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

Abstract

It is now generally accepted that vaccinia virus may be cultivated in a medium containing living tissue and that cell proliferation is not essential for multiplication of the virus. Also it is stated that survival and increase of virus cannot occur, except in the presence of living cells, for any appreciable period at incubator temperature. Although the earlier literature presents several accounts of successful cultivation on lifeless media, these have not withstood the criticism which improved methods of testing for the presence of virus have brought to bear on them. After considerable experience in culti­vating vaccinia virus both by tissue culture and on the rabbit kidney medium used by Maitland and Laing (1930), a short series of experiments was reported demonstrating the possibility of cultivating the virus in an apparently cell-free medium (Eagles and McClean, 1930). Further evidence substantiating these results and demonstrating considerable multiplication in cell-free media was presented by Eagles and McClean (1931). Other investigators, notably Rivers (1931) and Maitland, Laing and Lyth (1932) have so far been unable to secure similar results. In view of the fundamental importance of cultivating a virus in a lifeless medium, the failure of these later investigators to confirm our results, and in view of the criticism that the kidney extract medium may, in our case, have contained some residual cells or cell-fragments, a new series of experiments was undertaken. The method of preparing the culture medium has been altered with a view to securing one richer in cell substance while rendering the presence of viable cells remote. It has been suggested that the method of calculating the total amount of increase in virus through a series of sub­cultures by comparing unincubated control and culture at each step may give an erroneous idea of multiplication and that a more precise method might be adopted. Babbits are known to vary considerably in their response to vaccinia virus. This, however, does not satisfactorily explain the large and unexpected reduction in titre which has occasionally occurred when testing the virus content of the unincubated control. In the series here presented the method of calcu­lating increase of virus through a series of passages has been based on the theoretical potency of a seeding representing in all cases a 1-50 dilution of the previous subculture, which had been experimentally titrated to the end point.