ABSTRACT
Methods for measuring O(2) within living cells that rely on luminescent probes are hampered by several factors: local conditions of hydrophobicity, pH, ionic composition, dielectric constant, and photobleaching by free radical species. Use of a polymer-embedded luminophore should minimize these problems. Here we use a Ru(II) coordination complex embedded within 45 nm hydrodynamic diameter nanoparticles, and demonstrate that both phosphorescence intensity and lifetimes are O(2)-sensitive, both in aqueous suspensions and intracellularly (e.g. 4.06 versus 1.55 microseconds under anaerobic or aerobic conditions, respectively). Electroporation is necessary for incorporation of the nanoparticles into yeasts: it is more effective with the fission yeast, Schizosaccharomyces pombe, than for the budding yeast, Saccharomyces cerevisiae. However, electroporation was not required for particle uptake into a cultured human cell-line (mammary adenosarcoma MCF-7), although the intracellular distribution of the probe is more general to intracellular compartments when electroporation is employed. These procedures did not compromise vitality of cells over periods of 6 h, as judged by retention of structural characteristics evident in Nomarski interference or confocal microscopy images. Spatial resolution of intracellular structures defined by nanoparticle phosphorescence intensity imaging indicates potential usefulness of the application of lifetime imaging techniques for mapping of intracellular O(2) distributions.
Subject(s)
Acrylic Resins/chemistry , Intracellular Space/metabolism , Luminescent Agents/chemistry , Nanoparticles/chemistry , Organometallic Compounds/chemistry , Oxygen/metabolism , Phenanthrolines/chemistry , Aerobiosis , Anaerobiosis , Cell Line, Tumor , Electroporation , Humans , Luminescent Agents/metabolism , Luminescent Measurements , Molecular Imaging , Organometallic Compounds/metabolism , Phenanthrolines/metabolism , Saccharomyces cerevisiae/cytology , Schizosaccharomyces/cytologyABSTRACT
Identification of Salmonella serotypes is based on flagellar and somatic antigens. The absence of flagella may consequently affect complete identification of the serotype; here it is shown that Salmonella enterica serovar Typhimurium exhibits morphological differences dependent on the peptone constituents of the culture medium. Aflagellate salmonella were produced in certain media where the nutritional ingredient was casein-based peptone or gelatin-based peptone; in gelatin-based peptone, aggregates of salmonella were observed. However, in media containing soy-based peptone as the primary nutrient, salmonella displayed a normal flagellated morphology. Transfer of aflagellate salmonella from nutritionally poor media, with casein- or gelatin-based peptone, into rich nutrient broth allowed flagella synthesis, indicating that the aflagellate form is still able to produce flagella. Amino acid sequencing of the peptones producing aflagellate organisms showed a relatively low tyrosine concentration: only 0.03+/-0.01 g l(-1) for gelatin-based buffered peptone water, compared to 0.21+/-0.01 for soy-based buffered peptone water. Tyrosine is essential for flagellin, which is the subunit of the salmonella flagellar filament. The addition of 200 muM tyrosine to casein-based peptone media produced flagellate salmonella; 2 mM glucose was needed in addition to tyrosine to achieve a similar morphology in gelatin-based media. Therefore, culture media containing less than 1.20 g tyrosine l(-1), and of limited carbohydrate source, when used for serological testing of clinical isolates, may result in an incomplete serological identification.
