MEDIA MAKING AND APPARATUS 277 



J 7 or spores.— Proceed as above, but in decolorizing use alcohol t 

 part, in water 3 parts, instead of acid alcohol. 



GRAM S STAIN. 



Prepare a saturated solution of gentian violet in alcohol and 

 jkeep as a stock solution. 



Prepare fresh each time anilin oil water thus : 



'Anilin oil 1 c.c. 

 .Water 50 c.c. 



Shake violently ; filter repeatedly until clear. Mix three parts 

 ■of the anilin oil water with one part of the gentian violet solution — 

 thus making " anilin-oil-gentian-violet." 



Process. 



1. Prepare smears as usual. 



2. Stain in anilin-oil-gentian-violet, \ x / 2 minutes. 

 '3. Wash in water; smear now deep violet. 



4. Wash with Gram's solution (iodin 1, potassium iodide 2 a 

 water 300) ; smear now purple black. 



5. Decolorize with successive small quantities of 95% alcohol 

 until no more color comes away in the alcohol. 



6. Counterstain with Bismark brown (5% aqueous solution). 



7. Wash, dry and mount for examination. 



This rather important stain depends for its differential value 

 on the fact that whereas all species of bacteria, stained by anilin- 

 oil-gentian-violet, may be decolorized with alcohol, certain species, 

 if iodin be used to fix the stain, become undecolorizable, while others; 

 Jose the stain as readily after the iodin treatment as without it. 



