50 PARASITIC AMCEBJE OF MAN. 



The utmost care must be taken that everything 

 used in preparing this stain, and using it, is chemically 

 clean, and that none of the dishes or implements 

 used are moist and that they be sterilized before use. 



The stock solution consists of Azure II-eosin, 3 

 grams, and Azure II, 0.8 gram. These are dissolved, 

 by constantly shaking, in 250 c.c. of pure glycerine 

 at a temperature of 60 degrees C. After solution is 

 complete, 250 c.c. of absolute methyl alcohol (Kaul- 

 baum I) previously heated to 60 degrees C. is added. 

 This mixture is well shaken, allowed to stand at room 

 temperature for 24 hours, and then filtered into a 

 chemically clean, sterilized, air-tight bottle. During 

 the filtration the funnel must be covered in order to 

 protect the hydroscopic fluid from moisture and the 

 bottle containing the stock solution should be kept 

 in a dark place. 



The method of using this stain is as follows: 



Thin, even smears of the material containing the 

 amoebae are made upon cover slips and fixed while 

 wet in osmic acid vapor or sublimate alcohol, and 

 stained with the following solution: 



Stock solution 10 drops. 



Distilled water 10 c.c. 



1 per cent potass, carb. solution, 1 or 2 drops to each 

 10 c.c. of the above mixture. 



This staining solution is allowed to act for from 

 30 minutes to 6 hours, although fairly good prepara- 



