GRAM'S METHOD 103 



the best result is desired. Unfortunately Gram's method 

 is not applicable for all organisms, as many do not retain 

 their colour by the process. This disadvantage, however, 

 is counter- balanced by the fact that it forms a valuable 

 means of distinguishing organisms, and is always one of 

 the points to be noted in bacteriological diagnosis. Most 

 of the moulds, yeast, streptothrix and sarcina forms, and 

 cocci stain by it, though there are exceptions ; the spirilla 

 and protozoa do not stain by it, but as regards the bacilli 

 no rule can be laid down (see p. 105). Films are stained 

 for five to ten minutes, and sections for ten minutes to 

 half an hour, in anilin- or carbol- gentian violet solution. 

 The superfluous stain is then drained or blotted off, not 

 washed away, the specimen is rinsed with Gram's iodine 

 solution and is treated with fresh iodine solution for from 

 one-half to two minutes. 



GRAM'S IODINE SOLUTION 



Iodine .... 1 part 



Potassium iodide ..... 2 parts 



Distilled water 300 parts 



The purple colour of the gentian violet changes to a 

 dirty yellowish- brown, and sections become much like 

 a used tea-leaf. The specimens must not be passed on 

 to the next solution until they have assumed the brown 

 colour. Cover- glass specimens are best immersed in the 

 solution in a watch- glass, film side up. 



The specimens are removed from the iodine solution, 

 drained, and then immersed in alcohol, preferably methyl- 

 ated spirit. In this the purple colour of the gentian 

 violet returns and is dissolved out, so that they ultimately 

 become colourless ; this is aided by moving them gently 

 about, and for sections two or more baths of alcohol may 

 be an advantage, a fresh one being substituted when the 

 first has become deeply coloured. Films decolorise much 

 more readily than sections, and they should be removed 



