Carbon coated copper grids

Growth of Tn5 insertants was assessed by measuring optical density (OD) at 565 nm. Cultures with no severe growth defect were further screened for magnetic phenotype by determination of c_mag (36 (link)). For a set of mutants, cells were also assessed by optical microscopy for their swimming behavior, cell shape, and alignment in response to an externally applied magnetic field. Magnetosome morphology was analyzed with respect to size, shape, and numbers per cell by TEM analysis. For this, concentrated cells were adsorbed onto carbon-coated copper grids (Science Services, Munich, Germany) and imaged at 80 kV without negative staining in a TECNAI F20 microscope (FEI, Eindhoven, Netherlands). Ultrastructural analysis of mutants provided information on modifications in magnetosome biosynthesis as well as magnetosome organization.

Transmission electron microscopy of whole cells was performed with specimens directly deposited onto carbon-coated copper grids (Science Services, Munich, Germany). For TEM imaging a Jeol Jem 1400 + (Freising, Germany) was operated at an acceleration voltage of 80 kV. Image acquisition was performed with a Gatan Erlangshen ES500W CCD camera. Average particle sizes were measured by data processing with ImageJ software package v1.52i. For quantitative assessment of magnetite particle biomineralization at different timepoints, specifically at the process start, in the mid-exponential growth phase and at end of growth 1000 magnetosome particles per triplicate were counted and combined for evaluation.