Development of a chemically defined liquid medium for growth of Legionella pneumophila

Abstract

A chemically defined liquid medium was used to study the physiology and antigen [Ag] production of the Legionnaires disease bacterium. The medium contains basal salts, vitamins, .alpha.-ketoglutaric acid, pyruvate, 0.05% L-cysteine, 0.05% glutathione and a mixture of 20 additional amino acids, each of 0.01% final concentration, except serine, which was at 0.1%. The medium, in shake culture at 37.degree. C with increased CO2 at pH 6.5, supports the maximum rate of growth, the highest cell yields and the maximum cell surface Ag as distinguished by specific fluorescein isothiocyanate-conjugated antibody. CO2, pyruvate and .alpha.-ketoglutarate strongly stimulated growth. Cysteine and methionine were required for growth. Serine, threonine, histidine, tyrosine and tryptophan were energy sources. Glutathione substituted for cysteine, but cystine did not. The organisms did not glucose and polysaccharides, as judged by cell yields when these carbohydrates were present or absent. The chelators malate, citrate and EDT totally inhibited growth. .beta.-Mercaptoethanol, thioglycolate, dithiothreitol and Tween 80 (0.05%) inhibited growth strongly or completely. Catalase activity was extremely weak or absent. Morphology varied, depending upon conditions and phases of growth. Filamentous forms became chains of cigar-shaped bacilli fragmenting to pairs and becoming coccoidal in the late stationary phase of growth. The organism grew at 25, 30 and 37.degree. C. Although they varied in their growth characteristics, 10 isolates were passed for 5 transfers in the chemically defined broth, giving maximum rates of growth, cell yields and Ag production.