286 RESEARCH IN PROTOZOOLOGY 



thetic, but guinea-pigs and rats should be etherized. Chicks re- 

 quire no anesthesia. 



The third method of inoculation permits the introduction of in- 

 fective stages belonging to the asexual cycle, merozoites, as well 

 as mature oocysts. Merozoites are collected by scraping infected 

 portions of the intestine of a freshly killed host. The scrapings 

 are immediately suspended in warm (37° C.) Ringer's fluid, 

 strained through several thicknesses of cheesecloth to remove 

 debris which would clog the needle and are then aspirated into a 

 warm Luer syringe provided with a twenty gauge needle. The 

 syringe and contents are kept at incubator temperature until use. 

 Just before use, a drop of the contents is expressed upon a clean 

 slide and examined under the microscope to be sure that the mero- 

 zoites are present and viable (as evidenced by their motility). 

 Briefly, the procedure is (i) to prepare the inoculum (a suspen- 

 sion of either merozoites or oocysts), (2) anesthetize the re- 

 cipient, (3) prepare it for abdominal section, (4) expose the site 

 of injection (stomach, duodenum, jejunum, ileum, liver, etc.), (5) 

 inject inoculum^ and (6) repair the abdominal incision. 



It is many times necessary to perform inoculations quantita- 

 tively. This may be done by washing and concentrating a quantity 

 of sporulated oocysts. The suspension is tJwroiigJily shaken, and 

 a drop is placed in the chamber of an ordinary hemocytometer. 

 Only the sporulated oocysts from the entire ruled space (nine 

 square millimeters) are counted, and the number on this area is 

 divided by 0.9 which will give the number per cubic millimeter. 

 An average of at least ten counts should be taken. The suspension 

 may then be adjusted by dilution to a content of oocysts which is 

 convenient for computation, e.g., 10,000 oocysts per cubic cen- 

 timeter. This stock solution should be protected against growth 

 of bacteria or free-living protozoa by the addition of a drop or 

 two of strong formalin. The number of oocysts should be counted 

 at various intervals as a few of the unsegmented oocysts not 

 originally included in the count do go on to development, thereby 

 increasing the number, and there is a constant low rate of mor- 

 tality among the mature oocysts which tends to decrease the num- 

 ber. Coccidial oocysts are heavier than water and quickly fall to 

 the bottom of any solution whose specific gravity is i.oo or less. 

 Consequently it is necessary to shake a counted suspension before 

 withdrawing a sample, and if a syringe is used in injecting them, 



