STAINING METHODS 37 



c. Wash with water and decolorize with acetic alcohol (95 per 

 cent alcohol, two parts, 1 per cent acetic acid, one part) until only 

 a faint pink color remains. Finally wash thoroughly with water. 



d. Mount the cover glass in water and examine microscopi- 

 cally with the * objective. The spores should be red and the 

 rest of the film colorless or a very faint pink. If satisfactory, 

 pass on to section e ; if not, repeat steps b to d inclusive. 



e. Stain with weak methylene blue. 



f. Wash in water, dry, and examine under the microscope. 

 The spores should be red, the cells blue. Young spores are more, 

 readily decolorized than older ones. 



Exercise 62. Differential Staining 



1. Gram's method. This method depends upon the fact that 

 the protoplasm of some bacteria permits aniline-gentian-violet 

 and Gram's iodine solution, when consecutively used, to enter 

 into chemical combination, resulting in the formation of a blue- 

 black pigment which is practically insoluble in alcohol. Such 

 organisms are said to be Gram positive ; if the color is not held, 

 they are said to be Gram negative. 



a. Prepare aniline-gentian-violet as follows : 



Saturated alcoholic solution of gentian violet 11 cc. 



Absolute alcohol 10 cc. 



Aniline water l 100 cc. 



This solution does not keep well. 



b. Prepare Gram's iodine solution as follows : x 



Iodine 1 g. 



Potassium iodide , 2 g. 



Distilled water 300 cc. 



c. Prepare a cover-glass film and fix in the usual way. 



d. Stain with aniline-gentian-violet for three to five minutes. 



e. Drain off excess of stain and, without washing, cover the 

 film with Gram's iodine solution ; allow it to remain for one 

 to three minutes. The cover glass looks black at this point. 



1 Made by shaking together 5-6 cc. of aniline oil in 100 cc. of distilled water, 

 with subsequent filtering. 



