Figure 2.

Daunorubicin accumulation within WHO-2 pretreated with C and ECg. WHO-2 was treated with broth, C (256 mg/L), ECg (32 mg/L) and C and ECg (64 mg/L + 8 mg/L, 128 mg/L + 16 mg/L, 256 mg/L + 32 mg/L) and then cultured for 6 h at 37 °C in a heated, shaking, environmental chamber. Then, the bacteria were centrifuged at 3500× g for 5 min. After washing three times and resuspending, the bacterial suspension was adjusted to an OD₆₀₀ of 1.0. (A) Bacteria were incubated with daunorubicin (40 mg/L) in the dark at 37 °C for 30 min; 0.5 mL of bacteria was collected. Bacteria were washed three times and resuspended with PBS. Next, the bacteria were fixed on glass cover slips and observed under a 510 Meta confocal microscope (Zeiss, Göttingen, Germany); (B) Bacteria were incubated with daunorubicin (40 mg/L) in the dark at 37 °C for 2, 10, 20 and 30 min; 0.5 mL of bacteria was collected. Bacteria were washed three times and resuspended with phosphate buffered saline (PBS). Then, quantitative determination of daunorubicin accumulation in the absence and presence of C and ECg was performed using fluorospectrophotometry at an emission wavelength of 467 nm and an excitation wavelength of 588 nm.