A chemically defined liquid medium has been developed for the study of the physiology and
antigen 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 degrees C with increased CO2 at pH 6.5, supports the maximum rate of growth, the highest cell yields, and the maximum
cell surface antigen as distinguished by specific
fluorescein isothiocyanate-conjugated antibody. Studies during the development of this medium showed that CO2,
pyruvate, and
alpha-ketoglutarate strongly stimulated growth; that
cysteine and
methionine were required for growth; and that
serine,
threonine,
histidine,
tyrosine, and tryptophane were energy sources.
Glutathione substituted for
cysteine, but
cystine did not. The organisms did not use
glucose and
polysaccharides, as judged by cell yields when these
carbohydrates were present or absent. The
chelators malate,
citrate, and
ethylenediaminetetraacetic acid 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. In general, filamentous forms became chains of cigar-shaped bacilli fragmenting to pairs and becoming coccoidal in the late stationary
pha-e of growth. The organism grew at 25, 30, and 37 degrees C. Although they varied in their growth characteristics, 10 isolates were passed for five transfers in the chemically defined broth, giving maximum rates of growth, cell yields, and
antigen production.