DIFFERENTIAL STAINING OF FILMS 209 



2. Without washing, replace the violet by a modified Gram's iodine solu- 

 tion made as follows : 



Iodine, 1 gram. 



Potassium iodide, - 2 grams. 



Distilled water, - - 200 c.c. 



Allow the solution to act for 4-6 seconds, renewing it once or twice during 

 that period. 



3. Wash in distilled water. 



4. Decolourize with an acetone-alcohol solution : 



Absolute alcohol, - 5 volumes. 



Acetone, - 1 volume. 



Decolourization does not take place at once and is only fully manifested 

 after washing in distilled water. 



5. Wash in distilled water. 



6. Stain the ground work rapidly with an alcoholic solution of eosin : 



Saturated solution of eosin * in 95 per cent, alcohol, - 1 volume. 



Alcohol (95 per cent.), - 2 volumes. 



7. Wash, dry, mount, and examine as before. 



(8) Merieux's method. In the author's experience this method has never 

 given results equal to those obtained with the methods already described. 



1. Stain with carbol-violet as in (y). 



2. Treat with the following solution for 4-6 seconds, renewing the solution once 

 or twice during that time : 



Iodine, 1 gram. 



Potassium iodide, - 2 grams. 



Saturated solution of eosin 2 in 50 per cent, alcohol, - - 20 c.c. 

 Distilled water, - - 200 



3. Wash in distilled water. 



4. Decolourize in a 1 in 6 solution of acetone in alcohol (vide supra). 



5. Wash, dry, mount and examine. 



(e) Kuhne's method. 1. Stain for several minutes in carbol-blue (p. 138) 

 or in ammoniacal blue (p. 139). 



2. Wash in distilled water. 



3. Treat with Gram's iodine solution for 2-3 minutes. 



4. Wash in distilled water. 



5. Decolourize with a saturated solution of fluorescein in absolute alcohol. 



6. When the ground-work no longer appears blue, wash in absolute alcohol 

 then in clove oil and xylol, and mount in balsam. 



The bacteria appear violet on a background lightly tinted with fluorescein. 



B. Claudius' method. 



This method as already described on p. 136 can be used for staining smear 

 preparations. 



C. Methods available for staining organisms which are gram-negative. 



1. Blood-films. 



In double staining blood-films containing gram-negative organisms use is 

 made of the property possessed by the red cells of combining with eosin, and 

 also of the marked selective affinity shown by bacteria for the basic aniline 

 dyes. 



Note. The three methods about to be described are the original methods. They 

 have undergone many improvements which, being specially adapted to work on 

 the Ifematozoa, will be considered in the chapter (LVIII.) dealing with these 

 organisms. 



1 Alcohol-soluble eosin. 2 Water-soluble eosin. 



O 



