INTRODUCTION 9 



(a) Saturated alcoholic solution of gentian violet, 30 c.c. 



Aniline water, 100 c.c. 



As this solution does not keep well, only a small amount 

 should be made up. 



The aniline water is obtained by thoroughly shaking up 4 c.c. 

 of aniline with 100 c.c. of water, and then filtering through moist 

 filter paper until the liquid is clear and free from any oily drops ; 

 any aniline which remains in the bottom of the flask or tube 

 after shaking should not be poured on the filter. 



(b) Iodine . . . . . i gr. 



Potassium iodide . . . . 2 gr. 



Water . ... . . 300 c.c. 



Dissolve the potassium iodide in about 10 c.c. of the water 

 and add the iodine : when the latter is dissolved add the rest of 

 the water. 



In using the above solutions for staining purposes, the follow- 

 ing procedure should be adopted : 



(1) Float the cover-glass, film downwards^ on aniline-gentian- 

 violet solution in a watch-glass, and allow the stain to act for 

 five minutes. 



Take up with forceps and drain off the superfluous stain by 

 dipping the edge of the cover-glass on blotting-paper. 



(2) Then, immerse the cover-glass for 1-2 minutes in the 

 iodine solution in a watch-glass, film side up. The purple 

 colour of the gentian violet should change to a yellowish brown 

 tint. 



(3) Now, remove the cover-glass, taking care to note on 

 which side the bacteria are ; drain off the superfluous liquid and 

 dip the cover-glass into a vessel containing absolute alcohol for 

 10 seconds, and into a second vessel of absolute alcohol for 

 10-15 seconds ; when no more of the gentian violet stain is seen 

 to wash off, take it out and wash it in water, and examine either 

 in water or after drying in Canada balsam. Too long immersion 

 in alcohol must be avoided. 



