Viruses 397 



Thomas M. Rivers proposed the use of tissue cultures in 1931 for 

 growing microorganisms, especially viruses. This in vitro cultiva- 

 tion technic involves the use of a suspending fluid, like Tyrode's 

 solution, which contains mineral nutrients, glucose, and sometimes 

 blood serum; live tissue cells, including mouse or chick embryo, 

 skin, liver, and kidney; and the inoculum containing the suspected 

 virus. 



Cells of hatched chicks are completely resistant to the action of 

 many bacteria, rickettsiae, and viruses, while actively growing 

 embryonic tissue in chicks developing within the egg is particularly 

 susceptible to the action of many of these same microorganisms. 

 The abrupt change that takes place upon hatching of the chick 

 has not been explained satisfactorily. 



FERTILE EGGS 



While developing chick embryos were used by Ogston as early 

 as 1881 for the cultivation of bacteria, it was not until fifty years 

 later that Gerrit J. Ruddingh and his colleagues employed the 

 chorio-allantoic membrane (a membrane surrounding the chick) 

 of the developing chick for the cultivation of viruses. Since the 

 eggs of hens are generally free of microorganisms, nature has pro- 

 vided an ideal culture medium, as well as a suitable container, in 

 which to grow pure cultures of microbes. Fertile eggs are incu- 

 bated until the embryo is from four to fourteen days old. The age 

 of the developing embryo is a critical factor in the growth of 

 viruses, some of which will only grow during the early develop- 

 mental stage of the embryo and others of which prefer older cells 

 for their growth. 



The egg shell is cut preparatory to inoculation with virus by 

 means of a drill similar to those used by dentists. A square or 

 triangular-shaped piece of the shell is gently removed with a pair 

 of flamed forceps. Aseptic technic is practiced throughout this 

 entire procedure, with tincture of iodine or other suitable disin- 

 fectants being used to treat the shell of the egg before cutting out 

 the "window." The underlying shell membrane is kept intact until 

 a small hole is drilled at the air sac end of the egg. Ry applying 



