Computational protocol: P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality

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Protocol publication

[…] Cultures were grown at treated as in “arabinose survival curve at 1x108 CFU/mL” and were allowed to grow at 30°C for four hours (or overnight) after arabinose treatment. A 5 μL aliquot of each culture was removed and incubated with an equal volume of 10 μM DAPI for 5 minutes at room temperature. Then, 1–2 μL of the mixture was placed on a 1.5% agar pad, allowed to soak in, and flipped on to a glass microscope slide. All pictures were taken within a half hour of mounting.All the images were acquired on a commercial Olympus IX83 inverted microscope equipped with an Olympus UPLSAPO60XS, 1.42 NA silicone oil immersion objective and Hamamatsu C11440 charge-coupled device (CCD) camera operated by a personal computer (PC) running MetaMorph for Olympus software. Brightfield images were obtained using an X-Cite 120 halide arc lamp (Lumen Dynamics Group, Inc.) and Olympus IX3-LWUCDA motorized long working distance (LWD) condenser. The fluorescence excitation for DAPI was performed using the Olympus standard components, and the emission for DAPI was obtain using a 5060C-OFF-Zero (-ZERO pixel set mounted in cube, Semrock) filter set. Contrast of images was adjusted for publication using ImageJ or Adobe Photoshop. Cell length measurements were obtained using the MicrobeTracker plugin for Matlab [] and were converted to μm using the conversion factor 0.1083 μm/pixel. […]

Pipeline specifications

Software tools MetaMorph, ImageJ, MicrobeTracker
Application Microscopic phenotype analysis
Organisms Neisseria gonorrhoeae, Escherichia virus P1, Escherichia coli, Staphylococcus aureus, Bacteria, Homo sapiens
Chemicals Ciprofloxacin