258 MANUAL OF MICROBIOLOGICAL METHODS 



biotics in many chicken feeds may interfere with the growth of rickettsiae 

 and some viruses (Greiff and Pinkerton, 1951). Obtaining embryonated 

 eggs from a flock on an antibiotic-free diet may be difficult but necessary 

 for some types of experimental and diagnostic work. 



Preinoculation incubation. Fertile eggs set at 37.5°C develop satis- 

 factorily for most viral and rickettsial work. The setting date should be 

 recorded on the eggs or elsewhere so the embryo age can be determined as 

 required. While it is preferable to set the eggs soon after they are 

 received, this may be delayed for a day or two. If they are held, they 

 should be stored at a low temperature, GO-GS'^F, so as to minimize the 

 drop in fertility. Hatching eggs properly handled should yield 70 per 

 cent fertility or better. Proper attention should be paid to humidity and 

 air circulation. Regular hatching incubators with forced-air circulation 

 and dual thermostats usually are satisfactory. Dual thermostats so 

 arranged that the spare is cut in when the other fails and a warning light 

 or buzzer comes on will save several days of lost time, not to mention the 

 investment in eggs. Moisture content of the air should be maintained at 

 90 per cent saturation. 



Candling. After the eggs have been incubated for the required length 

 of time, which ordinarily will vary from 5-10 days, they are candled. 

 Five- to 7-day-old embryos are frequently employed when yolk-sac 

 inoculations are used, whereas when other areas are injected older 

 embryos, up to 10 days, are selected. An inexpensive commercial candler 

 or any apparatus causing a strong light to go through the egg without 

 scattering to blind the operator is satisfactory. If candling is carried on 

 with the proper equipment in a dark room, the air sac, embryo, and some 

 of the blood vessels can be recognized easily, especially when white- 

 shelled eggs are used. Usable eggs are chosen on the basis of the distinct 

 blood vessels and active embryos. For the beginner it is well to check 

 his discards and some of those he considers acceptable by breaking open 

 the shell and observing the actual situation which he has characterized 

 indirectly. 



Marking, Time can be saved by marking the eggs as they are candled. 

 Rotating the egg on the candler with the pencil following the air-sac 

 periphery outlines this area. A mark may be placed over the spot where 

 the embryo shadow is most visible. An additional mark can be made at 

 the intended point of inoculation for the allantoic sac. 



Windowing and Inoculation. If it is desired to inoculate the amniotic 

 sac or embryo under direct observation, or if the inoculum is to be applied 

 directly to the chorioallantoic membrane, the embryos should be win- 

 dowed. Any small motor tool that can be fitted with an emery disk may 

 be used for preparing the window in the shell. An area of about 1.5 cm 

 square is marked off approximately equidistant from each end of the 



