Several facts were known concerning the milk agent: For instance, we knew that it can pass through Seitz and Berkefeld filters (10) and is transmissible through milk. When injected into susceptible mice, it induces the formation of mammary tumors in 10 to 15 months. Attempts to shorten this latent period have so far been unsuccessful. Since none of the current serological or immunochemical methods are accurate enough to detect the MTA, bioassays remain the only available test. Through bioassays, it has been established that the cancerogenic power of milk, or infectivity, is heat labile and is completely destroyed in a little more than 1 hour at 37° C. (11). This could be a handicap for its cultivation; on the other hand, that it has been found stable in a range of hydrogen ion concentration varying from pR 5. to 10.2 (12) is an asset, because the pH. of tissue-culture media is usually adjusted to neutrality. Two other factors could be of consequence for successful cultivation: (a) the source of the agent, (6) the choice of a tissue substrate.

The milk from RIII mice has been found a potent source material. Injected into susceptible mice, it can induce tumors even after dilutions up to 10^-10 or 10^-12. The test mice used for all our bioassays are from our Columbia C57BL strain sensitive enough to respond to such high dilutions, yet completely agent-free, since not a single spontaneous tumor has been recorded in the colony in the past 12 years.

Such mice were indicated to provide the tissue substrate of the cultures, and since the milk agent has a special affinity for the mammary epithelium, the mammary epithelium had to be grown. For many years, the growth of this tissue met with considerable difficulties. In 1950, Hardy (13) demonstrated the possibility of obtaining differentiation in organ cultures of rudimentary mammary systems from the abdominal skin of mouse embryos. We adopted this method in our first experiments. Later on, however, the enzymatic treatment of adult mammary glands enabled us to grow sheets of mammary epithelium in conventional roller tubes (14).