yiROLOGICAL METHODS 267 



constant dose of challenge virus. If the reverse approach is desirable, 

 the serum may be held constant and the virus diluted by increments of 10. 

 The difference in infectivity titer between that in the presence of the 

 antiserum and that of the control can be attributed to the neutralizing 

 properties of the serum. 



Other methods for estimating the 50 per cent end point are also avail- 

 able (Finney, 1947; Irwin and Chaseman, 1939). 



Cultivation in Tissue Cultures 



Uses, Many of the kno\vn viruses and rickettsiae have been propa- 

 gated in tissue cultures. This method of culture has had a rather wide 

 adaptation for the virologist, including isolation, identification, study of 

 reproduction cycles, and cellular pathology. Large quantities of virus 

 have been cultivated to be used for vaccines and for other reasons. 

 Antibody neutralization of virus and screening of various chemicals 

 including antibiotics for their effects on viruses and rickettsiae may be 

 carried out in tissue culture. 



Materials. Tissue-culture methods change at such a rapid rate and 

 vary so much in complexity that it would be advisable for the beginner 

 to start with a simple method. After he has acquired some basic expe- 

 rience with this method, various original works and texts can be consulted 

 to extend his proficiency. Basically the ingredients of a tissue culture 

 are tissue and extracellular fluid. Tissues used will vary considerably 

 with the virus to be propagated. Embryonic tissue usually suffices 

 quite well, although testicular tissue and propagated cells of various 

 kinds may also be used. 



Chick embryo, because of its availability, is often used. The air-sac 

 ends of 10- to 12-day-old embryonated eggs are painted with tincture of 

 iodine, and the solution allowed to dry. Sterile forceps are used to 

 break and pull away the shell over the air sac. A different pair of forceps 

 is used to strip away the shell membrane, and the contents of the egg 

 are poured out into a sterile petri dish. The embryos are then collected 

 in a sterile receptacle prior to mincing. After the eyes of the embryos 

 are removed, the remainders are placed on a firm cutting surface such 

 as bakehte. With a sharp blade, e.g., razor blade, the embryos are cut 

 into tissue fragments approximately 1 mm square. The fragments are 

 transferred to a sterile receptacle, e.g., petri dish or watch glass, for 

 washing with salt solution containing no sodium bicarbonate. Three 

 washings usually suffice for removal of most of the blood cells. Certain 

 tissue, e.g., embryonic lung, can be selected and used exclusively for the 

 culture. 



The size of the flask or container used will determine roughly the 

 number of tissue fragments used. Tissue from each embryo can be sus- 



