Orca flash4.0 lt digital cc11440 42u cmos camera

Wild type F. tularensis LVS was cultured in CDM for 24, 48, 96, and 336-h in triplicate. At each time point 1 ml of bacteria were incubated for 30 min with 10 μg ml^–1 FM 4–64 fluorescent dye before being spotted onto pads of 1% agarose in PBS. Fluorescently labeled bacteria were spotted onto pads of 1% agarose in PBS and imaged using an Olympus IX73 microscope equipped with a 100x NA. 1.30 Phase objective and an ORCA-Flash4.0 LT+ Digital CC11440-42U CMOS camera (Hamamatsu). Exposure times of 50 ms were used for each channel, and images were processed using ImageJ (Schneider et al., 2012 (link)) where the dark and light points were set to the same levels for all images. Bacteria that had not been stained with FM 4–64 were used as a negative control for fluorescence.

Francisella tularensis LVS cells expressing emgfp were cultured in CDM for either 24 h (Culturable) or for 504 h (VBNC) at 37°C. Bacteria were normalized by optical density and diluted in fresh growth medium. Six replicates were aliquoted in a 96-well plate for incubation with shaking at 37°C with or without 100 μg/ml gentamicin in a BioTek Synergy H1 plate reader. Optical density and fluorescence were measured at 2-h intervals. To obtain fluorescence micrographs, F. tularensis LVS bacteria expressing emgfp were cultured in CDM for either 24 h (Culturable) or for 552 h (VBNC) at 37°C. Cultures were then incubated for 1 h with (+) or without (-) 100 μg ml^–1 gentamicin. Bacteria were spotted onto pads of 1% agarose in PBS and imaged using an Olympus IX73 microscope equipped with a 100x NA. 1.30 Phase objective and an ORCA-Flash4.0 LT+ Digital CC11440-42U CMOS camera (Hamamatsu). Exposure times of 50 ms were used for each channel, and images were processed using ImageJ (Schneider et al., 2012 (link)) where the dark and light points were set to the same levels for all images. F. tularensis LVS containing the empty vector pSC13 was used as a control for fluorescence.