The cultivation of vaccinia virus A new series of subcultures in cell-free medium

Abstract

A medium was prepared consisting of (1) saline extract of minced rabbit kidney rendered cell-free by alternate centrifugation and freezing-thawing, (2) fresh rabbit serum, and (3) Tyrode''s sol.;[long dash]cells (other than erythrocytes) could not be found in hanging drop preparations or in stained deposits. In this medium, vaccinia virus was cultivated through 10 generations, with total multiplication of 1020. The end point of a titration of the 10th subculture was greater than that of the original, although during subculture the original virus seeding had been diluted 1017 times. Such a condition could not be accounted for by mere survival, especially in view of the amount of dilution and the long periods of incubation at 37[degree]C. A series of 8 subcultures were carried out in the same medium, which had been frozen and thawed. Although freezing does not damage the medium to any great extent, increases in virus obtained thereafter are more irregular. It is suggested that substances are released from the cells during preparation of the medium which are, in the fresh state, essential to virus growth. The virus is not altered by cultivation in cell-free medium, as is evidenced by type of reaction produced in animals, by neutralization with antiviral serum, and by presence of masses of elementary bodies in passage subcultures.