NEW METHOD FOR GRAM STAIN 173 



alcohol decolorizes the Gram positive organisms very rapidly 

 a solution of the Gram precipitate in 75 per cent alcohol should 

 stain the Gram positive organisms almost as rapidly as the 

 gonococcus like organisms. If this dye-iodine compound could 

 be forced to remain in the gonococci we could stain the films with 

 a weak counter stain which would stain the staphylococci and 

 we woiild have the phenomenon of a reversed Gram stain. The 

 practical application of such a staining method in the examina- 

 tion of pus for gonococci is obvious. 



Unfortunately the addition of iodine to the dye saturates its 

 affinities so that an alcohol or acetone solution of the dye-iodine 

 precipitate does not stain cells. By simply exposing films of 

 organisms to the solution we can not determine whether the 

 molecules of the precipitate have penetrated into the gonococci 

 and not into the staphylococci. The staining power of the solu- 

 tion can be materially increased by the addition of alkaU but 

 we do not know what affect this has on the size of the molecules. 

 If the molecules are reduced in size they should enter the Gram 

 positive as well as the Gram negative organisms. If the size 

 of the molecular groups is not altered by the addition of the alkali 

 then the gonococci should be penetrated and stained and the 

 staphylococci not penetrated and not stained or only the surface 

 stained. Exposure to a counter stain or weak decolorizer and 

 counter stain should stain the staphylococci and not the 

 gonococci. 



ATTEMPT TO REVERSE THE GRAM PHENOMENON 



Experiment 1. Films of pure cultures of Staphylococcus 

 aureus, Neisseria catarrhalis and Bacterium typhosum were made 

 on a slide in physiological salt solution, air dried and fixed by 

 heat. A sufficient amount of iodine solution was added to a 

 quantity of an aqueous solution of methyl violet to cause a 

 maximum precipitation, the precipitate washed to remove excess 

 iodine and dried. The films were flooded with a saturated 

 alcoholic solution of this precipitate. A few drops of a strong 

 mixture of sodium bicarbonate, sodium phosphate and sodium 

 h3'droxide were added to the dye on the slide. After an exposure 



