NEW METHOD FOR GRAM STAIN 167 



decolorization of the Gram positive organism and the skilful 

 control of the decolorizer. With the best dj'es it is not necessary 

 to pay special attention to this process although the excess water 

 should always be removed by blotting. 



There are then, in so far as water is concerned, three facts 

 which should be understood, controlled and utilized in the making 

 of a Gram stain: 1, Water added to the decolorizer increases its 

 power of decolorizing the Gram positive organisms; 2, Water 

 added to the decolorizer slows down the rate at which the dye- 

 iodine precipitate is taken into solution; 3, A dye-iodine pre- 

 cipitate goes into solution before drying much more rapidly than 

 after drying. 



Since a small amount of water must be left in the film and the 

 addition of water to the decolorizer affects the results it is in- 

 advisable to use the decolorizer more than once as is sometimes 

 done when decolorization is brought about by placing slides in 

 Coi)lin jars. Also the decolorizer takes up but a small amount 

 of the precipitate and quickly becomes saturated and then 

 ceases to decolorize the Gram negative organism. 



EFFECT OF THE IODINE SOLUTION 



The addition of the iodine solution causes a heavy precipita- 

 tion of the dye. This precipitate is insoluble in water, but 

 readily soluble in alcohol or acetone and has no staining affinity 

 for cells. It is washed out of the Ciram negative organisms 

 more rapidly than out of the Gram positive organisms. It is 

 washed out of the Gram negative organisms more rapidly and 

 from the Gram positive organisms less rapidly than the unpre- 

 cipitated dye. According to the physical conception of the 

 Gram reaction, the molecules of the Gram precipitate are of 

 such a size that m solution in alcohol or acetone and in the 

 absence of water they do not as readily pass throiigh the limiting 

 membranes of Gram positive as of Gram negative organisms. 

 Neide (quoted by Benians, 1912) states that the potency of the 

 Gram reaction depends largely on the strength of the iodine 

 solution and on the length of the period of application. By 

 using a solution containing double the amount of iodine present 



