322 KENNETH N. ATKINS 



Wash thoroughly before decolorizing in alcohol for two minutes or until the al- 

 cohol is no longer colored. In thick smears, as pus, the decolorization may be 

 continued for ten minutes. 



Wash. Counterstain. Wash, dry and mount. 



In this proposed modification aniline sulphate is used in place 

 of aniline and sodium hydroxide is added to the iodine solution. 

 Otherwise there is no change in the solutions. Aniline sulphate 

 is a stable salt, both in its dry form and in solution. The ani- 

 line-sulphate-gentian-violet stain should keep indefinitely.' The 

 instant that the modified iodine solution is placed on the slide 

 preparation, stained with aniline-sulphate-gentian-violet, the 

 aniline sulphate is decomposed and free aniline liberated. The 

 effect then, is the staining of the bacteria according to Gram's 

 classical method with a solution that is prepared as used. 



The addition of a weak solution of sodium hydroxide to iodine 

 causes the formation of sodium-hypoiodite. In the solution 

 above, the amount of sodium hydroxide is not sufficient to con- 

 vert all of the iodine to sodium-hypoiodite and leaves an excess 

 of iodine in solution. The amount of free iodine present is about 

 the same as that present in the iodine solution of the Gram stain. 

 This solution contains sufficient hydroxyl-ion concentration to 

 set free aniline from aniline sulphate. 



Other aniline salts were tried, such as aniline acetate, oxalate, 

 nitrate and hydrochloride. The aniline acetate was discarded be- 

 cause of its instability. Difficulty was experienced in the use of 

 aniline nitrate and aniline hydrochloride. The bacteria did not 

 take the color of the gentian violet properly and the finished 

 preparation was decidedly lacking in brilliancy. Apparently 

 there was a decided difference in the penetrating power (if 

 the term may be properly used), of the aniline salts. Aniline- 

 oxalate-gentian-violet seemed to penetrate, i.e., stain the bac- 

 terial cells in the primary step of the Gram stain, fully as well 

 as aniline-gentian-violet. Aniline-sulphate-gentian-violet also 

 penetrated well but specimens stained with aniline hydrochloride 

 and aniline-nitrate-gentian-violet and washed, dried and exam- 

 ined before the addition of the iodine solution, appeared a light 

 purple color instead of a dark purple. This suggests the testing 



