2.2. Antibacterial Susceptibility Testing

Antibacterial susceptibility tests were carried out according to [12]. Single colonies of bacteria were picked from an agar plate and inoculated in Luria broth media. The bacterial suspension was incubated overnight. Bacterial suspension was adjusted to an equivalent of 0.5 McFarland's standard. The concentration of cells was adjusted to 1 × 10⁶ cfu/ml by diluting with media. Antibacterial effects of the compounds were tested using the broth microdilution assay. Each compound was dissolved in DMSO. Concentrations of 0 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/ml, and 100 μg/ml of the compounds were prepared from stock solution. Ciprofloxacin was used as the standard antibiotic. A volume of 100 μl of test compound and 100 μl of cells were added to wells onto a 96-well plate making a total of 200 μl in each well. This was followed by 24 hour incubation. After 24 hour incubation, MTT assay was carried out to determine the cell viability. A volume of 20 μl of MTT reagent was added to every well and incubated for 2 hours. A dark purple color indicated presence of viable cells. [13]. Absorbance was read at 590 nm using a Tecan Genios-Pro microplate reader (Tecan Group Ltd Mannedorf, Switzerland). Minimum inhibitory concentration (MIC) was determined, the lowest concentration that exhibited absence of viable cells seen as a yellow color of the MTT. Cell viability was calculated and expressed as a percentage.

Bactericidal properties of the test compounds were assessed using a time-kill assay [14]. The bactericidal properties were tested by broth microdilution, performed on 96-well plates. The plates were incubated at 37ºC, and absorbance was measured at 590 nm using a Tecan Genios-Pro microplate reader after time 2 hours, 4 hours, 8 hours, 24 hours, 28 hours, and 32 hours. The checkerboard assay was performed as described by Chang et al. [14]. Varying concentrations of curcumin (25, 50, and 100 μg/ml) and ciprofloxacin (0.0625, 0.125, 0.25, 0.5, and 1 μg/ml) were combined to investigate the effect of the combined drugs on P. aeruginosa and S. aureus.

Propidium iodide, a dye that is capable of binding to nucleic acids, was used to investigate the effects of the drugs on bacterial membranes as described by Moyo and Mukanganyama [15]. The dye is unable to enter viable cells. P. aeruginosa and S. aureus cells were suspended in 0.9% saline solution (OD600 = 1.5). The cell suspensions were exposed to different concentrations of the drugs, half the MIC (½ MIC), MIC, and twice the MIC (2 × MIC) in duplicate for 10 minutes. 1 ml of the bacterial suspension was centrifuged for 1 minute at 11 000 rpm. The pellet was washed with 1 ml 0.9% saline solution. A volume of 3 μl of propidium iodide was added to each sample, the solution was mixed, and samples were kept in the dark for 10 minutes. Fluorescence was measured at excitation and emission wavelengths of 544 nm and 612 nm, respectively, using a fmax microplate spectrofluorometer (Molecular Devices, Sunnyvale, USA). The controls used were untreated cells and 0.1% sodium dodecyl sulphate (SDS).

Cells were suspended in 0.9% saline solution (OD₆₀₀ = 1.5). Cell suspensions were exposed to drug curcumin at concentrations of 1/2 MIC, MIC, and 2 × MIC. Samples were incubated at 37ºC with shaking (120 rpm) for 120 minutes. A volume of 500 μl cell suspension was centrifuged at 7000 rpm for 2 minutes. The protein content was determined using Bradford's method. Briefly, 950 μl of coomassie brilliant blue G-250 was added to 50 μl of the supernatant. The color was allowed to develop for 10 minutes, and absorbance was measured at 590 nm using a Tecan Genios-Pro microplate reader. The controls used were 0.1% SDS and untreated cells. Bovine serum albumin (BSA) was used as a standard to determine protein concentration.