in Muller-formol solution. 

 Paraffin imbedding. 



2. Staining overnight in Orcein D. (Griibler), 0.1 



Officinal " schwef elsaure " (sulphuric acid), 2.0 



70 per cent, alcohol, 100.0 



3. Washing in 70 per cent, alcohol for a short time to re/nove 



the excess of orcein. 



4. Washing in water. 



5. Staining in polychrome methylene-blue ten minutes to two 



hours. 



6. Washing in distilled water. 



7. Thorough differentiation in glycerin-ether 1 : 2-5 water until 



the tissues become pale blue. 



8. Washing in distilled water. 



9. Seventy per cent, alcohol. 



10. Absolute alcohol. 



11. Xylol. 



12. Balsam. 



* "Centralbl. f. allg. Path. u. path. Anat." Bd. xiv, No. 14, p. 561. 



