serotype (strain NADL A-13) grew from inocula as small as two cells in liquid polysorbate 80 medium (P-80 medium), in P-60, P-40 and P-20 media, and in P-80 medium from which polysorbate, NH4CI or thiamine had been omitted. It grew well initially in vitamin Bi-deleted P-80 medium, but only with inocula as large as 26 × 10 cells per ml.

P-80 medium lacking both polysorbate and NH4CI supported light growth from small inocula, but the omission of thiamine and vitamin Bi in addition seriously affected the properties of the medium. Where readily detectable growth did not develop in liquid nutrient-deleted medium, viable organisms could often be demonstrated indirectly by subculture to semisolid medium, and their occurrence was influenced by the presence of albumin, thiamine, and vitamin B.

Growth on semisolid media was comparable with that in liquid media of similar composition. The absence of poly sorbate 80, thiamine, or vitamin B ! prevented the appearance of Dinger's zones of growth from small inocula.

Antigenic composition as measured by microscopic agglutination tests with homologous and heterologous antisera was not appreciably affected by repeated subculturing in various complete and incomplete media.

Homogenates of infected-hamster-kidney tissue in bovine serum-albumin diluent still contained viable organisms after 60 days' storage at 23-25°C. Organisms derived from this material after 3 and 16 days' storage showed no loss of virulence. Organisms grown in artificial culture showed no loss of virulence after storage in bovine albumin diluent or phosphate buffer for 7 days at 23-25°C.

Cultures of the organism survived without loss of virulence for 15 months in 13 semisolid media of differing complexity. Single colonies derived from five different solid media were grown in semisolid forms of the parent media and stored at 23-25°C for 10 months without loss of virulence.


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