Studies on Adenovirus Type 12 1. The tissue culture of adenovirus 12 by three methods

It is known that adenovirus type 12 nduces tumor in a high proportion of young hamsters, and also other types of adenoviruses do so to some extent. In the study of this virus the problems are posed the survival of the virus inoculated in animals, the presence or absence of the virus in the tumor developed by the virus, and the isolation of virus from man. It is, therefore, necessary to find out some appropriate methods to detect even a minute quantity of virus. With this view in mind, the authors conducted a series of experiments by the following three methods of successive tissue cultures using monolayer HeLa cells in culture infected with adenovirus 12 for the period of 7 days for each generation to see which of the three methods is most suitable. The results of this study are briefly summarized as follows 1. The tissue culture conducted for generations of HeLa cells with freezing-thawing techniques: The cells growing in monolayer in the test tubes were infected with adenovirus type 12 and cultured for 7 days as one generation. At the termination of each culture period the medium was changed with 1ml of fresh one. The first generation cells were frozen and thawed, cent-rifuged, and then the supernatant was placed into new monolayer HeLa cells. Subsequently, tissue culture was continued for generation after generation. In the doses of 1, and 10(-1) TCID(50) of virus used, 3 generations were needed to confirm proliferation of virus on the ground of cellular degeneration, and in the doses of 10(-2), 2×10(-3), and 10(-3) TCID(50), 4 generations were needed. 2. Virus culture through successive generations of infected cells: As in the preceding experiments the infected HeLa cells were cultured for 7 days, and after dispersing the cells in monolayer with trypsin solution, this first generation was divided into two equal parts, each of which was placed in the test tubes and cultured. Successive cultures were conducted in similar manner. The growth rate of virus in this series proved to be about the same as in the foregoing experiments. 3. Virus culture on slant agar: As in the preceding experiments the infected HeLa cells cultured for 7 days were dispersed with trypsin solution. This cell suspension of the first generation was centrifuged and the sediment, divided into about five equal masses of cells, was placed on slanted surface of agar, and cultured. Then one half the amount of the cells growing on the agar for 7 days was cultured again on the agar. The other half of the sediment was frozen-thawed with 1ml of the medium, centrifuged, and the supernatant was placed in the new monolayer to determine the proliferation of the virus. This method also gave the results not so different from those by the two preceding methods. From these results it is concluded that the tissue culture conducted on the slant agar is the most suitable method for the detection of virus growth because it is easy to manipulate and simple to maintain successive generations.