180 VICTOR BURKE 



clear. Proper control of this process will reduce the amount of 

 decolorizer used to a minimum. Placing the decolorizer in a 

 Coplin jar and dipping the slide up and down in it will not give 

 satisfactory results. 



F. Drying after the decolorizer is essential as the aqueous 

 counter stain mixing with the decolorizer has an effect on the 

 Gram positive organisms. The smear can now be exainined for 

 Gram positive organisms. The oil can then be washed off with 

 xj'lene, the slide dried and the counter stain applied. 



G. Various counter stains can be used but I prefer Safranin 

 O, or neutral red as recommended by Jensen. With dyes giving 

 a poor Gram reaction it is necessary to reduce the counter stain- 

 ing to a minimum. 



H. Washing should be sufficient to remove the dye from the 

 surface of the organisms, flushing for a few seconds will suffice. 



CONCLUSIONS 



1. Steaming the iodine solution on the slide or increasing the 

 period of exposure does not cause the Gram negative organism 

 to resist decolorization. 



2. Drying the film after exposure to the iodine solution greatly 

 delays decolorization of the Gram negative organisms. 



3. There are two critical steps in the Gram staining method; 

 (a) the removal of the water after the iodine solution and (b) the 

 decolorization. The control of these steps determines to a 

 large extent the amount of differentiation between the Gram 

 positive and Gram negative organisms. 



4. The addition of water to the decolorizer, either on the slide 

 or in the bottle, retards the decolorization of the Gram negative 

 organisms and increases the rate of decolorization of the Gram 

 positive organisms. 



5. Acetone is superior to absolute alcohol as a decolorizer. 

 It decolorizes the Gram negative organisms and debris on the 

 slide much more rapidly and the Gram positi\"e organisms more 

 slowly than absolute alcohol. 



6. Mounting the fihns in distilled water, tap water or physio- 

 logical salt solution does not affect the staining reaction. Mount- 



