192 STUDY OF INDIVIDUAL BACTERIA-STAINING 



compound is insoluble in alcohol, and other bacteria do not 

 contain this substance. Consequently when bacteria are 

 stained by Gram's method (given below), those that con- 

 tain this chemical remain colored, while if it is not present 

 the dye is washed out by the alcohol and the bacteria are 

 colorless and may be stained by a contrast stain. The bac- 

 teria which stain by this method are said to "take Gram's" 

 or to be "Gram-positive," while those that decolorize are 

 called "Gram-negative." The method is: 



1 . Prepare the film as above given. 



2. Stain with fresh anilin gentian violet 1 minute. 



3. Wash in tap water. 



4. Cover with Gram's solution (1 minute). 



5. Wash in tap water. 



6. Wash with 95 per cent, alcohol three times or until no 

 more color comes out. 



7. Dry and examine. 

 Gram's solution is: 



I 1 part 



KI . 2 parts 



H2O . . 300 parts 



This method is excellent for differentiating Gram-posi- 

 tive and Gram-negative organisms on the same slide. First 

 stain by this method and after washing with alcohol stain 

 with a counter-stain, carbol-fuchsin diluted ten to fifteen 

 times with water is excellent. The Gram-positive bacteria 

 are violet and the Gram-negative are red. 



It is also of great value in staining Gram-positive bac- 

 teria in tissues, but the sections should be stained about five 

 minutes in the anilin gentian violet and be left about two 

 minutes in the Gram solution. Sections are to be counter- 

 stained in Bismarck brown, dilute eosin or safranin solutions 

 and cleared in oil of bergamot, lavender or origanum and 

 not in clove oil or carbol-xylol, as these latter dissolve out 

 the dye from the bacteria. 



Staining of Spores in the Rod. — Prepare the films as usual. 

 Cover with carbol-fuchsin, using plenty of stain so that it 

 will not dry on the slide ; heat until vapor arises, not to boil- 

 ing; cool until the stain becomes cloudy and heat again 



