60 MANUAL OF HUMAN PROTOZOA 



with distilled water, prepare 0.5-1% slightly alcoholic solu- 

 tion which will be readv for immediate use and which can 

 be used repeatedly. 



The smears are now placed vertically in iron alum solu- 

 tion and left in it for 1-3 hours. Wash with running water 

 for 5 minutes and rinse in distilled water. Immerse the 

 smears in haematoxylin solution for 1-3 hours. Smears are 

 now washed for 5 minutes in running water and placed 

 face up in a Petii dish containing a weak (0.25%) iron 

 alum solution for decolorization which must be controlled 

 under a compound microscope. If all smears are of 

 uniformly the same thickness and meant for the same pro- 

 tozoa, one of the smears may be watched continuously. If 

 the smear contains, for example, the trophozoites of En- 

 tamoeba histolytica, the whole amoeba will look black at 

 the beginning, but the cytoplasm becomes gradually de- 

 colorized, leaving the nucleus and the cytoplasmic inclu- 

 sions still dark. As the process is continued, further de- 

 colorization takes place, and finally the nucleus may ap- 

 pear as a ring. Often it is necessary to control the last stage 

 of the process under a high dry objective. For this purpose, 

 flood the well in a depression slide with the iron alum solu- 

 tion and place a representative smear over it with the 

 smeared side facing downward. Such a smear could be 

 observed with a high dry objective and will be better con- 

 trolled. Optimum staining will be attained by repeated 

 practice. 



When the decolorization is completed, the smears are 

 washed gently in running water for about 15 minutes in 

 order to insure the complete washing of the iron alum. 

 Rinse in distilled water and transfer to 30%, 50%, and 70% 



