Figure 4. σB -dependent expression of EGFP is increased in L. monocytogenes EGD-e wild-type integrant in stationary phase and by osmotic stress. Cells of integrants bearing Plmo2230::egfp fusion were grown from starting OD600 = 0.05 up to either exponential (OD600 = 0.6) or stationary phase (16 h) in BHI or BHI supplemented with 0.5 M NaCl. A similar number of cells (equivalent to 1 ml of OD600 = 0.6 and diluted 1:2 by the fixing procedure) was diluted 1:10 in PBS and concentrated on 10 mm diameter, 0.2 μm pore polycarbonate membrane (Millipore) by filtration. For each experimental condition three biological replicates were analyzed and a minimum ten randomly selected fields of each membrane were captured with a CCD camera attached to Nikon Eclipse E600 with B-2A and ND8 filters used at a fixed exposure time of 2 sec with representative fields shown (A). Fluorescence levels of cells were quantified with automated image processing of microscopic fields by counting numbers of detectable particles (B) and their relative fluorescence intensities (C) with ImageJ 1.44 software with appropriate manipulations as described by others.39,40