3ccd digital camera c7780

For chromosome segregation analyses (Figure 2), cells were fixed and stained as described (Carrasco et al., 2004 (link)). To obtain exponentially growing cells, overnight cultures were inoculated in LB rich medium. The recO16, ΔrecA, ΔrarA, ΔrecJ, ΔrecQ, ΔrecX, or ΔrecU mutants in the pcrA-ssrA sspB (pcrA_T) context (Table 1) were grown unperturbed in LB medium to OD₅₆₀ = 0.2 (37°C). IPTG (500 μM) was added to half of the culture, and both cultures were incubated (60 min, 37°C). Then, cells were collected, subjected to fixation with 2% formaldehyde, and finally stained with 4′,6′-diamino-2-phenylindole (DAPI) (1 μg/ml). Samples were visualized and photographed by fluorescence microscopy with a Hamamatsu 3CCD Digital Camera C7780 coupled to a BX61 Olympus fluorescence microscope, equipped with an 100x immersion oil lens and a DAPI filter (U-MNU2).
The ImageJ software (NIH) was used to merge the phase contrast and DAPI-fluorescence images, which allowed us to distinguish the septum, and thus determine the filamentation event and was also used to determine the cell length. Blind scoring was performed on captured images as described (Carrasco et al., 2004 (link)).

Cell samples for immunofluorescence microscopy were obtained and processed as described [34 (link)]. Cell samples were fixed with methanol/acetic acid (4:1) and adhered to poly-L-lysine pre-treated coverslips, and permeabilized with lysozyme (100 μg/ml, 2 min). Non-specific binding sites were first blocked by incubating cells in 2% bovine albumin (BSA, Serva) in PBS (20 min), followed by incubation (overnight, 4°C) with purified antibodies diluted in blocking solution. FtsZ, ZipA⁺, FtsA⁺, FtsA*, FtsQ, FtsK and FtsN proteins were detected with antibodies MVC2, MVC1, MVC3, MVC9, MVC6, MVG1 (S3 Table). Unbound primary antibodies were removed by extensive washing, followed by incubation with secondary antibody Alexa 594-conjugated anti-rabbit antibody (Invitrogen A-11037) (S3 Table) to detect the proteins (red signal). The nucleoids were stained with DAPI (25μg/ml) (Sigma D9542). Cover slips were mounted in Vectashield medium (Vector Laboratories) and sealed.
Cells were imaged with a Hamamatsu 3CCD Digital Camera C7780 coupled to a BX61 Olympus fluorescence microscope, equipped with an 100x immersion oil lens. The filters used to detect the red signal (proteins) or the blue signal (nucleoids) were U-MWTY2 and U-MNU2 respectively. The images were captured and deconvolved with SimplePCI imaging software. Intensity levels and image overlay were adjusted using Adobe Photoshop CS3.

For chromosome segregation analyses, cells were fixed and stained as described [41 (link)]. To obtain exponentially growing cells, overnight cultures were inoculated in LB rich medium. Cells were grown unperturbed in LB medium to OD₅₆₀ = 0.2 with shaking at 37 °C. IPTG (500 μM) was added to half of the culture, and both cultures were further incubated (60 min, 37 °C). Then, cells were collected, subjected to fixation with 2% formaldehyde, and finally stained with 4′,6′-diamino-2-phenylindole (DAPI) staining (1 μg/mL). Samples were visualized and photographed by fluorescence microscopy with a Hamamatsu 3CCD Digital Camera C7780 (Hamamatsu, Japan) coupled to a BX61 Olympus fluorescence microscope (Tokyo, Japan), equipped with a 100× immersion oil lens and a DAPI filter (U-MNU2).
The ImageJ software (NIH, Bethesda, MD, USA) was used to merge the phase contrast and DAPI-fluorescence images, which allowed us to distinguish the septum, and thus determine the filamentation event, and was also used to determine the cell length. Blind scoring was performed on captured images as described [41 (link)].