XIV. 



X RAYS AND X IRRADIATION 475 



upon a dilution technique (see 17,28,29,4-5). Work with vaccinia 

 VH'US is of this type. Vaccinia virus for irradiation is extracted from 

 an infected rabbit testis, generally preserved in glycerin, by grinding 

 the testis in sand and diluting it with a 1/10 physiological salt solu- 

 tion, as, for instance, Locke's. iVfter centrifuging this ground solu- 

 tion a portion of it, say, 0.75 ml. of the supernatant, may be trans- 

 ferred to suitable dishes and exposed to the effects of X rays. Dishes 

 for this exposure have been very satisfactorily made out of ordinarj^ 

 Parawax by simply boring out a flat hollowed depression in the wax 

 and putting the virus solution in this depression. The area irradiated 

 should be sufficient so that the depth of solution is slight and correc- 

 tion for absorbing energy reduced to a minimum. Since X rays are 

 more lethal to bacteria than to viruses, the technique if properly 

 handled will keep the material essentially biologically sterile for other 

 organisms than the vaccinia. The reduction in the virus particles 

 due to irradiation may be determined by titration experiments, in 

 which 0.1 ml. of the irradiated or controlled virus suspension is inocu- 

 lated intradermally into rabbits in separately marked spots. 



The rabbit is first shaved, then the body surface marked off in 

 squares; as many as thirty or forty squares may be possible on a 

 good-sized rabbit. In the center of the square, 0.1 milhliter of the 

 virus suspension is inoculated into the corium. Negative and posi- 

 tive takes will be noted. The virus will not spread but will cause a 

 swelling and reddening of the area for a positive take. Negatives 

 will show^ little swelling. The different treated solutions and con- 

 trols should be distributed over the surface of the rabbit, either at 

 random or with some prearranged design capable of easy statistical 

 evaluation, so that there is no place where infection interaction inter- 

 feres with the interpretation of the data. Positive tests show that 

 virus particles are present. Negative tests show that all the virus 

 particles have been destroyed in the sample inoculated. Only this 

 type of data can be obtained from such material. An estimate of 

 the number of virus particles in the treated and control solutions 

 can be obtained by diluting the solutions and getting the dilution at 

 which approximately 30-50% of the inoculation tests are positive 

 and the rest negative. The dilution factor, the percentage of 

 positives, and the assumption that the particles are distributed as in 

 the Poisson series make it possible to estimate the number of virus 

 particles in the original solutions undei- test. Comparison of these 

 numbers for differently treated solutions gives the X-ray effect. 



