NEW METHOD FOR GRAM STAIN 161 



as anilin oil and phenol results in a loss in the stability of the 

 solution. The stability of the solution has been found to vary 

 with the method of mixing the ingredients together. The 

 original anilin gentian violet as described by Gram may deterio- 

 rate in a few days. Sterling's modified solution lasts several 

 months. Other modifications are said to keep indefinitely. 

 Perhaps the most promising of these is the one recently described 

 by Atkins (1920) in which anilin sulphate replaces the anilin water 

 in the primary stain and NaOH is added to the iodine solution. 



One of the objections to the use of the Gram stain has been 

 the unsatisfactory or unstable nature of this primary solution. 

 Until the discovery of Jensen (1912) that an aqueous solution of 

 methyl violet gives very satisfactory results it was assumed that 

 a mordant such as anilin oil was a necessary factor in making 

 a good Gram stain. Aqueous solutions of methyl violet are 

 coming into general use in Europe. That this has not occurred 

 in America is due to the difficulty of obtaining methyl violet 

 6b, as recommended by Jensen and to the fact that the American 

 dyes have given unsatisfactory results. With the production 

 of better domestic dye stuffs or improved methods of using the 

 present products we believe that the favored primary stain will 

 eventually be an aqueous solution which will be stable over a 

 long period of time. This stability, combined with ease of prep- 

 aration, are factors in its favor. 



The present paper describes the results of experiments to 

 compare aqueous solutions of various domestic dye stuflfs and 

 their value as substitutes for the anilin gentian violet solution 

 in the Gram staining method.^ The dyes used gave variable, 

 and in most cases such unsatisfactory results that the experiments 

 were extended to cover an analysis of the different factors deter- 

 mining the Gram reaction in the hope of modifying the staining 

 method in such a way that satisfactory results could be obtained 

 with more of the domestic dye products. The experiments 

 mcluded the determination of the effect of heat and of acid and 

 alkali added to the primary stain on the slide, the effect of 



' This work was begun by Mrs. Pearl M. Smith and continued by the writer. 



