466 RESEARCH IN PROTOZOOLOGY 



tube, and is centrifugalized for 3 minutes. The supernate is decanted and 

 the sediment is examined for cysts. 



The concentration method of De Rivas is said to be very efficient 

 for the cysts of intestinal protozoa. It is carried out as follows : 



A small quantity (one or two grams) of feces is added to from five to 

 ten cubic centimeters of five per cent acetic acid in a test tube and is thor- 

 oughly shaken. After the mixture is homogeneous it is filtered through sev- 

 eral thicknesses of cheesecloth and five cubic centimeters of the filtrate is 

 added to an equal quantity of ether in a fifteen cubic centimeter centrifuge 

 tube. The tube is closed and is shaken horizontally, and then is centrifuged 

 for a few minutes. The contents of the tube separate into four layers: the 

 etherial extract at the top, the detritus plug near the middle, next the acetic 

 acid solution, and lastly the sediment. The sediment is examined for cysts. 



Other methods for concentrating coccidial oocysts are given in 

 Chapter XXXI. 



Observation of Killed and Fixed Protozoa 

 Killed protozoa may be examined in smears or films or in cut 

 sections. 



Smears. Intestinal material may be smeared upon glass slides as 

 indicated above. If a permanent preparation is not required the 

 smear may be made in a solution containing iodine (one per cent 

 iodine in two per cent potassium iodide) which will tend to render 

 nuclei and flagella a trifle more conspicuous than they appear in a 

 fresh living preparation. 



If permanent preparations are required the smear may be 

 "fixed" and stained. ''Fixing" the organisms involves the pre- 

 cipitation of their protein constituents and is accomplished 

 by immersing the slide or coverslip upon which the smear is 

 made in one of such fluids as Schaudinn's,^ Bouin's,® Carnoy's/ 



° Schaudinn's solution 



Saturated aqueous solution of mercuric bi- 

 chloride 65 parts 



95% ethyl alcohol 33 parts 



Glacial acetic acid 2 to 5 parts 



' Bouin's solution 



Saturated aqueous solution of picric acid. . 75 parts 



Formalin 25 parts 



Glacial acetic acid 5 parts 



^ Carnoy's solution 



Glacial acetic acid i part 



Absolute alcohol 6 parts 



Chloroform 3 parts 



