Fmax microplate spectrofluorometer

Evaluation of the ability of chondrillasterol to disrupt P. aeruginosa cells and cause leakage of nucleic acids was done according to the method of El-Nakeeb [35 (link)] with some modifications. P. aeruginosa (ATCC 27853) was grown overnight. The overnight culture was centrifuged at 2068 g for 4 min in a Hettich Rotofix 32 centrifuge (Tuttlingen, Germany) and the supernatant discarded. The pellet was suspended in 0.9% sterile saline until (OD₆₀₀ = 1.5). The suspensions were exposed to 100 μg/mL and 50 μg/mL of chondrillasterol and incubated at 37 °C with shaking (2 g-force) for 10 min in a Lab-Companion incubator (SI300 Incubated shaker, Jeiotech, Korea). The controls used were cells exposed to 0.1% SDS for the positive control and untreated cells served as the negative control. From each sample, 1 mL aliquots were centrifuged at 20427 g-force for 1 min (Hettich Rotofix 32 centrifuge, Tuttlingen, Germany). The pellet was washed with 0.9% saline solution and suspended in 3 mL of saline. A volume of 3 μL propidium iodide were added to each sample and the solution was mixed. The samples were kept in the dark for 10 min after which fluorescence was measured at excitation and emission wavelengths of 544 nm and 612 nm respectively using an fmax microplate spectrofluorometer (Molecular Devices, Sunnyvale, USA).

Propidium iodide is a dye that is capable of binding to nucleic acids. The dye is unable to enter viable cells, making it useful for determining the effects of plant extracts on bacterial membranes. B. cereus and E. coli cells were suspended in 0.9% saline solution (OD₆₀₀ = 1.5). The cell suspensions were exposed to plant extracts at concentrations of the MIC and double the MIC in duplicate for 10 minutes. The bacteria (1 mL) were centrifuged for 1 minute at 11000 ×g (Centrifuge 5415C, Eppendorf, Berlin, Germany). The pellet was washed with 1 mL 0.9% saline solution. Three microliters of propidium iodide was added to each sample and the solution was mixed. The samples were kept in the dark for 10 min. Fluorescence was measured at excitation and emission wavelengths of 544 nm and 612 nm, respectively, using an f_max microplate spectrofluorometer (Molecular Devices, Sunnyvale, USA). The controls used were nontreated cells, 3% DMSO, 0.1% SDS, and kanamycin.

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 (link)]. 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).