VIROLOGICAL METHODS 259 



egg and on the embryo side. The square and slightly beyond is dis- 

 infected by painting it with tincture of iodine. With the use of the 

 grinding tool the shell is ground through along the pencil line, care being 

 taken not to penetrate to the choiioallantois. A small slit is ground over 

 the air sac so the air can escape when the membrane is to be dropped. 



After the slit and square have been cut, the remainder of the work may 

 be done under a glass hood to reduce contamination from air-borne 

 bacteria. First the shell is removed with sterile forceps. Then the shell 

 membrane is wet with a 70 per cent ethanol solution containing 0.1 per 

 cent thymol. This membrane is pierced carefully with a sterile lance so 

 as not to injure the underlying chorioallantois. With the vacuum 

 broken, the contents of the egg settle by gravity, displacing the air from 

 the normal air sac. With sterile forceps the shell membrane is then 

 removed, leaving a square aperture through which the embryo and 

 embryonic membranes can be observed. The shell surrounding the hole 

 is again disinfected by carefully painting it with a swab partially soaked. 

 with tincture of iodine. One must be cautious not to allow the iodine 

 solution to enter the embryo. Finally a small square of transparent 

 gummed tape is stuck on the egg to cover the hole. 



Dropping the chorioallantois successfully requires considerable prac- 

 tice. If the membranes are torn, the embryo usually has lost its useful- 

 ness. Any unnecessary manipulation is likely to cause hemorrhage of or 

 nonspecific lesions on the membrane. Windowed eggs require consider- 

 able time for preparation but allow the worker subsequently to inspect 

 the results of his inoculations under direct observation and permit 

 definitive inoculation of specific sites. 



Amniotic sac. Using a 27-gauge 12-in. side-delivery needle, it is 

 possible to inoculate the amniotic sac without windowing the egg. The 

 embryo is prepared as for allantoic sac inoculation with one hole made 

 directly over the embryo. Introducing the needle directly toward the 

 embryo, which is visualized by the aid of a candler, one can be successful 

 in about 90 per cent of the attempts. It is advisable to practice several 

 times, using a methylene blue solution to determine one's aptness. One- 

 tenth-milliliter inoculum may be used on the membrane, and 0.05 ml in 

 the amniotic sac. More or less may be used as the experiment dictates. 



Yolk sac. Yolk-sac inoculation is commonly used for the propagation 

 of rickettsiae and certain viruses, e.g., those of the meningopneumonitis 

 group. A single hole drilled over the air sac is adequate. Usually a 

 large-bore needle is used for the injection. A IJ^-in. 20-gauge needle is 

 suitable. With the egg in a vertical position, large end up, the needle is 

 pushed through the hole and straight downward. To check the accuracy 

 of position a slight pull on the syringe plunger causes yolk to appear in 

 the syringe. The inoculum can then be discharged. 



