282 RESEARCH IN PROTOZOOLOGY 



standing at least three hours, the supernatant fluid is decanted 

 through the top of the funnel, the sediment being carefully re- 

 tained. Water is added to make up the original volume, the funnel 

 is shaken vigorously, and the contents are permitted to sediment 

 as before. If time allows, it is well to continue this washing by 

 sedimentation until the supernatant fluid appears to be perfectly 

 clear after three hours' standing. Two sedimentations will, however, 

 suffice to free the oocysts of most of the light suspended matter 

 which is present with them. After the final decantation, the original 

 volume is restored with a boiled saturated solution of common salt 

 and is thoroughly shaken. The increased specific gravity of the 

 liquid containing the oocysts causes the organisms to float. After 

 this solution has stood for an hour, it is drawn off from the outlet at 

 the bottom of the funnel until the layer of floating particles reaches 

 the stopcock. This material is saved in the funnel and is again 

 diluted with water. Enough water must be added so that the 

 retained volume is increased at least three times. The specific 

 gravity of the solution is now reduced below that of the oocysts 

 so that they will sink to the bottom. They are permitted to sedi- 

 ment overnight, after which the oocyst-containing layer may be 

 drawn off from the bottom outlet either directly in receptacles for 

 sporulation or into fifty cubic centimeter centrifuge tubes for 

 further concentration. 



Sporulation. Investigations upon the coccidia of birds or of 

 mammals usually involves the experimental infection of animals 

 to act as hosts. To successfully infect new animals, it is necessary 

 to have mature oocysts, that is, oocysts which contain their final 

 number of viable sporozoites within their sporocysts. Oocysts 

 passed by animals during the course of the infection are unseg- 

 mented. They contain a single contracted (if the individual organ- 

 ism has been fertilized) mass of protoplasm. Under suitable con- 

 ditions of moisture, oxygen tension, and absence- of bacterial 

 putrefaction this protoplasmic mass will fractionate into a number 

 of sporoblasts (specific for the species) and these sporoblasts 

 will again subdivide into a number of sporozoites. The three 

 following methods of sporulating oocysts have been used by the 

 writer with success : 



The usual method of accomplishing this sporulation is to place 

 feces containing immature oocysts into a solution which will pre- 

 vent growth of bacteria without injuring the coccidia. A five per 



