Quantifying Bacterial Polyamine Levels

Polyamines were measured using the Total Polyamine Assay Kit (MAK349, Sigma). A 100 µL aliquot of the indicated bacterial cultures was collected, centrifuged, washed with 1× PBS, and resuspended in PBS. Bacteria were lysed with 1:10 vol/vol chloroform, vortexed, and incubated at room temperature for 2 h. The solution was centrifuged, and the top aqueous layer was collected. Following the manufacturer’s instructions, 1.0 µL of the collected sample was mixed with the Total Polyamine Assay Kit reagents, incubated at 37 °C for 30 min, and read using a CLARIOStar plate reader using end point fluorescence (λ_ex = 535 nm/λ_em = 587 nm). Polyamine concentrations were determined by comparing values to a standard curve constructed from known concentrations of putrescine. Values were then normalized to OD₆₀₀ measurements taken from the original bacterial cultures.