PROTOZOA METHODS. 191 



then run through 80 percent, 95 percent, and 100 percent alcohol, 

 five minutes in each, thence into clove oil, or cedar oil, keeping all 

 reagents carefully covered, and leave till the object is thoroughly 

 penetrated. This latter process may take five to ten minutes. 



If, on putting your objects into the clearing medium, the 

 latter exhibits a milky-white appearance, the material is not 

 sufficiently dehydrated, and must be returned to 100 percent 

 alcohol. 



After clearing is completed, put the object on a clean slide 

 with a little balsam and cover. 



The material not treated with borax-carmine may be run 

 back through 50 percent and 35 percent alcohols to water, to 

 which a few drops of hematoxylin have been added, or put from 

 water into alum-carmine. The former stain, if dense, should not 

 require over twenty to thirty minutes, but objects must be left 

 in alum-carmine ten to twenty hours. When a good color is 

 obtained, run the material through the grades of alcohol, from the 

 lowest to the highest (five minutes in each), and mount as in the 

 case of the borax-carmine objects. 



Objects stained in alum-carmine will probably not overstain; 

 but excess of hematoxylin should be extracted with acidulated 

 alcohol when the 70 percent grade is reached, after which it is 

 very essential that all of the acid be removed by repeated changes 

 of 70 percent alcohol. Otherwise the objects will fade. 



Protozoa Methods. A simple method for preparing such 

 forms as Paramsecium is as follows: 



Kill in Sublimate Acetic (a small watch-glass or concave slide 

 is a good container), let settle, run to 70 percent alcohol, drop 

 on a slide smeared with albumen fixative, let it remain a minute, 

 then thrust the slide into 70 percent alcohol. Stain, after 

 running to grade of alcohol or water in which stain is made 

 (hsematoxylin or picro-cannine is good), dehydrate, clear, and 

 mount as usual. 



