Coverslip

A single colony was inoculated in 1 ml of LBM supplied with 50 μg/ml gentamicin (if necessary), and grown at 37°C with 250 rpm shaking for overnight. The overnight culture was diluted 100-fold in 5 ml of LBM and grown at 37°C with 250 rpm shaking to an OD₆₀₀ of ~0.4. Next, 1 ml of the cell culture was collected by centrifugation at 3,000 × g for 2 min at room temperature and concentrated by 25-fold in fresh LBM. 10 μl of cells were then mixed with 10 μl of appropriate phage strains to reach an appropriate MOI, followed by incubation at 30°C for 10 min to allow for phage infection. The infection mixture was further diluted by 10-fold into 50 μl of fresh LBM at room temperature. 1 μl of the diluted culture was gently placed onto a piece of agarose pad (~1 mm thick) with 1:5 diluted LBM, arabinose (0.8%), and DAPI (5 μg/ml; Invitrogen™, No. D1306). A coverslip (No.1.5, Fisher Scientific) was gently laid over the agarose pad and the sample was imaged under the fluorescence microscope at 30°C within a cage incubator to maintain temperature and humidity.

1 mL of host cell MG1655 was grown in M9 minimal medium (11.3 g/L M9 salts, 1 mM MgSO₄, 0.5 μg/mL thiamine HCl, 0.1% casamino acids, 100 μM CaCl₂) supplemented with 0.4% maltose (M9M) at 37°C for overnight. The overnight culture was subsequently diluted 1:100 into 5 mL of M9M and grown at 37°C with 265 rpm shaking until OD₆₀₀ of ~0.3. 1 ml of cells were then concentrated by centrifugation at 2000×g for 2 minutes at room temperature, and resuspended in ice-cold M9M to OD₆₀₀ of ~3. 10 μL of phage stock was mixed with 10 μL of the resuspended cells to reach an appropriate API, followed by incubation on ice for 30 min and an additional 5 min incubation at 35°C water bath to trigger phage DNA ejection. The phage-cell mixture was then diluted by 10 fold into 50 μL of M9M at room temperature. 1 μL of the diluted mixture was placed onto a 1.5% agarose pad of M9M (~1 mm thick) until visibly dry (~1 min). A coverslip (No.1, Fisher Scientific) was gently laid over the mixture and the sample was imaged under the fluorescence microscope at 30°C within a cage incubator (InVivo Scientific, St. Louis, MO). For the phage DNA reporter movies, the same protocol was performed but with the reporter host strain (MG1655 seqA-mKO2 Cm^RΔdam::Kan^R) [12 (link)].