Bacterium Inoculums preparations for the MIC and MBC experiments
From the previous isolated and identified MRSA and VRSA S. aureus isolates, four MRSA+VRSA and four MRSA+VSSA isolates were chosen for MIC and MBC experiments. With a sterile loop, a loopfull from each chosen isolate was inoculated from the BHI slant into MH broth then incubated with shaking at 37°C overnight for 24 hours. The concentrations of these suspensions were adjusted to be equal to the 0.5 McFarland standards by adding a sterile saline. Test and standard tubes were compared against a white background with a contrasting black line. Then the Suspensions concentrations were adjusted by spectrophotometer to an optical density of 0.10 at 625 nm to obtain the concentration of 1×108 CFU/ml. Finally, the cultures suspensions were then diluted to be equal to the concentration of 5×105 CFU/ml. Antimicrobial agents' preparations for the MIC and MBC experiments i. Dodonaea angustifolia The D. angustifolia plant was kindly obtained from Professor Doctor Fayez Zaki, Professor of entomology, biological control, plant protection, National Research Centre. The plant extract was extracted according to the method described in (Abd El-Moez et al., 2014) with some modification; briefly, the plant was collected, washed under tap water, rinsed with distilled water, dried in drying hot air oven at 40°C, and grinded. 50 grams of the shade resulting material were taken and put with continuous shaking in 500ml absolute ethanol for 24 hours. The extract was then filtered through a 22 μm filter paper then the resulting filtrate was combined and dried using a rotatory evaporator at 40°C. The previous procedure was repeated three times then 100 mg of the resulting powder materials were dissolved in 1ml dimethyl sulfoxide solvent (DMSO: Water, 2:4 v/v) to prepare the stock solution of 100 mg/ml. Then this stock will be 10 fold diluted to obtain 10 mg/ml then two-fold serial dilutions were done using MH broth to prepare the following concentrations (5, 2.5, 1.25, 0.625, 0.3, 0.15, 0.08, 0.04, 0.02 and 0.01 mg/ml). Fig 1 Dodonaea angustifolia plant ii. Honey Two 500 ml bottles of honey were purchased and used in this study; the first one was processed well-identified …show more content…
angustifolia extract, honey and AuNPs against the eight chosen S. aureus isolates (four MRSA+VRSA and four MRSA+VSSA) were analyzed through determination of their minimum inhibitory concentrations (MIC) values, minimum bactericidal concentrations (MBC) values, and MBC/MIC ratio by broth micro-dilution method using 96-well micro-titer plates. To each 50 μL of the antibacterial agent dilution, 50 μL of adjusted bacterial concentration inoculum (5 × 105 CFU/mL) were added in the 96-well micro-titer plates, the growth control wells contained MH broth medium with tested bacterial concentrations and sterility control wells contained only MH broth medium as shown in figure 2. The plates were then covered to ensure that the bacteria were not dehydrated. Then the plates were incubated at 37°C for 18 to 20 hours. The lowest concentration of each antibacterial agent that inhibited the bacterial growth was considered as the MIC (CLSI, 2006). After the MIC determination, aliquots of 100 μL from each well that does not show any bacterial growth after incubation were streaked onto BHI agar plates followed by incubation at 37°C for 20 hours. The lowest concentration that kills 100% of the initial bacterial population showing no colonies on the BHI agar was recorded as the
From the previous isolated and identified MRSA and VRSA S. aureus isolates, four MRSA+VRSA and four MRSA+VSSA isolates were chosen for MIC and MBC experiments. With a sterile loop, a loopfull from each chosen isolate was inoculated from the BHI slant into MH broth then incubated with shaking at 37°C overnight for 24 hours. The concentrations of these suspensions were adjusted to be equal to the 0.5 McFarland standards by adding a sterile saline. Test and standard tubes were compared against a white background with a contrasting black line. Then the Suspensions concentrations were adjusted by spectrophotometer to an optical density of 0.10 at 625 nm to obtain the concentration of 1×108 CFU/ml. Finally, the cultures suspensions were then diluted to be equal to the concentration of 5×105 CFU/ml. Antimicrobial agents' preparations for the MIC and MBC experiments i. Dodonaea angustifolia The D. angustifolia plant was kindly obtained from Professor Doctor Fayez Zaki, Professor of entomology, biological control, plant protection, National Research Centre. The plant extract was extracted according to the method described in (Abd El-Moez et al., 2014) with some modification; briefly, the plant was collected, washed under tap water, rinsed with distilled water, dried in drying hot air oven at 40°C, and grinded. 50 grams of the shade resulting material were taken and put with continuous shaking in 500ml absolute ethanol for 24 hours. The extract was then filtered through a 22 μm filter paper then the resulting filtrate was combined and dried using a rotatory evaporator at 40°C. The previous procedure was repeated three times then 100 mg of the resulting powder materials were dissolved in 1ml dimethyl sulfoxide solvent (DMSO: Water, 2:4 v/v) to prepare the stock solution of 100 mg/ml. Then this stock will be 10 fold diluted to obtain 10 mg/ml then two-fold serial dilutions were done using MH broth to prepare the following concentrations (5, 2.5, 1.25, 0.625, 0.3, 0.15, 0.08, 0.04, 0.02 and 0.01 mg/ml). Fig 1 Dodonaea angustifolia plant ii. Honey Two 500 ml bottles of honey were purchased and used in this study; the first one was processed well-identified …show more content…
angustifolia extract, honey and AuNPs against the eight chosen S. aureus isolates (four MRSA+VRSA and four MRSA+VSSA) were analyzed through determination of their minimum inhibitory concentrations (MIC) values, minimum bactericidal concentrations (MBC) values, and MBC/MIC ratio by broth micro-dilution method using 96-well micro-titer plates. To each 50 μL of the antibacterial agent dilution, 50 μL of adjusted bacterial concentration inoculum (5 × 105 CFU/mL) were added in the 96-well micro-titer plates, the growth control wells contained MH broth medium with tested bacterial concentrations and sterility control wells contained only MH broth medium as shown in figure 2. The plates were then covered to ensure that the bacteria were not dehydrated. Then the plates were incubated at 37°C for 18 to 20 hours. The lowest concentration of each antibacterial agent that inhibited the bacterial growth was considered as the MIC (CLSI, 2006). After the MIC determination, aliquots of 100 μL from each well that does not show any bacterial growth after incubation were streaked onto BHI agar plates followed by incubation at 37°C for 20 hours. The lowest concentration that kills 100% of the initial bacterial population showing no colonies on the BHI agar was recorded as the