NEW METHOD FOR GRAM STAIN 165 



tion of the cell wall but may be due in part to the presence of 

 acid. 



The application of these facts in the study of slow growing 

 organisms is obvious. Its value in the examination of pus and 

 body fluids is also evident and has been discussed in a separate 

 paper (Burke, 1921). 



The sodium bicarbonate solution may be omitted from the 

 Gram stain if the best dyes are used. We have found that 3 

 to 8 drops of a 5 per cent solution of sodium bicarbonate solution 

 is usually sufficient to insure good results. If too much sodium 

 bicarbonate solution is added a heavy precipitate forms and there 

 is an almost complete separation of the dye from the water. This 

 should be avoided. A film forms where a drop of strong sodium 

 bicarbonate hits the dye. This disappears as the dye and sodium 

 bicarbonate are thoroughly mixed. 



Sodium bicarbonate should not be added to the stock solution 

 of the stain as there results a rapid breaking down of the solution. 

 Whether the sodium bicarbonate is just as effective when added 

 to the iodine solution was not determined. 



EFFECT OF WATER ON DECOLORIZATION 



The presence of water on the slide to which the decolorizer 

 is added has a marked effect on the rate and extent of decoloriza- 

 tion. The rate and degree of decolorization in the presence of 

 water depends to some extent, according to the physical concep- 

 tion of the reaction, upon the action of the water on the cell wall 

 of the Gram positive organisms and upon the fact that the precip- 

 itate formed by the dye and the iodine goes into solution in the 

 decolorizer more rapidly if kept moist than when allowed to 

 dry. It follows that in making a stain it is advisable to remove 

 as much water as possible from the slide without allowing the 

 dye precipitate to become dry before adding the decolorizer. 

 Also that after decolorization of the Gram negative organisms 

 is complete water or an aqueous counter stain should not be 

 added to the sUde until the decolorizer has evaporated or been 

 removed. The addition of water to the decolorizer decreases its 

 power to take up the dye precipitate into solution and increases 

 its rate of decolorization of the Gram positive organisms. This 



