STAINING 205 



If it is desired to preserve the preparation it may then be 

 mounted in balsam. This is not necessary, as they keep 

 just as well, provided the immersion oil is removed. To 

 do this, fold a piece of filter paper so that at least three 

 thicknesses result. Lay this on the slide and press firmly 

 several times, when the surplus oil will be taken up by the 

 paper. Slides not mounted in balsam are more apt to 

 become dusty than those that are. This is the only disad- 

 vantage. 



Gram's Method of Staining.— It has been ascertained that 

 some bacteria contain a substance, possibly a protein, 

 which forms a compound with gentian violet and iodine, 

 which compound is insoluble in alcohol, and other bacteria 

 do not contain this substance. Consequently when bac- 

 teria are stained by Gram's method (given below), those 

 that contain 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 

 bacteria which stain by this method are said to "take 

 Gram's" or to be "Gram-positive," while those that decol- 

 orize are called "Gram-negative." The method is: 



1. Prepare the film as above given. 



2. Stain with fresh anilin gentian violet one minute. 



3. Wash in tap water. 



4. Cover with Gram's solution one 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 " 



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 counterstain, carbol-fuchsin diluted ten to fifteen 

 times with water is excellent. The Gram-positive bacteria 

 are violet and the Gram-negative are red. 



