Bacteria Live/Dead Staining Kit

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	Incubate both samples at room temperature for 1 hour, mixing every 15 minutes. The final dye concentration in suspension should be 5 μM DMAO and 4 μM EthD-III

Protocol tips
Incubate both samples at room temperature for 1 hour, mixing every 15 minutes. The final dye concentration in suspension should be 5 μM DMAO and 4 μM EthD-III

Publication protocol

Besides using the MBC and MIC assays, we also used the bacteria live/dead staining kit (PromoCell GmbH, Germany) to visualize the antimicrobial selectivity of PMBs against a mixture of different families of bacteria. The staining kit contains fluorescent dyes DMAO (ex/em ∼490/520 nm) and EthD-III (ex/em ∼530/∼ 635 nm) in DMSO. With an appropriate mixture of DMAO and EthD-III, bacteria with intact cell membranes (i.e., live) stain fluorescent green, whereas bacteria with damaged cell membranes (i.e., dead) stain fluorescent red. In a typical test, bacteria were first grown to mid log phase (OD600 = 0.5–0.7), harvested by centrifugation at 6000 rcf for 5 min, and washed with sterile PBS buffer (pH = 7.4, 150 mM NaCl, 10 mM NaH2PO4) twice. The bacterial cells were then resuspended in PBS buffer. A mixed suspension of E. coli and S. aureus was prepared by mixing both bacteria at an OD600 ratio of 1/1. The bacteria mixture was diluted to OD = 0.2 and incubated with sPMB or L-rPMB solutions for 3 h. The ratio of the amount of polymer versus the amount of bacteria was adjusted to be roughly the MBCE. coli/(5 × 105 CFU/mL). The mixed bacteria incubated with PBS buffer without PMBs were used as controls. After the incubation, the mixed bacteria were washed and resuspended with Tris buffer (pH = 7.4, 150 mM NaCl, 10 mM Tris). The staining kit was applied to the suspension and incubated for 15 min following the instruction provided by the manufacture. The final dye concentration in suspension was 5 μM DMAO and 4 μM EthD-III. The bacteria were washed and resuspended with Tris buffer. Ten μL of suspension was imaged under a Nikon laser scanning confocal microscope (Nikon T1-E microscope with A1 confocal and STORM super-resolution modules) using a 100× oil-immersion objective lens

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Manufacturer protocol

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