160 VICTOR BURKE 



The control of the decolorizer requires experience. Some of 

 the methods used advocate exposure of the smear to the decolor- 

 izer for a definite period of time, other methods recommend 

 exposure to the decolorizer as long as any of the stain comes out. 

 The skilled technician modifies both of these methods as the 

 occasion arises. The time of exposure to the decolorizer must 

 vary with the type of smears to be examined and depends upon 

 the excellence of the violet stain and decolorizer used and upon 

 the nature of the treatment accorded the smear between the 

 removal of the iodine solution and the appUcation of the decolor- 

 izer. It has not heretofore been recognized that the extent of 

 the blotting and accompanying drying of the smear after the 

 iodine solution greatly influences the decolorization of the Gram 

 negative organisms. The process of blotting after the iodine 

 constitutes one of the two critical steps in the Gram stain and will 

 be discussed in detail later. Aside from the blotting after the 

 iodine and the control of the decolorizer, the balance of the 

 process can be carried on by an untrained assistant. 



No one method so far advanced has been shown to be dis- 

 tinctly superior to all the other methods. The Committee on 

 the Descriptive Chart of the Society of Bacteriologists has not 

 yet selected any one method as the standard method to be used 

 in the study and description of pure cultures. This committee 

 has presented three methods for the consideration and criticism 

 of the members of the Society. (Conn, H. J., 1919; Atkins, 

 K. N., 1920.) While not recommending any one complete 

 method it does recommend that 100 per cent alcohol be used as 

 the decolorizer, that the films be made with distilled water and 

 that the gentian violet, iodine solution and alcohol be drained 

 or blotted off but not washed off of the fihn. 



The different methods so far advanced vary in the making of 

 the solutions, the strength of the solutions and the time periods 

 of exposure. The most important modifications of the method 

 as presented by Gram have resulted from efforts to improve the 

 primary stain by increasing the penetration and intensity of the 

 stain and the permanency of the solution. In most cases in- 

 creasing the intensity of the stain by the use of mordants such 



