208 GUIDANCE IN MAKING PERMANENT PREPARATIONS. 



into acidulated alcohol till the color assumes a brilliant appear- 

 ance, but do not allow it to fade too far. Wash in 70 percent and 

 then run through 80 percent and 95 percent alcohol five min- 

 utes each and mount directly in upural. 



If balsam is to be used, continue from the 95 percent alcohol 

 to 100 percent alcohol, five minutes, 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 lat- 

 ter exhibits a milky-white appearance, the material is not suffi- 

 ciently 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 Paramaecium 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 

 (haematoxylin or picro-carmine is good), dehydrate, clear, and 

 mount as usual. 



