APPENDIX 459 



away. 5. Wash in water, blot off superfluous water, and set aside to dry. 

 If thought desirable, the preparation may be counter-stained by the 

 application of a very weak solution of Ziehl-Neelsen. 



The method of Gram enables us to classify bacteria into two great 

 groups. Certain organisms when coloured with a basic stain in aniline 

 or carbolic acid solution, and afterwards treated with a special mordant of 

 which iodine is the base, resist decolorisation by means of absolute 

 alcohol or other like reagent. Others, on the contrary, when treated in 

 the same fashion, readily give up their stain and decolorise when treated 

 with such reagents. The Bacillus anthracis may be taken as a type of the 

 former, the Bacillus typhosus of the latter. 



Nicolle's Modification of Gram's Method, used in the staining of 



diphtheria bacillus, consists in substituting carbolic acid for aniline water, 

 and in the use of a stronger iodine solution, and of acetone in the 

 decolorising fluid. Take 10 c.c. saturated alcoholic solution of gentian- 

 violet, and add 100 c.c. of a 1 per cent, solution of carbolic acid. The 

 iodine solution consists of iodine 1 gramme, potassium iodide 2 grammes, 

 and distilled water, 200 c.c. Place the film in the stain for five minutes, 

 then pass directly into iodine solution for five seconds, and decolorise by 

 passing rapidly through a mixture of one volume of acetone with four 

 volumes of absolute alcohol. This removes all unfixed stains at once. The 

 specimen is then dehydrated in xylol, allowed to dry, and mounted in 

 balsam. 



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

 carbol-fuchsin : 



Basic fuchsin . . . . . . .1 gramme 



Absolute alcohol . . . . . . 10 c.c. 



Carbolic acid . . . . . .5 grammes 



Distilled water , - -.. . > .. . 100 c.c. 



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 : () The cover-glass with the dried film upon it is immersed 

 in the hot stain for one to three minutes. (6) Remove the cover-glass 

 from the carbol-fuchsin, and, after washing in water, place it in a capsule 

 containing a 33 per cent, solution of nitric acid to decolorise 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, (d) Now 

 place the cover-glass for a minute or two in a saturated aqueous solution 

 of methylene-blue, which will counter-stain the decolorised ground 

 substance blue, (e) Wash in water, (f) 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 a primary stain (carbol-fuchsin). 



This stain may be used for the bacillus of tubercle, with the 

 modification necessary to separate the bacillus of tubercle from other 

 organisms (leprosy, etc.) with similar staining properties. This modifica- 

 tion is to wash in absolute alcohol, after the carbol-fuchsin stain has been 



