262 MANUAL OF MICROBIOLOGICAL METHODS 



lation is on the chorioallantoic membrane of a windowed egg, the mem- 

 brane can be examined daily for lesions by carefully peeling back the 

 gummed tape. An ophthalmoscope may aid in differentiating shell dust 

 from opaque lesions. Punched eggs can be candled daily or twice daily 

 to learn the viability of the embryo. Some viruses kill the embryo, while 

 others do not. Control eggs of the same age (uninoculated or inoculated 

 with the same diluent used for the experimental eggs) should be included. 

 Examination of these controls will help interpret findings among the 

 experimental eggs. When possible a sufficient number of eggs should be 

 inoculated to allow one to sample and pass a certain number each day. 

 Under such circumstances it is possible to determine the gross state of the 

 embryo and membranes as well as test for hemagglutinin and comple- 

 ment-fixing antigen. Determining the appropriate time interval and the 

 material to be passed on to new embryos requires experience and judg- 

 ment. Any significant positive finding should suggest passing the 

 material involved. Pooling membranes and embryo may be tried in the 

 absence of a significant finding. Each fluid passage should be accom- 

 panied by culturing for bacteria and, when indicated, pleuropneumonia 

 organisms. Smears should be made of the membranes, stained with 

 Giemsa's and Macchiavello's stains, and a search made for larger viruses, 

 rickettsiae, and inclusion bodies. It is also advisable to use a saline 

 extract of the pooled materials as antigen in the complement-fixation test 

 against known antibodies and patients' acute and convalescent sera. 

 Where sufficient antibody and antigen are available, a checkerboard 

 complement-fixation test is advisable to avoid the Neisser-Wechsberg 

 phenomenon (see Chap. IX). 



Harvesting. The procedure for harvesting the fluids and membranes 

 from embryonated eggs will vary with the method of inoculation and the 

 information the investigator wishes to obtain. It is easy to rupture a 

 vessel during harvest; therefore, it is a common procedure to expose 

 embryos to 4°C overnight to congeal the blood, thus avoiding hemorrhage 

 into the fluid one expects to harvest. Allantoic fluid harvest from 

 punched eggs can be accomplished quite easily. The shell over the air 

 sac is swabbed with tincture of iodine. After the solution has dried, the 

 shell is broken and removed, exposing the sclerotic membrane under the 

 air sac. The membrane may be clarified with a few drops of 95 per cent 

 ethanol or stripped away with sterile forceps prior to harvesting the fluid. 

 It is well to examine each egg before harvesting. The odor and appear- 

 ance of the eggs contents have definite meaning to the experienced investi- 

 gator. A number of bacteria and molds growing in the egg give off 

 characteristic odors. Pseiidomonas aeruginosa commonly produces its 

 soluble green pigment to alert one to its presence. Death of the embryo 

 and disintegration of the membranes where they are not anticipated 



