2.4. Antibacterial assays
The antibacterial assay of compounds 1–16 except 8 was evaluated
against Staphylococcus aureus ATCC 25922, Klepsiella pneumoniae ATCC
13883, Enterococcus faecalis ATCC 10541, Escherichia coli ATCC 11775
and Pseudomonas aeruginosa ATCC 27853. All the bacteria were obtained
from the American Type Culture Collection (Rockville, USA). The
antibacterial assay was carried out as described in the literature elsewhere
[16]. The preparation of bacterial inocula was done by using
18 h old overnight bacterial cultures prepared in Nutrient Agar. A few
colonies of bacteria were collected aseptically with a sterile loop and
introduced into 10 ml of sterile 0.90% saline solution. The concentration
of the suspensionwas then standardized by adjusting the optical density
to 0.10 at 630 nm, corresponding to bacterial cell suspension of 108
colony-forming units/mL (CFU/mL) [17]. This cell suspension was diluted
100 times to obtain 106 CFU/mL before use. The compounds were
dissolved in DMSO and then added to bacteria suspension to obtain the
final concentration of 5% (v/v) DMSO or less. Serial twofold dilutions
from 200 μg/mL of the compounds were performed in 96-well microtiter
plates. Each well contained 100 μL of sample of each concentration.
Into each well was then introduced 100 μL of the bacterial suspension.
The final concentration range of the test compounds was 100–0.781
μg/mL, and the plates were incubated at 37 °C for 24 h. After incubation,
the wells were examined for growth of microorganisms by measuring
optical density (OD) value of the wells. Each experiment was repeated
three times and Norfloxacin, bacteria suspension of 5% (v/v) DMSO
were used as a positive control and a blank control, respectively. By comparing
to OD values, we can point out MIC values of these compounds
among the selected concentration range and MIC N 100 μg/mL was
considered to be inactive.
2.4. Antibacterial assaysThe antibacterial assay of compounds 1–16 except 8 was evaluatedagainst Staphylococcus aureus ATCC 25922, Klepsiella pneumoniae ATCC13883, Enterococcus faecalis ATCC 10541, Escherichia coli ATCC 11775and Pseudomonas aeruginosa ATCC 27853. All the bacteria were obtainedfrom the American Type Culture Collection (Rockville, USA). Theantibacterial assay was carried out as described in the literature elsewhere[16]. The preparation of bacterial inocula was done by using18 h old overnight bacterial cultures prepared in Nutrient Agar. A fewcolonies of bacteria were collected aseptically with a sterile loop andintroduced into 10 ml of sterile 0.90% saline solution. The concentrationof the suspensionwas then standardized by adjusting the optical densityto 0.10 at 630 nm, corresponding to bacterial cell suspension of 108colony-forming units/mL (CFU/mL) [17]. This cell suspension was diluted100 times to obtain 106 CFU/mL before use. The compounds weredissolved in DMSO and then added to bacteria suspension to obtain thefinal concentration of 5% (v/v) DMSO or less. Serial twofold dilutionsfrom 200 μg/mL of the compounds were performed in 96-well microtiterplates. Each well contained 100 μL of sample of each concentration.Into each well was then introduced 100 μL of the bacterial suspension.The final concentration range of the test compounds was 100–0.781μg/mL, and the plates were incubated at 37 °C for 24 h. After incubation,the wells were examined for growth of microorganisms by measuringoptical density (OD) value of the wells. Each experiment was repeatedthree times and Norfloxacin, bacteria suspension of 5% (v/v) DMSOwere used as a positive control and a blank control, respectively. By comparingto OD values, we can point out MIC values of these compoundsamong the selected concentration range and MIC N 100 μg/mL wasconsidered to be inactive.
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