158 Methods of Observing Micro-organisms 



Counterstain if desired; wash off the counterstain with water; 



Dry; 



Mount in Canada balsam. 



NicoUe* suggests the following modification of the Gram 

 technic : 



(o) For Cover-glass Specimens: 



1. Stain for one to five minutes in a warm solution made as follows: 10 cc. 



of saturated alcoholic sojution of gentian violet, 100 cc. of a i per cent, 

 aqueous solution of carbolic acid. 



2. Immerse from four to six seconds in the iodine-iodide of potassium solu- 



tion, 



3 . Decolorize in a mixture of 3 parts of absolute alcohol and 1 part of acetone. 



4. Counterstain if desired. 

 (i) For Sections: 



1. Stain the nuclear elements of the tissue with carmine. For this NicoUe 



prefers Orth's carmine solution (5 parts of Orth's carmine with 1 part 

 of 95 per cent, alcohol). 



2. Stain in the carbol-gentian violet, as indicated above. 



3. Immerse for four to six seconds in the iodine-iodide of potassium solution. 



4. Differentiate with absolute alcohol containing 0.33 per cent, (by volume) 



of acetone. 



5. Treat with 95 per cent, alcohol containing some picric acid until the 



tissue is greenish yellow (one to five seconds), 



6. Dehydrate with absolute alcohol. 



7. Clear with xylol or other appropriate reagent. 



8. Mount in balsam. 



Eosin and Methylene-blue (Mallory) make a beautiful contrast 

 tissue stain for routine work, and also demonstrate the presence 

 of most bacteria. The success of the method seems to depend largely 

 upon the quahty of the reagents used and a careful study of their 

 effects. Hardeniag in Zenker's fluid is highly recommended as a 

 prehminary. The details as given by Mallory are as follows: 



1. Stain paraffin sections in a 5 to 10 per cent, aqueous solution of eosin 



from 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, car- 



bonate of potassium i, water 100) diluted i : 10 with water, from 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 cyto- 

 plasm, etc., red. 



Zielerf recommends for the staining of the typhoid, glanders and 

 other difficultly stainable bacteria, the following method of demon- 

 stration in the tissues: 



* "Ann. de I'Inst. Pasteur," 1895, EC. 



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



