Titrations of Mammary Tumor Virus in Fresh and Treated RIII Milk and Milk Fractions2

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Abstract
Freezing and thawing 1 time destroyed more than 99% of the bioactivity of a sample of undiluted skim milk, and freezing and thawing 5 times destroyed more than 99.9%. Freezing after a 100-fold dilution in phosphate-buffered saline was less destructive. Incubation at 37°C for 18 hours had little effect on the bioactivity of either undiluted or diluted milk. A correlation was found between the content of B particles and bioactivity of fractions taken after centrifugation of milk concentrates on preformed gradients of Ficoll. Fractions were assayed by observation of tumor incidence in C57BL/Haag mice and nodule incidence in BALB/c mice. The two methods gave comparable results. Although the B particles and the bioactivity were concentrated in one area of the centrifuge tube, no part of the tube was void of either. The bioactivity of any sample could be determined only by titration to the endpoint. There was no relationship between the tumor incidence or nodule incidence and concentration of infectious particles, except where the dilutions approached the endpoint. The tumor incidence of the principal virion zone (No. 3) increased from 10% at 10−1 dilution to 55% at 10−7 dilution. In convection-free or twice-repeated centrifugations of fresh milk, supernatants free of bioactivity were obtained.

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