960 THE ANIMAL VIRUSES 



in the testicle, but subsequently transfers were made without difficulty, and the virus 

 reached its maximum nmltiplication in the testicle after 4 or 5 days ; it remained 

 stationary in amount till the 8th day, and then decreased till after 5 weeks its pre- 

 sence could no longer be detected. By testicular cultivation, the virus did not lose 

 its affinity for the skin ; both the testicular and the skin strains gave similar re- 

 actions in the skin, cornea, and testicles of rabbits, and in the skin of human beings. 

 In 1925 Parker and Nye succeeded in growing the vaccinia and herpes viruses in 

 tissue cultures prepared with normal rabbit testis and plasma. This was confirmed 

 by Carrel and Rivers in 1927 working with the vaccinia viru;. Infected rabbit 

 testicle was ground up in a mortar, added to chick-embryo pulp, left for 

 24 hours in the ice-chest, and then inoculated into a Carrel flask containing 

 a coagulum of hen plasma. The cultures were washed every 2 or 3 days with 

 Tyrode's solution, and were nourished with a dilute fowl-embryo extract. After 

 periods varying from 1 to 4 weeks the contents of the flasks were withdrawn, ground 

 up in a mortar, and titrated on rabbits by the intradermal method. It was found 

 that the cultures, which were seeded with 25 to 250 intradermal units of virus 

 per ml., contained after incubation for a week between 10,000 and 100,000 units 

 per ml., showing that actual multiplication of the virus had occurred. Haagen 

 (1928) reported a modified method of in vitro cultivation of vaccinia virus in 

 the presence of rabbit testicle, rabbit plasma, and rabbit spleen extract. Using 

 this method, he carried the virus through 37 passages in a period of about 8 months. 

 During the first 5 days of each subculture the virus multiplied about 1,000 times, 

 and during the whole period its virulence remained approximately constant. 

 Findlay (1928) reported similar success in the cultivation of the fowl-pox virus 

 by Carrel's method. Craciun and Oppenheimer (1926) cultivated vaccinia virus 

 in association with embryonic guinea-pig's cornea, and carried through nine suc- 

 cessive transfers during a period of 71 days. Finally the in vitro cultivation of 

 vaccinia virus in the apparent absence of proliferating cells was reported by the 

 Maitlands (1928). Infected rabbit testicle was ground up, diluted with Tyrode's 

 solution, added to minced hen's kidney, placed in the cold room for 4 hours, and 

 then cultivated in a Carrel flask containing hen's serum. The flasks were incubated 

 at 37° C, and subcultures made about once a week. Four passages were made 

 in all. A 1/625 dilution of the last subculture, which represented a dilution of 

 1/625 X 10^ of the primary inoculum, produced vaccinia on inoculation into 

 rabbits ; the original inoculum was active only in a 1/2500 dilution. Living 

 tissue was, of course, not excluded, but no evidence of its multiplication was 

 obtained. Various modifications of the Maitland technique, such as cultivation 

 in agar slant cultures (Kurotchkin 1939), wide tubes (Findlay and MacCallum 

 1940), and roller tubes (Feller et al. 1940) have been successfully introduced. 



The function of the tissue cells in the cultivation of viruses has not been estab- 

 lished, though much is known about the various factors that influence growth 

 (see Hallauer 1938). Zinsser and Schoenbach (1937) observed that development 

 of the equine encephalitis virus reached its maximum in 3 to 4 days, whereas 

 that of Rickettsia was considerably later — 6th to 8th day. They therefore drew 

 the tentative conclusion that the metabolism of viruses and of rickettsise differed, 

 the former multiplying best during the period of active growth of the tissue cells, 

 the latter after the cells had begun to die off. Maitland and Laing (1941) have 

 brought evidence to suggest that with vaccinia virus the cells exert two separate 

 functions, one to initiate growth, the other to maintain growth once the virus 



