The problems raised by this avian neoplasm are precisely those of the problem of cancer in general and were they solved, the knowledge of carcinogenesis in general would have been advanced enormously.

It is to the great credit of Bryan and his collaborators to have obtained relatively purified reproducible preparations of Rous virus (68, 69), a remarkable achievement in itself, since the difficulties of the purification of the virus are extreme (70-72), and to have demonstrated, in a series of outstanding experiments, the relationship between the amount of recoverable virus in the tumor and the amount of the agent used as inoculum (78, 74)- Tumors produced with small amounts of virus yielded little or no extractable agent, and it could be demonstrated further by electron microscopy that a high level of infectivity of extracts of the tumor was paralleled morphologically by a large number of intracellular viral particles (61, 75). This not only provided the advantage of bringing additional proof that the particles observed in the electron microscope were indeed the Rous etiologic agent but demonstrated that experimental conditions could be devised to yield tumors in which otherwise virus particles could be difficult to detect but which now contained an abundance of the agent. It could be clearly seen that, in growths yielding extracts below a certain infective titer, no particles, or very few of them, could be found in the electron microscope, while a remarkably large number of particles could be demonstrated in tumors from which extracts of high infectivity were obtained. The data in this area of study are being confirmed now on a much larger scale.

From the point of view of human tumors, these observations are of great theoretical importance, since it is a reasonable hypothesis that tumors under "natural" conditions in man probably compare with the low-titer group. At any rate, the experimental findings already described account for the phenomenon of the absence or scarcity of particles in all previous studies of the Rous sarcoma. Thus, the notion of "virus-enriched" preparations for electron microscopy was imposed upon electron micros-copists in a realistic way. Such material became available in studies in vivo in the Rous sarcoma as a result of the efforts of a team who had devoted themselves during years of research to the complete study of that tumor. But practical drawbacks of this system, the need for great quantities of birds, and especially the insecurity due to the presence in these of 10 percent latent contamination with lymphomatosis virus, need not be emphasized. For these and other reasons, the great advance of the last few years has been the demonstration that a parallel enrichment in the content of tumor viruses in cells may be achieved through tissue culture (76). From the special point of view of the electron microscopist the advantages of this method are many, in addition to the increase in particles:

1. Better preservation of the infrastructure of cells may be obtained.

2. Phenomena may be observed with precise timing after infection, and a cytologic cycle of virus characteristics may become apparent.

3. Biologic tests may be effected in parallel with electron microscopy on samples in which homogeneity of the virus preparation is well assured.

The reason that only few virus-induced tumor systems have been studied up to now in this combination is explained by the difficulty of developing susceptible cell strains, that of finding optimum conditions foi virus multiplication, and the often unsatisfactory degree of purification of tumor viruses.

Five systems have been investigated thus far in tissue culture: the Shope fibroma (56), polyoma (14-17), mouse mammary cancer (50, 77), myeloblastosis (37, 88), and the Rous sarcoma (29, 40, 78). The fundamental findings with certain of these systems will certainly have been considered in detail in the section of this symposium devoted to tissue culture and will not be dwelt upon here, except for the problem of the Rous sarcoma. It may be recalled, though, before discussion of the details of the work with Rous-virus infected cells, that it was through tissue culture that polyoma virus was observed morphologically for the first time, and that proof could be given, also, for the first time, that the particles observed in mouse mammary cancer in the electron microscope were the causal agent. In myeloblastosis, likewise, the contribution of tissue culture has been enormous. Results in this instance are of prime importance, since they are concerned with a system extensively studied and in which biologic and biochemical tools are at hand as in no other virus-induced tumor (79-81).