APPENDIX 339 



sionally used in place of the aniline gentian-violet, and there are 

 other slight modifications of the method. 



Ziehl-Neelsen Method. Here the primary stain is a solution of 

 carbol-f uchsin : 



Fuchsin i part 



Absolute alcohol lo parts 



5 per cent, aqueous solution of carbolic acid. . . loo parts 



It is best to heat the dye in a sand-bath, in order to distribute 

 the heat evenly. The various stages in the staining process are 

 as follows: {a) The cover-glass with the dried film upon it is 

 immersed in the hot stain for one to three minutes, ij?) Remove 

 the cover-glass from the carbol-fuchsin, and place it in a capsule 

 containing a 25 percent, solution of sulphuric acid to decolourise 

 it. Here its redness is changed into a slate-grey colour, {c) 

 Wash in water, and alternately in the acid and water, until it is 

 of a faint pink colour. (^) Now place the cover-glass for a 

 minute or two in a saturated aqueous solution of methylene-blue, 

 which will counter-stain the decolourised ground substance blue. 

 {e) Wash in water. (/) Dehydrate by rinsing in methylated spirit, 

 dry, and mount. A pure culture of bacteria will not necessarily 

 require the counter-stain (methylene-blue). Sections of tissue 

 may require twenty to thirty minutes in the primary stain (carbol- 

 fuchsin). This stain is used for tubercle and leprosy. With a 

 little practice the staining of the bacillus of tubercle when present 

 in pus or sputum becomes a very simple and accurate method of 

 diagnosis. A small particle of sputum or pus is placed between 

 . two clean cover-glasses and thus pressed between the thumb and 

 finger into a thin film. This is readily dried and stained as 

 above, the bacillus of tubercle appearing as a delicately-beaded 

 red rod with a background of blue. 



Bacteriological Diagnosis. The following points must be ascer- 

 tained in order to identify any particular micro-organism: 



(i) Its morphology, bacillus, coccus, spirillum, etc.; the pres- 

 ence or absence of involution forms. 



(2) Motility by the unstained cover-glass preparation (** hang- 

 ing drop "); note presence of flagella. 



