Experiments, relevant to growth in milk, were done to delineate the aerobic and anaerobic growth of Listeria species on selected sugars in several media. All species grew on glucose aerobically, forming lactic acid and (or) acetic acid. Anaerobically, only lactic acid was formed; cell yields were 80% of those obtained aerobically. When incubated aerobically, small amounts (1.5 microns/mL) of isovaleric acid, 2-hydroxyisovaleric acid, and trace amounts of isobutyric acid were formed. These products were characteristically formed by 26 strains representing all the species of Listeria. Added leucine stimulated isovaleric acid formation. Anaerobic fermentations of glucose could be followed by 60 to 80% cell lysis; less lysis occurred in air. Anaerobically, only hexoses and pentoses supported growth; aerobically, maltose and lactose supported growth of some strains, but sucrose did not support growth of any strain tested. Listeria grayi and Listeria murrayi utilized the galactose and glucose moieties of lactose for growth; Listeria monocytogenes and Listeria innocua used only the glucose moiety. Glucosamine and N-acetylglucosamine supported aerobic and anaerobic growth as well as glucose, and their presence stimulated the utilization of lactose by "lactose-negative" strains. Analyses of cultures grown at 5 degrees C in sterile milk treated with glucose oxidase supported the conclusion that the glucose of the milk was the major, if not the limiting, substrate that supported growth.

The amino acids required for growth and as energy sources by 10 strains of Legionella pneumophila were determined by using a chemically defined medium. All strains required arginine, cysteine, isoleucine, leucine, threonine, valine, methionine, and phenylalanine or tyrosine. Most strains (7 of 10) required serine, and two strains had to be supplied proline before growth could be established. All 10 strains used serine and, to a lesser extent, threonine as the sole sources of

Growth of Legionella species in a defined medium deficient in iron did not result in the production of phenolic or hydroxamate siderophores which could be detected by chemical or biological assay methods. Growth of a variety of other gram-negative organisms under the same conditions resulted in the production of both hydroxamate and phenolate siderophores. The iron-deficient medium limited growth of the Legionella species more severely than it did the growth of the other gram-negative organisms. We have concluded that Legionella species do not make the commonly recognized siderophores, probably because they are restricted in their growth to those environments in which inorganic iron is readily available or is supplied in a form bound to an unknown carrier.

Serial passage of six strains of Legionella pneumophila and one strain of Pseudomonas aeruginosa in a liquid chemically defined medium deficient in trace metals resulted in the death of five L. pneumophila strains and very limited growth in the remaining strain and the P. aeruginosa strain. Addition of either iron or magnesium restored growth to almost normal levels in ail of the strains when early-passage inocula were used. A low concentration of magnesium stimulated growth with cobalt, copper, iron, manganese, molybdenum, vanadium, or zinc. When a complete defined medium containing trace metals was used, growth was inhibited by adding the chelators ethylenediaminetetraacetic acid, citrate, or 2,2'-bipyridyl. Chelator inhibition was partly or fully relieved with either calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, nickel, vanadium, or zinc. P. aeruginosa differed from L. pneumophila in that it required higher concentrations of each chelator to inhibit growth and that its growth was stimulated by only four metals: calcium, iron, magnesium, and zinc. A trace-metal supplement for L. pneumophila was designed which included all metals stimulating growth in these experiments and which proved to be sufficient for optimal growth of all the strains.

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.