102 APPLIED BACTERIOLOGY 



Gram's Method. — This is one of the most valuable and 

 widely-used differential staining processes. Gram's method 

 is used as an aid to the diagnosis of a large number of 

 micro-organisms. The process can be applied equally well 

 to cover-glass preparations and to sections. The cover-glass 

 or section is first stained with aniline gentian violet, and 

 then decolourised with iodine solution. A precipitate is 

 formed with the colouring matter, which adheres to the 

 organisms, but can be easily washed out of the tissues. 

 The bacillus of cholera, typhoid, glanders, the spirilla of 

 recurrent fever, the gonococcus and the Pneumococcus 

 Friedlanderi are amongst the organisms which yield up 

 their colour, and therefore do not stain by Gram's 

 method. 



The process is as follows : (1) Stain the cover-glass or 

 section in Ehrlich's aniline gentian violet for ten minutes. 

 (2) The section is then immersed without washing in iodine 

 solution (1 gramme of iodine and 2 grammes of potassium 

 iodide are dissolved in 300 c.c. of water) for one to two 

 minutes. (3) Wash in alcohol until no more colour comes 

 away. (4) Counter-stain in an aqueous solution of eosine. 

 (5) Dehydrate in alcohol. (6) Clear in xylol or oil of cloves, 

 transfer to the slide with the section-lifter, lay out flat, 

 blot off the excess of oil, add a drop of balsam, and mount. 



Gram-Giinther Method. — Giinther has modified Gram's 

 original method by giving the preparation a washing with 

 a 8 per cent, solution of hydrochloric acid for a few seconds 

 after the first alcoholic washing. By this treatment cleaner 

 and brighter preparations are said to be obtained. Botkin 

 recommends that the section should be washed in aniline 

 water, after staining with gentian violet, and before im- 

 mersing in the iodine solution. 



It is very important to note that every pigment is not 

 suitable for this method. Fuchsine, methylene blue, and 



