SPECIAL STAINING 185 



"Gram-positive" may in part or wholly lose their "Gram- 

 positive" properties. 



Two theories, one chemical the other physical, have been 

 offered in explanation of the mechanism of the Gram method 

 of staining. In the chemical theory it is believed that, 

 through the intervention of the iodine, the gentian-violet 

 is linked inseparably to the protoplasm of "Gram-positive" 

 bacteria and is not so linked in the "Gram-negative" species. 

 The physical theory assumes differences in permeability 

 of either the bacterial envelope or the bacterial protoplasm. 

 In those species that are highly permeable the precipitation 

 resulting from the interaction between the iodine and the 

 gentian-violet occurs so deeply within the bacterial structure 

 that it is not readily washed out by the final alcohol bath, 

 this would be the case with the "Gram-positive" species; 

 while in the case of the "Gram-negative" species, assumed 

 to be less permeable, the precipitation is upon their sur- 

 faces and is readily removed by the final rinsing in alcohol. 



Glacial Acetic Acid Method. Another method that may 

 be employed for demonstrating the presence of the cap- 

 sule surrounding certain organisms is to prepare the 

 cover-slips in the ordinary way, then cover the layer 

 of bacteria upon them with glacial acetic acid, which 

 is instantly poured off (not washed off with water), and 

 the aniline-water gentian-violet solution dropped upon 

 them; this is allowed to remain three or four minutes, is 

 poured off, and a few drops more are added, and lastly the 

 slip is washed in a solution of sodium chloride of from 0.6 

 to 0.7 per cent, in strength; but at times it must be stronger, 

 occasionally as concentrated as 1.5 to 2 per cent. The reason 

 for this is that if the slips be washed in water, or in salt- 

 solution that is too weak, the mucin capsule that has been 



