208 THE EXAMINATION OF MATERIAL 



may be placed on the film and the preparation examined at once with an 

 oil-immersion lens. 



Films made on cover-glasses should first be examined in water, and if satis- 

 factory they can then be mounted in balsam after drying and clearing in 

 clove oil and xylol. 



In preparations stained as above the groundwork is pink and the micro- 

 organisms violet. Decolourization must be continued until all traces of 

 violet have disappeared from the groundwork (fig. 154). 



FIG. 154. Gram's stain. 

 Scraping from gum stained by Gram's method (oc. 2, obj. -n>th, Zeiss). 



Blood-films stained by Gram's method give very beautiful preparations. 

 When dealing with the blood of birds the action of the alcohol must be 

 continued until all but the nuclei of the red cells are decolourized and must 

 be stopped short of complete decolourization. so that after counter-staining 

 with eosin the protoplasm of the red cells is stained red while the nuclei of 

 the red cells and the micro-organisms are stained violet. 



Note. Vesuvin can be used as a counter-stain instead of eosin. 



Vesuvin, - 5 grams. 



Distilled water, - - 100 c.c. 



Micro-organisms which retain the stain by Gram's method are then stained deep 

 violet while gram-negative organisms and the nuclei of the leucocytes are dark 

 brown and the protoplasm of the leucocytes light brown. 



(ft) Method recommended. 1. Flood the film with carbol-gentian-violet 

 (p. 138). Stain for about a minute. 



2. Without washing, replace the violet with Gram's iodine and stain for 

 1-2 minutes. 



3. Wash in distilled water. 



4. Decolourize with absolute alcohol. 



Instead of using absolute alcohol alone the process may be hastened by washing first 

 with alcohol then with aniline oil and again with alcohol. But it should be pointed 

 out that aniline oil is a very powerful decolourizing agent and should therefore only 

 be allowed to act for a few seconds. 



5. Wash in distilled water. 



6. Counterstain with an aqueous solution of eosin as before. 



7. Wash, dry, mount and examine as above (a). 



(y) Nicolle's method. 1. Stain with carbol-gentian-violet (p. 138) for 

 20-30 seconds. 



