Axiocam digital

B.B. burgdorferi cells (5 × 10⁷ spirochetes/ml) were observed under a dark-field microscope (Zeiss Axio Imager M1), and images were captured using an AxioCam digital camera. Swarm plate motility assays were performed as described (Sultan et al., 2015 (link)). Approximately 1 × 10⁷ cells in a 5 µl volume were spotted onto 0.35% agarose plate containing plating BSK medium diluted 1:10 in Dulbecco’s phosphate buffered saline. Since B. burgdorferi is a slow growing organism (8–12 hr generation time), plates were incubated for 5 days at 35°C in a 2.5% CO₂ humidified incubator. To determine cell morphology, growing B. burgdorferi cells were observed under a dark-field microscope (Zeiss Axio Imager. M1).

E. coli K12 cultures and ∆ClpB mutant cultures were spun down at 3200× g during 5 min at 4 °C and pellets were dissolved in 1 mL of PBS. Then, 20 µL of the bacterial solution were spread on slides, fixed by ethanol, and dried at 37 °C for 15 min. Immunocytochemistry was performed using triton solution (1%) to permeabilize bacteria followed by blocking solution (PBS, 5% Bovine serum albumin, 1% Triton solution, 0.2% Sodium Azide). Mouse monoclonal antibody against ClpB (1:50, Delphi Genetics) was incubated overnight at 4 °C and revealed with a secondary anti-mouse antibody (1:200) conjugated to fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch) for 1.5 h at room temperature. Two drops of 4′,6-diamidino-2-phenylindole (DAPI) (Sigma) were added to each slide to counterstain the bacterial DNA. Microscopy was performed and images were taken using a Zeiss Imager Z1 fluorescence microscope equipped with an Apotome and an AxioCam digital camera (Zeiss, Oberkochen, Germany). Immunopositive cells were counted at 100× magnification from 5 different slides per group.

Animals were immobilized in 0.01% tetramisole hydrochloride (Sigma‐Aldrich) on 4% agar pads and visualized using a Zeiss Axioskop equipped with epifluorescence and DIC/Nomarski optics, and images were collected with an AxioCam digital camera. To discriminate dying MN fluorescence from endogenous autofluorescence, a Zeiss filter set 09 was used. This setting allowed the observation of intestinal cell autofluorescence in yellow and apoptotic fluorescence‐positive dying cells in green.

Zeiss AxioImager A1 microscope was used to examine immunofluorescent stained sections, and images were captured using an Axiocam digital camera with Zeiss Axiovision computer software version 4.8. Images were transferred to Photoshop for sizing and annotation and exported at TIFF flies.