100 APPLIED BACTERIOLOGY 



case of sputum). The stain must be warmed until steam 

 just rises ; it must on no account be allowed to boil, other- 

 wise a precipitation of the colouring matter will take place. 

 If preferred, the cover-glass can be held in a pair of forceps, 

 and a few drops of the warm stain dropped on from a 

 pipette ; the stain is kept warm by very gentle heating over 

 a flame. The cover-glass is now well washed in water, 

 and then held in 25 per cent, sulphuric acid until just 

 decolourised ; the cover-glass is again well washed in water, 

 and then counter-stained in Loffler's methylene blue for 

 thirty seconds, again washed in water, dried between filter- 

 paper, warmed slightly until quite dry, and, lastly, mounted 

 in balsam. 



The tubercle bacilli will be stained red, and the lung 

 debris dark blue. 



The method for staining the tubercle and leprosy bacillus 

 in section by this process is as follows : (1) Stain the 

 sections in warm carbol-fuchsine solution for ten minutes. 

 (2) Einse in water. (3) Decolourise in 25 per cent, sulphuric 

 acid and water, transferring from one to the other alter- 

 nately until decolourised. (4) Einse in water. (5) Counter- 

 stain in Loffler's methylene blue solution for three minutes. 

 (6) Dehydrate in absolute alcohol. (7) Clear in xylol or oil 

 of cloves for five minutes, transfer to slide, blot off excess of 

 clearing agent, and mount with a drop of balsam. 



Grain'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. 



