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Using the filter paper disk diffusion assay technique as a tool in our antibiotic screening program, occasional samples have been encountered that were without inhibitory activity but induced a marked stimulation of the test organism, Bacillus subtilis, ATCC 6633. This effect was manifested as a zone of heavy growth in the region of the plate surrounding the disk saturated with the sample under test and was sharply demarcated from a less dense background. Low power microscopic examination of the area revealed larger surface colonies in the stimulated area. Within this stimulated zone, occasional subsurface colonies could be seen that were comparable in size with the smaller colonies in the area outside the stimulated zone, suggesting that stimulation of growth occurred only under conditions of strict aerobiosis. When an extract of Streptomyces sp mycelium became available that consistently produced the effect, an attempt was made to isolate the stimulatory factor. It was found that an acid extract of the ash of the sample contained the active principle. Tests with various inorganic ions revealed that manganese salts SUMMARY Manganese has been shown to stimulate sporulation of several species of the genus Bacillus. Sporulation is specifically dependent upon the presence of manganese at a concentration greater than that required for full vegetative growth of BaciUus subtilis, ATCC 6633, which was studied further as a representative of the genus.

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The mammary tumor virus (MuMTV) in the milks of 7 mouse strains and substrains was titrated for infectivity in 4 strains. The data indicated that: 1) Each strain shed a different MuMTV and some genetic strains carried two MuMTV's, each discernible by its mouse strain preference in infectivity tests. 2) Less than 5% of RIIIf and about 10% of Af mice shed detectable MuMTV antigen in their milks after the third parturition. After the sixth parturition, 33% of RIIIf and 50% of Af, and after the ninth parturition, 60% of RIIIf and 90% of Af mice shed viral antigen in their milks. The MuMTV's in milks of high-parity mothers were most infectious in mouse strains different from those most susceptible to MuMTV in RIII and A milks of low-parity mothers. Therefore, RIII and A mice each harbored two viruses, one that was removed by foster-nursing and the other that was not. 3) The susceptibility incidence of RIIIfC57BL mice to RIII virus changed gradually from about 10% in 1970 to about 70% in 1975. Susceptibility of C57BL mice to RIII virus did not change appreciably over this period, and the natural tumor incidence in RIIIfC57BL remained unchanged (about 10%). In addition to their susceptibility to RIII virus, C57BL mice were also susceptible to GR virus; they were relatively resistant to other strains tested. They were especially resistant to RIIIf virus, to which Af and BALB/c mice were very susceptible. 4) Approximately 90% of C3HfC57BL and C3HfBALB/c mice shed antigen in their milks after the third parturition, although the tumor incidence was less and occurred later than in C3H mice. No clear-cut differences could be detected in infectivities between low-parity C3H milk and high-parity C3Hf milks tested in several assay strains.

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