Cultivation of Bacterium tularense in Simplified Liquid Media

Abstract

Liquid media constructed from either gelatin or casein hydrolysates, or from amino acids, readily supported growth of Bact. tularense when they were supplemented with accessory factors. These media are accurately reproducible and contain neither protein nor unknown substances of large mol. weight. They are suitable for large scale cultivation of the organisms for chem. analysis. In basal media without supplements, visible growth was not obtained with inocula smaller than 1,000,000 organisms. The significant accessory factors were in liver-cake extract, blood cell extract, and biotin concentrate. Pantothenic acid, pimelic acid, nicotinic acid and biotin, in mixture permitted limited growth, but they were inadequate substitutes for liver-cake or blood cell extracts. The unknown potent factors in liver-cake and blood cell extracts were thermostable and dialyzable. Although all necessary accessory factors are not yet chemically definable, close approximations to complete media must have been reached inasmuch as inocula of approx. 1-15 organisms to 5 ml. of media were sufficient to initiate sustained growth. The indispensability of cystine or cysteine was confirmed. The NaCl tolerance of the organism was high. Slightly above 1% was optimal, and 2% NaCl was not inhibitory. Virulence of cultures was maintained at or near maximal after prolonged continuous cultivation in liquid media. The production of a soluble toxin by Bact. tularense could not be demonstrated. Growth in liquid media produced steadily increasing alkalinity.