33^ APPENDIX 



glass, drop by drop, until precipitation commences. Cover-glass 

 preparations are floated in this mixture for fifteen to thirty minutes, 

 then washed for a few seconds in dilute nitric acid (one part nitric 

 acid to two of water), and then rinsed in distilled water. The 

 stain is removed from everything except the bacilli; but the 

 ground substance can be after-stained brown if the bacilli are 

 violet, or blue if they have been stained red" (Crookshank, 

 Bacteriology and Infective Diseases^ p. 89). 



Gram' s Method. The primary stain in this method is a solu- 

 tion of aniline gentian-violet (saturated alcoholic solution of 

 gentian-violet 30 cc, aniline water 100 cc), which stains both 

 ground substance and bacteria in purple. The preparation is 

 next immersed in the following solution for half a minute or a little 

 more: 



Iodine i part 



Potassium iodide 2 parts 



Distilled water 300 parts 



In this short space of time the iodine solution acts as a mord- 

 ant of the purple colour in the bacteria, but not in the ground 

 substance. Hence, if the preparation be now (when it has as- 

 sumed a brown colour) washed in alcohol (methylated spirit), the 

 ground substance slowly loses its colour and becomes clear. But 

 the bacteria retain their colour, and thus stand out in a well- 

 defined manner. Cover-glass preparations decolourise more 

 quickly than sections of hardened tissue, and they should only be 

 left in the methylated spirit until no more colour comes away. 

 The preparation may now be washed in water, dried, and 

 mounted for microscopic examination, or it may be double- 

 stained, that is, immersed in some contrast colour which will 

 lightly stain the ground substance. Eosin or Bismarck brown 

 are commonly used for this purpose. The former is applied for 

 a minute or two, the latter for five minutes, after which the 

 specimen is passed through methylated spirit (and preferably 

 xylol also) and mounted. The result is that the bacteria appear 

 in a dark purple colour on a background of faint pink or brown. 

 Carbol-thionine blue, picro-carmine, and other stains are occa- 



