108 STAINING METHODS 



5. Wash thoroughly in water. 



6. Counterstain in eosin, i per cent, aqueous solution. 



7. Wash. 



8. Dry and mount. 

 (Spores blue, bacilli red.) 



DIFFERENTIAL METHODS OF STAINING. 



Gram's Method. This method depends upon the 

 fact that the protoplasm of some bacteria permits 

 aniline gentian violet and Lugol's iodine solution, when 

 applied consecutively, to enter into a chemical combina- 

 tion which results in the formation of a new blue-black 

 pigment, only very sparingly soluble in absolute 

 alcohol. Such organisms are said to " stain by Gram,'* 

 or to be "Gram positive." 



1. Prepare a cover-slip film and fix in the usual way. 



2 . Stain in aniline gentian violet three to five minutes. 

 Filter as much aniline water on to the cover-slip as 



it will hold ; then add the smallest quantity <3f alcoholic 

 solution of gentian violet which suffices to saturate the 

 aniline water and form a "bronze scum" upon its 

 surface if too much of the alcoholic gentian violet is 

 added the alcohol present redissolves this scum. 



To prepare aniline water,- pour 4 or 5 c.c. aniline oil into a 

 stoppered bottle and add distilled water, 100 c.c. Shake vigour- 

 ously and filter immediately before use. The excess of oil sinks 

 to the bottom of the bottle and may be used again. 



3. Wash in water. 



4. Treat with Lugol's iodine solution until the film 

 is black or dark brown. 



To do this treat with iodine solution for a few seconds, 

 wash in water, and examine the film over a piece of 

 white filter paper. Note the colour. Repeat this proc- 

 ess until the film ceases to darken with the fresh appli- 

 cation of iodine solution. 



Lugol's solution is prepared by dissolving 



Iodine i gramme 



Iodide of potassium 3 grammes 



In distilled water 300 c.c. 



