COMPOUND STAINING 59 



stain is then dropped in from a drop bottle. The dilute solution 

 should be filtered on to the preparation and allowed to remain in 

 contact for from two to five minutes according to the potency of 

 the stain. For this filtration a convenient little apparatus is shown 

 on Fig. 7. It consists of a piece of stout copper wire turned to 

 a circle at each end and with an over-all length when finished of 

 about 5 inches. When required for use a circular filter paper of 

 the smallest size is folded and inserted in one of the loops as shown. 

 The ring will be found to grasp the paper better if the inner surface 

 of the loop has been slightly roughened by means of a blunt knife 

 or a file. 



Compound Staining and Special Stains 



(«) Gram's method of staining* {Nicolles modification) — 

 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, 



AH bacteria then can be grouped in two great classes, viz. : — 



1. Those which retain the stain when treated by the method of 

 Gram. 



2. Those which do not. 



In the original method of Gram, aniline-gentian-violet was the 

 stain invariably employed, but this is gradually being discarded in 

 favour of Nicolle's carbol-gentian-violet as more stable and of 

 equal efficacy in result 



The staining fluid consists of 10 c.c. of saturated alcoholic 

 solution of gentian-violet added to 100 c.c. of i per cent, carbolic 

 acid solution. 



The mordant is composed as follows : — 



Gram's Iodine Solution. 

 Iodine ...... i gramme. 



Potassium iodide . . . . .2 grammes. 



Distilled water ..... 200 c.c. 



The film is prepared and fixed in the usual manner. The pro- 

 cedure then is as follows : — 



k 



