Orca flash 2.8 camera

The initial cultures of X. citri wt and X. citri pMAJIIc-envC were prepared by cultivating bacteria in 5.0 mL of NB medium for approximately 16 h at 30 °C and 200 rpm. The cultures were then diluted to an OD 600 nm of 0.1 using fresh NB medium for a final volume of 5.0 mL and subsequently cultivated under the same conditions until an OD 600 nm of 0.3 was reached. At this point, arabinose was added to the medium to a final concentration of 0.05%, and the cultures were maintained at 30 °C and 200 rpm. After a minimum of 2 h of induction, 5 µL drops of cell cultures were placed on agarose-covered microscope slides for direct microscope observation [21 (link)]. For chromosome visualization, X. citri wt, ΔenvC and ΔenvC pMAJIIc-envC cells were cultivated under the same conditions described above and stained with DAPI using the protocol described in Ref [22 (link)]. Bacterial visualization was conducted using an Olympus BX61 microscope equipped with a monochromatic OrcaFlash2.8 camera (Hamamatsu, Japan) and TxRed and DAPI filters. Data collection and analysis were carried out using the CellSens Version 11 software (Olympus). Statistical analyses were conducted using GraphPad Prism version 6.

Wild‐type Xcc, and the mutants Xcc amy::pPM2a‐zapA and Xcc parB::pAPU3 were cultivated as described in the growth curve section. Six flasks were prepared for each strain to be tested and when the cultures reached the OD_600 nm of ~0.4, three flasks were subjected to a 6 h temperature shift at 20°C, 37°C, 40°C, 42°C, or 45°C, while the remaining three flasks were kept at 30°C to serve as internal experimental control. Right at the end of the shifts, three slides were prepared, one for each of the triplicates, for microscope analyses (20 μL of cell culture were dropped onto 1X PBS/1% agarose‐covered slides). Cells were visualized in the magnification of 100× using an Olympus BX‐61 microscope equipped with an orca‐flash2.8 camera (Hamamatsu, Higashi‐ku, Hamamatsu City, Japan). Images were captured and processed using the software CellSens Dimension ver. 11 (Olympus Latin America, INC., Miami, Florida, USA).

Mid-log cultures of leptospires were observed by dark-field microscopy with or without 1% methylcellulose (Sigma), using ×20 to ×200 magnification with an Olympus BX-53 139 microscope connected to a Hamamatsu Orca Flash 2.8 camera. Images were taken with the CellSense Software (Olympus) and videos were recorded with the MicroManager 1.4 software (μManager). Images were post-treated and analyzed with the ImageJ software (ImageJ). Three images of each strain containing approximately 90 bacteria were used to measure cell length. Motility of Leptospira strains was also evaluated in triplicate onto 0.5% agar EMJH plates by inoculating 5 μl of mid-log phase cultures (OD_450nm of approximately 0.3). Plates were incubated at 29°C for 10 days.