FIG. 1.

Characteristics of L. pneumophila exiting the exponential phase of growth. (A) Correspondence between the OD₆₀₀ and the growth phase of L. pneumophila AYET broth cultures. An exponential-phase culture was diluted 1:350 (▵), 1:120 (×), 1:35 (□), or 1:16 (○) and incubated at 37°C for 15 h; then the OD₆₀₀ of each subculture was determined at the times shown. Results from four representative cultures were superimposed such that their growth curves overlapped; similar results were obtained in several other experiments. (B) L. pneumophila sodium sensitivity was determined by plating cultures with the OD₆₀₀s shown on CYET medium without (open symbols) and with (closed symbols) 100 mM NaCl. Three experiments performed in duplicate are represented by different symbols (triangles, circles, and squares). (C) L. pneumophila cytotoxicity was judged by the capacity of viable macrophages to reduce the colorimetric dye Alamar blue after a 1-h incubation with bacteria obtained from AYET cultures grown to OD₆₀₀s of 0.374 (○), 1.519 (▵), 1.882 (□), 2.151 (•), and 2.231 (▴). The means of values for triplicate samples are shown; standard errors ranged from 1.5 to 8%. The multiplicity of infection (MOI) was calculated by plating the respective broth culture on CYET. The means of values for triplicate samples are shown; standard errors ranged from 2.5 to 14%. (D) Osmotic resistance of L. pneumophila cultures grown to the OD₆₀₀s indicated was determined by incubation for 1 h in AYET that did or did not contain 0.3 M KCl, dilution into water, and then plating of duplicate samples on CYET to quantify the CFU. Results from each of four experiments are represented by different symbols (triangles, squares, circles, and diamonds). (E) The ability of L. pneumophila grown to the indicated OD₆₀₀s to evade macrophage lysosomes was quantified by fluorescence microscopy, using the endocytic probe Texas red-ovalbumin. Data were obtained from four experiments, in which the number of phagosomes scored was 35 (◊), 75 (□), 50 (○), or 100 (▵). As a positive control, bacteria grown to an OD₆₀₀ of >2 and then killed with formalin were analyzed; a mean of 67% (standard error = 13) of such particles colocalized with Texas red-ovalbumin. (F) The capacity of L. pneumophila grown to the OD₆₀₀s indicated to enter and survive in macrophages was defined as protection from gentamicin added 2 h after infection. The mean percentage of infectious L. pneumophila was determined for duplicate or triplicate samples in four experiments, each represented by a different symbol.